Expression and purification of ELP-intein-tagged target proteins in high cell density E. coli fermentation.

PubMed

Fong, Baley A; Wood, David W

2010-10-19

Elastin-like polypeptides (ELPs) are useful tools that can be used to non-chromatographically purify proteins. When paired with self-cleaving inteins, they can be used as economical self-cleaving purification tags. However, ELPs and ELP-tagged target proteins have been traditionally expressed using highly enriched media in shake flask cultures, which are generally not amenable to scale-up. In this work, we describe the high cell-density expression of self-cleaving ELP-tagged targets in a supplemented minimal medium at a 2.5 liter fermentation scale, with increased yields and purity compared to traditional shake flask cultures. This demonstration of ELP expression in supplemented minimal media is juxtaposed to previous expression of ELP tags in extract-based rich media. We also describe several sets of fed-batch conditions and their impact on ELP expression and growth medium cost. By using fed batch E. coli fermentation at high cell density, ELP-intein-tagged proteins can be expressed and purified at high yield with low cost. Further, the impact of media components and fermentation design can significantly impact the overall process cost, particularly at large scale. This work thus demonstrates an important advances in the scale up of self-cleaving ELP tag-mediated processes.

Expression and purification of ELP-intein-tagged target proteins in high cell density E. coli fermentation

PubMed Central

2010-01-01

Background Elastin-like polypeptides (ELPs) are useful tools that can be used to non-chromatographically purify proteins. When paired with self-cleaving inteins, they can be used as economical self-cleaving purification tags. However, ELPs and ELP-tagged target proteins have been traditionally expressed using highly enriched media in shake flask cultures, which are generally not amenable to scale-up. Results In this work, we describe the high cell-density expression of self-cleaving ELP-tagged targets in a supplemented minimal medium at a 2.5 liter fermentation scale, with increased yields and purity compared to traditional shake flask cultures. This demonstration of ELP expression in supplemented minimal media is juxtaposed to previous expression of ELP tags in extract-based rich media. We also describe several sets of fed-batch conditions and their impact on ELP expression and growth medium cost. Conclusions By using fed batch E. coli fermentation at high cell density, ELP-intein-tagged proteins can be expressed and purified at high yield with low cost. Further, the impact of media components and fermentation design can significantly impact the overall process cost, particularly at large scale. This work thus demonstrates an important advances in the scale up of self-cleaving ELP tag-mediated processes. PMID:20959011

Effects of plant density on recombinant hemagglutinin yields in an Agrobacterium-mediated transient gene expression system using Nicotiana benthamiana plants.

PubMed

Fujiuchi, Naomichi; Matsuda, Ryo; Matoba, Nobuyuki; Fujiwara, Kazuhiro

2017-08-01

Agrobacterium-mediated transient expression systems enable plants to rapidly produce a wide range of recombinant proteins. To achieve economically feasible upstream production and downstream processing, it is beneficial to obtain high levels of two yield-related quantities of upstream production: recombinant protein content per fresh mass of harvested biomass (g gFM -1 ) and recombinant protein productivity per unit area-time (g m -2 /month). Here, we report that the density of Nicotiana benthamiana plants during upstream production had significant impacts on the yield-related quantities of recombinant hemagglutinin (HA). The two quantities were smaller at a high plant density of 400 plants m -2 than at a low plant density of 100 plants m -2 . The smaller quantities at the high plant density were attributed to: (i) a lower HA content in young leaves, which usually have high HA accumulation potentials; (ii) a lower biomass allocation to the young leaves; and (iii) a high area-time requirement for plants. Thus, plant density is a key factor for improving upstream production in Agrobacterium-mediated transient expression systems. Biotechnol. Bioeng. 2017;114: 1762-1770. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Yield improvement of heterologous peptides expressed in yps1-disrupted Saccharomyces cerevisiae strains.

PubMed

Egel-Mitani; Andersen; Diers; Hach; Thim; Hastrup; Vad

2000-06-01

Heterologous protein expression levels in Saccharomyces cerevisiae fermentations are highly dependent on the susceptibility to endogenous yeast proteases. Small peptides, such as glucagon and glucagon-like-peptides (GLP-1 and GLP-2), featuring an open structure are particularly accessible for proteolytic degradation during fermentation. Therefore, homogeneous products cannot be obtained. The most sensitive residues are found at basic amino acid residues in the peptide sequence. These heterologous peptides are degraded mainly by the YPS1-encoded aspartic protease, yapsin1, when produced in the yeast. In this article, distinct degradation products were analyzed by HPLC and mass spectrometry, and high yield of the heterologous peptide production has been achieved by the disruption of the YPS1 gene (previously called YAP3). By this technique, high yield continuous fermentation of glucagon in S. cerevisiae is now possible.

The effect of high concentrations of glufosinate ammonium on the yield components of transgenic spring wheat (Triticum aestivum L.) constitutively expressing the bar gene.

PubMed

Áy, Zoltán; Mihály, Róbert; Cserháti, Mátyás; Kótai, Éva; Pauk, János

2012-01-01

We present an experiment done on a bar(+) wheat line treated with 14 different concentrations of glufosinate ammonium-an effective component of nonselective herbicides-during seed germination in a closed experimental system. Yield components as number of spikes per plant, number of grains per spike, thousand kernel weight, and yield per plant were thoroughly analysed and statistically evaluated after harvesting. We found that a concentration of glufosinate ammonium 5000 times the lethal dose was not enough to inhibit the germination of transgenic plants expressing the bar gene. Extremely high concentrations of glufosinate ammonium caused a bushy phenotype, significantly lower numbers of grains per spike, and thousand kernel weights. Concerning the productivity, we observed that concentrations of glufosinate ammonium 64 times the lethal dose did not lead to yield depression. Our results draw attention to the possibilities implied in the transgenic approaches.

Efficient production of soluble recombinant single chain Fv fragments by a Pseudomonas putida strain KT2440 cell factory.

PubMed

Dammeyer, Thorben; Steinwand, Miriam; Krüger, Sarah-C; Dübel, Stefan; Hust, Michael; Timmis, Kenneth N

2011-02-21

Recombinant antibody fragments have a wide range of applications in research, diagnostics and therapy. For many of these, small fragments like single chain fragment variables (scFv) function well and can be produced inexpensively in bacterial expression systems. Although Escherichia coli K-12 production systems are convenient, yields of different fragments, even those produced from codon-optimized expression systems, vary significantly. Where yields are inadequate, alternative production systems are needed. Pseudomonas putida strain KT2440 is a versatile biosafety strain known for good expression of heterologous genes, so we have explored its utility as a cell factory for production of scFvs. We have generated new broad host range scFv expression constructs and assessed their production in the Pseudomonas putida KT2440 host. Two scFvs bind either to human C-reactive protein or to mucin1, proteins of significant medical diagnostic and therapeutic interest, whereas a third is a model anti-lysozyme scFv. The KT2440 antibody expression systems produce scFvs targeted to the periplasmic space that were processed precisely and were easily recovered and purified by single-step or tandem affinity chromatography. The influence of promoter system, codon optimization for P. putida, and medium on scFv yield was examined. Yields of up to 3.5 mg/l of pure, soluble, active scFv fragments were obtained from shake flask cultures of constructs based on the original codon usage and expressed from the Ptac expression system, yields that were 2.5-4 times higher than those from equivalent cultures of an E. coli K-12 expression host. Pseudomonas putida KT2440 is a good cell factory for the production of scFvs, and the broad host range constructs we have produced allow yield assessment in a number of different expression hosts when yields in one initially selected are insufficient. High cell density cultivation and further optimization and refinement of the KT2440 cell factory will achieve additional increases in the yields of scFvs.

Novel, high-yield red blood cell production methods from CD34-positive cells derived from human embryonic stem, yolk sac, fetal liver, cord blood, and peripheral blood.

PubMed

Olivier, Emmanuel; Qiu, Caihong; Bouhassira, Eric E

2012-08-01

The current supply of red blood cells expressing rare blood groups is not sufficient to cover all the existing transfusion needs for chronically transfused patients, such as sickle cell disease homozygous carriers, because of alloimmunization. In vitro production of cultured red blood cells is slowly emerging as a possible complement to the existing collection-based red blood cell procurement system. The yield of cultured red blood cells can theoretically be maximized by amplifying the stem, progenitor, or precursor compartment. Here, we combined methods designed to expand these three compartments to optimize the yield of cultured red blood cells and found that exposing CD34(+) cells to a short pulse of cytokines favorable for erythroid differentiation prior to stem cell expansion followed by progenitor expansion produced the highest yield of erythroid cells. This novel serum-free red blood cell production protocol was efficient on CD34(+) cells derived from human embryonic stem cells, 6-8-week yolk sacs, 16-18-week fetal livers, cord blood, and peripheral blood. The yields of cells obtained with these new protocols were larger by an order of magnitude than the yields observed previously. Globin expression analysis by high-performance liquid chromatography revealed that these expansion protocols generally yielded red blood cells that expressed a globin profile similar to that expected for the developmental age of the CD34(+) cells.

Technology of compact MAb and its application for medicinal plant breeding named as missile type molecular breeding.

PubMed

Putalun, Waraporn

2011-03-01

Single chain fragment-variable (scFv) enhanced solasodine glycoside accumulation in Solanum khasianum hairy root cultures transformed by the ScFv solamargine (As)-scFv gene. The scFv protein was expressed at a high level in inclusion bodies of E. coli. After being renatured, the scFv protein was purified in a one-step manner by metal chelate affinity chromatography. The yield of refolded and purified scFv was 12.5 mg per 100 ml of cell culture. The characteristics of the As-scFv expressed in E. coli and transgenic hairy roots were similar to those of the parent monoclonal antibody (MAb). The expression of scFv protein provides a low cost and a high yield of functional scFv antibody against solamargine. The full linear range of the ELISA assay using scFv was extended from 1.5-10 µg/ml. The expressed anti-solamargine scFv protein could be useful for determination of total solasodine glycoside content in plant samples by ELISA. Solasodine glycoside levels in the transgenic hairy root were 2.3-fold higher than that in the wild-type hairy root based on the soluble protein level and binding activities. The As-scFv expressed in S. khasianum hairy roots enhanced solasodine glycosides accumulation and provide a novel medicinal plant breeding methodology that can produce a high yield of secondary metabolites.

High-yield, in vitro protein expression using a continuous-exchange, coupled transcription/ translation system.

PubMed

Martin, G A; Kawaguchi, R; Lam, Y; DeGiovanni, A; Fukushima, M; Mutter, W

2001-10-01

The Rapid Translation System (RTS 500) (Roche Molecular Biochemicals) is a high-yield protein expression system that utilizes an enhanced E. coli lysate for an in vitro transcription/translation reaction. In contrast to conventional transcription/translation, this system allows protein expression to continue for more than 24 h. We demonstrated the utility of the RTS 500 by expressing different soluble and active proteins that generally pose problems in cell-based expression systems. We first expressed GFP-lunasin, a fusion protein that, because of its toxicity, has been impossible to produce in whole cells. The second protein we expressed, human interleukin-2 (IL-2), is generally difficult to produce, either as the native molecule or as a GSTfusion protein, in a soluble form in bacteria. Finally, we demonstrated the capacity of the RTS 500 to co-express proteins, by the simultaneous production of GFP and CAT in a single reaction. This new technology appears to be particularly usefulfor the convenient production of preparative amounts (100-900 microg) of proteins that are toxic or insoluble in cell-based systems.

Ectopic expression of Arabidopsis Target of Rapamycin (AtTOR) improves water-use efficiency and yield potential in rice

NASA Astrophysics Data System (ADS)

Bakshi, Achala; Moin, Mazahar; Kumar, M. Udaya; Reddy, Aramati Bindu Madhava; Ren, Maozhi; Datla, Raju; Siddiq, E. A.; Kirti, P. B.

2017-02-01

The target of Rapamycin (TOR) present in all eukaryotes is a multifunctional protein, regulating growth, development, protein translation, ribosome biogenesis, nutrient, and energy signaling. In the present study, ectopic expression of TOR gene of Arabidopsis thaliana in a widely cultivated indica rice resulted in enhanced plant growth under water-limiting conditions conferring agronomically important water-use efficiency (WUE) trait. The AtTOR high expression lines of rice exhibited profuse tillering, increased panicle length, increased plant height, high photosynthetic efficiency, chlorophyll content and low Δ13C. Δ13C, which is inversely related to high WUE, was as low as 17‰ in two AtTOR high expression lines. These lines were also insensitive to the ABA-mediated inhibition of seed germination. The significant upregulation of 15 stress-specific genes in high expression lines indicates their contribution to abiotic stress tolerance. The constitutive expression of AtTOR is also associated with significant transcriptional upregulation of putative TOR complex-1 components, OsRaptor and OsLST8. Glucose-mediated transcriptional activation of AtTOR gene enhanced lateral root formation. Taken together, our findings indicate that TOR, in addition to its multiple cellular functions, also plays an important role in response to abiotic stress and potentially enhances WUE and yield related attributes.

VARIABILITY AND CHARACTER ASSOCIATION IN ROSE COLOURED LEADWORT (PLUMBAGO ROSEA Linn.)

PubMed Central

Kurian, Alice; Anitha, C.A.; Nybe, E.V.

2001-01-01

Forty five plumbago rosea accessions collected from different parts of Kerala state were evaluated for variability in morphological and yield related characters and plumbagin content. Highly significant variation was evident for all the characters studied except leaf size indicating wide variability in the accessions. Accessions PR 25 and PR 31 appear to be promising with respect to root yield and high plumbagin content. Character association revelated significant and positive correlation of all the characters except leaf size with yield. Hence, selection of high yielding types could easily be done based on visual characters expressing more vegetative growth but with reduced leaf size. PMID:22557037

Tightly-Coupled Plant-Soil Nitrogen Cycling: Comparison of Organic Farms across an Agricultural Landscape

PubMed Central

Bowles, Timothy M.; Hollander, Allan D.; Steenwerth, Kerri; Jackson, Louise E.

2015-01-01

How farming systems supply sufficient nitrogen (N) for high yields but with reduced N losses is a central challenge for reducing the tradeoffs often associated with N cycling in agriculture. Variability in soil organic matter and management of organic farms across an agricultural landscape may yield insights for improving N cycling and for evaluating novel indicators of N availability. We assessed yields, plant-soil N cycling, and root expression of N metabolism genes across a representative set of organic fields growing Roma-type tomatoes (Solanum lycopersicum L.) in an intensively-managed agricultural landscape in California, USA. The fields spanned a three-fold range of soil carbon (C) and N but had similar soil types, texture, and pH. Organic tomato yields ranged from 22.9 to 120.1 Mg ha-1 with a mean similar to the county average (86.1 Mg ha-1), which included mostly conventionally-grown tomatoes. Substantial variability in soil inorganic N concentrations, tomato N, and root gene expression indicated a range of possible tradeoffs between yields and potential for N losses across the fields. Fields showing evidence of tightly-coupled plant-soil N cycling, a desirable scenario in which high crop yields are supported by adequate N availability but low potential for N loss, had the highest total and labile soil C and N and received organic matter inputs with a range of N availability. In these fields, elevated expression of a key gene involved in root N assimilation, cytosolic glutamine synthetase GS1, confirmed that plant N assimilation was high even when inorganic N pools were low. Thus tightly-coupled N cycling occurred on several working organic farms. Novel combinations of N cycling indicators (i.e. inorganic N along with soil microbial activity and root gene expression for N assimilation) would support adaptive management for improved N cycling on organic as well as conventional farms, especially when plant-soil N cycling is rapid. PMID:26121264

Tightly-Coupled Plant-Soil Nitrogen Cycling: Comparison of Organic Farms across an Agricultural Landscape.

PubMed

Bowles, Timothy M; Hollander, Allan D; Steenwerth, Kerri; Jackson, Louise E

2015-01-01

How farming systems supply sufficient nitrogen (N) for high yields but with reduced N losses is a central challenge for reducing the tradeoffs often associated with N cycling in agriculture. Variability in soil organic matter and management of organic farms across an agricultural landscape may yield insights for improving N cycling and for evaluating novel indicators of N availability. We assessed yields, plant-soil N cycling, and root expression of N metabolism genes across a representative set of organic fields growing Roma-type tomatoes (Solanum lycopersicum L.) in an intensively-managed agricultural landscape in California, USA. The fields spanned a three-fold range of soil carbon (C) and N but had similar soil types, texture, and pH. Organic tomato yields ranged from 22.9 to 120.1 Mg ha-1 with a mean similar to the county average (86.1 Mg ha-1), which included mostly conventionally-grown tomatoes. Substantial variability in soil inorganic N concentrations, tomato N, and root gene expression indicated a range of possible tradeoffs between yields and potential for N losses across the fields. Fields showing evidence of tightly-coupled plant-soil N cycling, a desirable scenario in which high crop yields are supported by adequate N availability but low potential for N loss, had the highest total and labile soil C and N and received organic matter inputs with a range of N availability. In these fields, elevated expression of a key gene involved in root N assimilation, cytosolic glutamine synthetase GS1, confirmed that plant N assimilation was high even when inorganic N pools were low. Thus tightly-coupled N cycling occurred on several working organic farms. Novel combinations of N cycling indicators (i.e. inorganic N along with soil microbial activity and root gene expression for N assimilation) would support adaptive management for improved N cycling on organic as well as conventional farms, especially when plant-soil N cycling is rapid.

Expression of the Nitrate Transporter Gene OsNRT1.1A/OsNPF6.3 Confers High Yield and Early Maturation in Rice.

PubMed

Wang, Wei; Hu, Bin; Yuan, Dingyang; Liu, Yongqiang; Che, Ronghui; Hu, Yingchun; Ou, Shujun; Liu, Yongxin; Zhang, Zhihua; Wang, Hongru; Li, Hua; Jiang, Zhimin; Zhang, Zhengli; Gao, Xiaokai; Qiu, Yahong; Meng, Xiangbing; Liu, Yongxin; Bai, Yang; Liang, Yan; Wang, Yiqin; Zhang, Lianhe; Li, Legong; Sodmergen; Jing, Haichun; Li, Jiayang; Chu, Chengcai

2018-03-01

Nitrogen (N) is a major driving force for crop yield improvement, but application of high levels of N delays flowering, prolonging maturation and thus increasing the risk of yield losses. Therefore, traits that enable utilization of high levels of N without delaying maturation will be highly desirable for crop breeding. Here, we show that OsNRT1.1A (OsNPF6.3), a member of the rice ( Oryza sativa ) nitrate transporter 1/peptide transporter family, is involved in regulating N utilization and flowering, providing a target to produce high yield and early maturation simultaneously. OsNRT.1A has functionally diverged from previously reported NRT1.1 genes in plants and functions in upregulating the expression of N utilization-related genes not only for nitrate but also for ammonium, as well as flowering-related genes. Relative to the wild type, osnrt1.1a mutants exhibited reduced N utilization and late flowering. By contrast, overexpression of OsNRT1.1A in rice greatly improved N utilization and grain yield, and maturation time was also significantly shortened. These effects were further confirmed in different rice backgrounds and also in Arabidopsis thaliana Our study paves a path for the use of a single gene to dramatically increase yield and shorten maturation time for crops, outcomes that promise to substantially increase world food security. © 2018 American Society of Plant Biologists. All rights reserved.

Expression of the Nitrate Transporter Gene OsNRT1.1A/OsNPF6.3 Confers High Yield and Early Maturation in Rice[OPEN

PubMed Central

Wang, Wei; Hu, Bin; Liu, Yongqiang; Che, Ronghui; Hu, Yingchun; Zhang, Zhihua; Wang, Hongru; Li, Hua; Jiang, Zhimin; Zhang, Zhengli; Gao, Xiaokai; Qiu, Yahong; Meng, Xiangbing; Liu, Yongxin; Bai, Yang; Liang, Yan; Wang, Yiqin; Zhang, Lianhe; Li, Legong; Sodmergen; Jing, Haichun

2018-01-01

Nitrogen (N) is a major driving force for crop yield improvement, but application of high levels of N delays flowering, prolonging maturation and thus increasing the risk of yield losses. Therefore, traits that enable utilization of high levels of N without delaying maturation will be highly desirable for crop breeding. Here, we show that OsNRT1.1A (OsNPF6.3), a member of the rice (Oryza sativa) nitrate transporter 1/peptide transporter family, is involved in regulating N utilization and flowering, providing a target to produce high yield and early maturation simultaneously. OsNRT.1A has functionally diverged from previously reported NRT1.1 genes in plants and functions in upregulating the expression of N utilization-related genes not only for nitrate but also for ammonium, as well as flowering-related genes. Relative to the wild type, osnrt1.1a mutants exhibited reduced N utilization and late flowering. By contrast, overexpression of OsNRT1.1A in rice greatly improved N utilization and grain yield, and maturation time was also significantly shortened. These effects were further confirmed in different rice backgrounds and also in Arabidopsis thaliana. Our study paves a path for the use of a single gene to dramatically increase yield and shorten maturation time for crops, outcomes that promise to substantially increase world food security. PMID:29475937

High-yield recombinant expression and purification of marginally soluble, short elastin-like polypeptides.

PubMed

Bahniuk, Markian S; Alshememry, Abdullah K; Unsworth, Larry D

2016-12-01

The protocol described here is designed as an extension of existing techniques for creating elastin-like polypeptides. It allows for the expression and purification of elastin-like polypeptide (ELP) constructs that are poorly expressed or have very low transition temperatures. DNA concatemerization has been modified to reduce issues caused by methylation sensitivity and inefficient cloning. Linearization of the modified expression vector has been altered to greatly increase cleavage efficiency. The purification regimen is based upon using denaturing metal affinity chromatography to fully solubilize and, if necessary, pre-concentrate the target peptide before purification by inverse temperature cycling (ITC). This protocol has been used to express multiple leucine-containing elastin-like polypeptides, with final yields of 250-660 mg per liter of cells, depending on the specific construct. This was considerably greater than previously reported yields for similar ELPs. Due to the relative hydrophobicity of the tested constructs, even compared with commonly employed ELPs, conventional methods would not have been able to be purify these peptides.

Optimization of supercritical carbon dioxide extraction of Piper Betel Linn leaves oil and total phenolic content

NASA Astrophysics Data System (ADS)

Aziz, A. H. A.; Yunus, M. A. C.; Arsad, N. H.; Lee, N. Y.; Idham, Z.; Razak, A. Q. A.

2016-11-01

Supercritical Carbon Dioxide (SC-CO2) Extraction was applied to extract piper betel linn leaves. The piper betel leaves oil was used antioxidant, anti-diabetic, anticancer and antistroke. The aim of this study was to optimize the conditions of pressure, temperature and flowrate for oil yield and total phenolic content. The operational conditions of SC-CO2 studied were pressure (10, 20, 30 MPa), temperature (40, 60, 80 °C) and flowrate carbon dioxide (4, 6, 8 mL/min). The constant parameters were average particle size and extraction regime, 355pm and 3.5 hours respectively. First order polynomial expression was used to express the extracted oil while second order polynomial expression was used to express the total phenolic content and the both results were satisfactory. The best conditions to maximize the total extraction oil yields and total phenolic content were 30 MPa, 80 °C and 4.42 mL/min leading to 7.32% of oil and 29.72 MPa, 67.53 °C and 7.98 mL/min leading to 845.085 mg GAE/g sample. In terms of optimum condition with high extraction yield and high total phenolic content in the extracts, the best operating conditions were 30 MPa, 78 °C and 8 mL/min with 7.05% yield and 791.709 mg gallic acid equivalent (GAE)/g sample. The most dominant condition for extraction of oil yield and phenolic content were pressure and CO2 flowrate. The results show a good fit to the proposed model and the optimal conditions obtained were within the experimental range with the value of R2 was 96.13% for percentage yield and 98.52% for total phenolic content.

The growing impact of lyophilized cell-free protein expression systems

PubMed Central

Hunt, J. Porter; Yang, Seung Ook; Wilding, Kristen M.; Bundy, Bradley C.

2017-01-01

ABSTRACT Recently reported shelf-stable, on-demand protein synthesis platforms are enabling new possibilities in biotherapeutics, biosensing, biocatalysis, and high throughput protein expression. Lyophilized cell-free protein expression systems not only overcome cold-storage limitations, but also enable stockpiling for on-demand synthesis and completely sterilize the protein synthesis platform. Recently reported high-yield synthesis of cytotoxic protein Onconase from lyophilized E. coli extract preparations demonstrates the utility of lyophilized cell-free protein expression and its potential for creating on-demand biotherapeutics, vaccines, biosensors, biocatalysts, and high throughput protein synthesis. PMID:27791452

Magnetic cell sorting purification of differentiated embryonic stem cells stably expressing truncated human CD4 as surface marker.

PubMed

David, Robert; Groebner, Michael; Franz, Wolfgang-Michael

2005-04-01

Embryonic stem (ES) cells offer great potential in regenerative medicine and tissue engineering. Clinical applications are still hampered by the lack of protocols for gentle, high-yield isolation of specific cell types for transplantation expressing no immunogenic markers. We describe labeling of stably transfected ES cells expressing a human CD4 molecule lacking its intracellular domain (DeltaCD4) under control of the phosphoglycerate kinase promoter for magnetic cell sorting (MACS). To track the labeled ES cells, we fused DeltaCD4 to an intracellular enhanced green fluorescent protein domain (DeltaCD4EGFP). We showed functionality of the membrane-bound fluorescent fusion protein and its suitability for MACS leading to purities greater than 97%. Likewise, expression of DeltaCD4 yielded up to 98.5% positive cells independently of their differentiation state. Purities were not limited by the initial percentage of DeltaCD4(+) cells, ranging from 0.6%-16%. The viability of MACS-selected cells was demonstrated by reaggregation and de novo formation of embryoid bodies developing all three germ layers. Thus, expression of DeltaCD4 in differentiated ES cells may enable rapid, high-yield purification of a desired cell type for tissue engineering and transplantation studies.

High Level Expression and Purification of Recombinant Proteins from Escherichia coli with AK-TAG

PubMed Central

Luo, Dan; Wen, Caixia; Zhao, Rongchuan; Liu, Xinyu; Liu, Xinxin; Cui, Jingjing; Liang, Joshua G.; Liang, Peng

2016-01-01

Adenylate kinase (AK) from Escherichia coli was used as both solubility and affinity tag for recombinant protein production. When fused to the N-terminus of a target protein, an AK fusion protein could be expressed in soluble form and purified to near homogeneity in a single step from Blue-Sepherose via affinity elution with micromolar concentration of P1, P5- di (adenosine—5’) pentaphosphate (Ap5A), a transition-state substrate analog of AK. Unlike any other affinity tags, the level of a recombinant protein expression in soluble form and its yield of recovery during each purification step could be readily assessed by AK enzyme activity in near real time. Coupled to a His-Tag installed at the N-terminus and a thrombin cleavage site at the C terminus of AK, the streamlined method, here we dubbed AK-TAG, could also allow convenient expression and retrieval of a cleaved recombinant protein in high yield and purity via dual affinity purification steps. Thus AK-TAG is a new addition to the arsenal of existing affinity tags for recombinant protein expression and purification, and is particularly useful where soluble expression and high degree of purification are at stake. PMID:27214237

Increased phytochrome B alleviates density effects on tuber yield of field potato crops.

PubMed

Boccalandro, Hernán E; Ploschuk, Edmundo L; Yanovsky, Marcelo J; Sánchez, Rodolfo A; Gatz, Christiane; Casal, Jorge J

2003-12-01

The possibility that reduced photomorphogenic responses could increase field crop yield has been suggested often, but experimental support is still lacking. Here, we report that ectopic expression of the Arabidopsis PHYB (phytochrome B) gene, a photoreceptor involved in detecting red to far-red light ratio associated with plant density, can increase tuber yield in field-grown transgenic potato (Solanum tuberosum) crops. Surprisingly, this effect was larger at very high densities, despite the intense reduction in the red to far-red light ratios and the concomitant narrowed differences in active phytochrome B levels between wild type and transgenics at these densities. Increased PHYB expression not only altered the ability of plants to respond to light signals, but they also modified the light environment itself. This combination resulted in larger effects of enhanced PHYB expression on tuber number and crop photosynthesis at high planting densities. The PHYB transgenics showed higher maximum photosynthesis in leaves of all strata of the canopy, and this effect was largely due to increased leaf stomatal conductance. We propose that enhanced PHYB expression could be used in breeding programs to shift optimum planting densities to higher levels.

MicroRNA-34a regulation of endothelial senescence

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ito, Takashi; Yagi, Shusuke; Yamakuchi, Munekazu, E-mail: munekazu_yamakuchi@urmc.rochester.edu

2010-08-06

Research highlights: {yields} MicroRNA-34a (miR-34a) regulates senescence and cell cycle progression in endothelial cells. {yields} MiR-34a expression increases during endothelial cell senescence and in older mice. {yields} SIRT1 is a miR-34a target gene in endothelial cells. {yields} SIRT1 mediates the effects of miR-34a upon cell senescence in endothelial cells. -- Abstract: Endothelial senescence is thought to play a role in cardiovascular diseases such as atherosclerosis. We hypothesized that endothelial microRNAs (miRNAs) regulate endothelial survival and senescence. We found that miR-34a is highly expressed in primary endothelial cells. We observed that miR-34a expression increases in senescent human umbilical cord vein endothelialmore » cells (HUVEC) and in heart and spleen of older mice. MiR-34a over-expression induces endothelial cell senescence and also suppresses cell proliferation by inhibiting cell cycle progression. Searching for how miR-34a affects senescence, we discovered that SIRT1 is a target of miR-34a. Over-expressing miR-34a inhibits SIRT1 protein expression, and knocking down miR-34a enhances SIRT1 expression. MiR-34a triggers endothelial senescence in part through SIRT1, since forced expression of SIRT1 blocks the ability of miR-34a to induce senescence. Our data suggest that miR-34a contributes to endothelial senescence through suppression of SIRT1.« less

TPS1 terminator increases mRNA and protein yield in a Saccharomyces cerevisiae expression system.

PubMed

Yamanishi, Mamoru; Katahira, Satoshi; Matsuyama, Takashi

2011-01-01

Both terminators and promoters regulate gene expression. In Saccharomyces cerevisiae, the TPS1 terminator (TPS1t), coupled to a gene encoding a fluorescent protein, produced more transgenic mRNA and protein than did similar constructs containing other terminators, such as CYC1t, TDH3t, and PGK1t. This suggests that TPS1t can be used as a general terminator in the development of metabolically engineered yeast in high-yield systems.

Inducing mutations through γ-irradiation in seeds of Mucuna pruriens for developing high L-DOPA-yielding genotypes.

PubMed

Singh, Susheel Kumar; Yadav, Deepti; Lal, Raj Kishori; Gupta, Madan M; Dhawan, Sunita Singh

2017-04-01

To develop elite genotypes in Mucuna pruriens (L.) DC with high L-DOPA (L-3, 4 dihydroxyphenylalanine) yields, with non-itching characteristics and better adaptability by applying γ-irradiation. Molecular and chemical analysis was performed for screening based on specific characteristics desired for developing suitable genotypes. Developed, mutant populations were analyzed for L-DOPA % in seeds through TLC (thin layer chromatography), and the results obtained were validated with the HPLC (High performance liquid chromatography). The DNA (Deoxyribonucleic acid) was isolated from the leaf at the initial stage and used for DNA polymorphism. RNA (Ribonucleic acid) was isolated from the leaf during maturity and used for expression analysis. The selected mutant T-I-7 showed 5.7% L-DOPA content compared to 3.18% of parent CIM-Ajar. The total polymorphism obtained was 57% with the molecular marker analysis. The gene expression analysis showed higher fold change expression of the dopadecarboxylase gene (DDC) in control compared to selected mutants (T-I-7, T-II-23, T-IV-9, T-VI-1). DNA polymorphism was used for the screening of mutants for efficient screening at an early stage. TLC was found suitable for the large-scale comparative chemical analysis of L-DOPA. The expression profile of DDC clearly demonstrated the higher yields of L-DOPA in selected mutants developed by γ-irradiation in the seeds of the control.

Expression of the Arabidopsis thaliana BBX32 gene in soybean increases grain yield.

PubMed

Preuss, Sasha B; Meister, Robert; Xu, Qingzhang; Urwin, Carl P; Tripodi, Federico A; Screen, Steven E; Anil, Veena S; Zhu, Shuquan; Morrell, James A; Liu, Grace; Ratcliffe, Oliver J; Reuber, T Lynne; Khanna, Rajnish; Goldman, Barry S; Bell, Erin; Ziegler, Todd E; McClerren, Amanda L; Ruff, Thomas G; Petracek, Marie E

2012-01-01

Crop yield is a highly complex quantitative trait. Historically, successful breeding for improved grain yield has led to crop plants with improved source capacity, altered plant architecture, and increased resistance to abiotic and biotic stresses. To date, transgenic approaches towards improving crop grain yield have primarily focused on protecting plants from herbicide, insects, or disease. In contrast, we have focused on identifying genes that, when expressed in soybean, improve the intrinsic ability of the plant to yield more. Through the large scale screening of candidate genes in transgenic soybean, we identified an Arabidopsis thaliana B-box domain gene (AtBBX32) that significantly increases soybean grain yield year after year in multiple transgenic events in multi-location field trials. In order to understand the underlying physiological changes that are associated with increased yield in transgenic soybean, we examined phenotypic differences in two AtBBX32-expressing lines and found increases in plant height and node, flower, pod, and seed number. We propose that these phenotypic changes are likely the result of changes in the timing of reproductive development in transgenic soybean that lead to the increased duration of the pod and seed development period. Consistent with the role of BBX32 in A. thaliana in regulating light signaling, we show that the constitutive expression of AtBBX32 in soybean alters the abundance of a subset of gene transcripts in the early morning hours. In particular, AtBBX32 alters transcript levels of the soybean clock genes GmTOC1 and LHY-CCA1-like2 (GmLCL2). We propose that through the expression of AtBBX32 and modulation of the abundance of circadian clock genes during the transition from dark to light, the timing of critical phases of reproductive development are altered. These findings demonstrate a specific role for AtBBX32 in modulating soybean development, and demonstrate the validity of expressing single genes in crops to deliver increased agricultural productivity.

Improved expression of recombinant plant-made hEGF.

PubMed

Thomas, David Rhys; Walmsley, Amanda Maree

2014-11-01

The yield of recombinant hEGF was increased approximately tenfold through a range of optimisations. Further, the recombinant protein was found to have biological activity comparable to commercial hEGF. Human epidermal growth factor (hEGF) is a powerful mitogen that can enhance the healing of a wide range of injuries, including burns, cuts, diabetic ulcers and gastric ulcers. However, despite its clinical value, hEGF is only consistently used for the treatment of chronic diabetic ulcers due to its high cost. In this study, hEGF was transiently expressed in Nicotiana benthamiana plants and targeted to the apoplast, ER and vacuole. Several other approaches were also included in a stepwise fashion to identify the optimal conditions for the expression of recombinant hEGF. Expression was found to be highest in the vacuole, while targeting hEGF to the ER caused a decrease in total soluble protein (TSP). Using a codon optimised sequence was found to increase vacuolar targeted hEGF yield by ~34 %, while it was unable to increase the yield of ER targeted hEGF. The use of the P19 silencing inhibitor was able to further increase expression by over threefold, and using 5-week-old plants significantly increased expression compared to 4- or 6-week-old-plants. The combined effect of these optimisations increased expression tenfold over the initial apoplast targeted construct to an average yield of 6.24 % of TSP. The plant-made hEGF was then shown to be equivalent to commercial E. coli derived hEGF in its ability to promote the proliferation of mouse keratinocytes. This study supports the potential for plants to be used for the commercial production of hEGF, and identifies a potential limitation for the further improvement of recombinant protein yields.

Genetic Engineering of Maize (Zea mays L.) with Improved Grain Nutrients.

PubMed

Guo, Xiaotong; Duan, Xiaoguang; Wu, Yongzhen; Cheng, Jieshan; Zhang, Juan; Zhang, Hongxia; Li, Bei

2018-02-21

Cell-wall invertase plays important roles in the grain filling of crop plants. However, its functions in the improvement of grain nutrients have not been investigated. In this work, the stable expression of cell-wall-invertase-encoding genes from different plant species and the contents of total starch, protein, amino acid, nitrogen, lipid, and phosphorus were examined in transgenic maize plants. High expressions of the cell-wall-invertase gene conferred enhanced invertase activity and sugar content in transgenic plants, leading to increased grain yield and improved grain nutrients. Transgenic plants with high expressions of the transgene produced more total starch, protein, nitrogen, and essential amino acids in the seeds. Overall, the results indicate that the cell-wall-invertase gene can be used as a potential candidate for the genetic breeding of grain crops with both improved grain yield and quality.

Comparative 2D-DIGE proteomic analysis of bovine mammary epithelial cells during lactation reveals protein signatures for lactation persistency and milk yield.

PubMed

Janjanam, Jagadeesh; Singh, Surender; Jena, Manoj K; Varshney, Nishant; Kola, Srujana; Kumar, Sudarshan; Kaushik, Jai K; Grover, Sunita; Dang, Ajay K; Mukesh, Manishi; Prakash, B S; Mohanty, Ashok K

2014-01-01

Mammary gland is made up of a branching network of ducts that end with alveoli which surrounds the lumen. These alveolar mammary epithelial cells (MEC) reflect the milk producing ability of farm animals. In this study, we have used 2D-DIGE and mass spectrometry to identify the protein changes in MEC during immediate early, peak and late stages of lactation and also compared differentially expressed proteins in MEC isolated from milk of high and low milk producing cows. We have identified 41 differentially expressed proteins during lactation stages and 22 proteins in high and low milk yielding cows. Bioinformatics analysis showed that a majority of the differentially expressed proteins are associated in metabolic process, catalytic and binding activity. The differentially expressed proteins were mapped to the available biological pathways and networks involved in lactation. The proteins up-regulated during late stage of lactation are associated with NF-κB stress induced signaling pathways and whereas Akt, PI3K and p38/MAPK signaling pathways are associated with high milk production mediated through insulin hormone signaling.

ADAM17 limits the expression of CSF1R on murine hematopoietic progenitors

PubMed Central

Becker, Amy M.; Walcheck, Bruce; Bhattacharya, Deepta

2014-01-01

All-lymphoid progenitors (ALPs) yield few myeloid cells in vivo, but readily generate such cells in vitro. The basis for this difference remains unknown. We hypothesized that ALPs limit responsiveness to in vivo concentrations of myeloid-promoting cytokines by reducing expression of the corresponding receptors, potentially through post-transcriptional mechanisms. Consistent with such a mechanism, ALPs express higher levels of Csf1r transcripts than their upstream precursors, yet show limited cell surface protein expression of CSF1R. ALPs and other hematopoietic progenitors deficient in ADAM17, a metalloprotease that can cleave CSF1R, display elevated cell surface CSF1R expression. Adam17−/− ALPs, however, fail to yield myeloid cells upon transplantation into irradiated recipients. Moreover, Adam17−/− ALPs yield fewer macrophages in vitro than control ALPs at high concentrations of M-CSF. Mice with hematopoietic-specific deletion of Adam17 have grossly normal numbers of myeloid and lymphoid progenitors and mature cells in vivo. These data demonstrate that ADAM17 limits CSF1R protein expression on hematopoietic progenitors, but that compensatory mechanisms prevent elevated CSF1R levels from altering lymphoid progenitor potential. PMID:25308957

Performance benchmarking of four cell-free protein expression systems.

PubMed

Gagoski, Dejan; Polinkovsky, Mark E; Mureev, Sergey; Kunert, Anne; Johnston, Wayne; Gambin, Yann; Alexandrov, Kirill

2016-02-01

Over the last half century, a range of cell-free protein expression systems based on pro- and eukaryotic organisms have been developed and have found a range of applications, from structural biology to directed protein evolution. While it is generally accepted that significant differences in performance among systems exist, there is a paucity of systematic experimental studies supporting this notion. Here, we took advantage of the species-independent translation initiation sequence to express and characterize 87 N-terminally GFP-tagged human cytosolic proteins of different sizes in E. coli, wheat germ (WGE), HeLa, and Leishmania-based (LTE) cell-free systems. Using a combination of single-molecule fluorescence spectroscopy, SDS-PAGE, and Western blot analysis, we assessed the expression yields, the fraction of full-length translation product, and aggregation propensity for each of these systems. Our results demonstrate that the E. coli system has the highest expression yields. However, we observe that high expression levels are accompanied by production of truncated species-particularly pronounced in the case of proteins larger than 70 kDa. Furthermore, proteins produced in the E. coli system display high aggregation propensity, with only 10% of tested proteins being produced in predominantly monodispersed form. The WGE system was the most productive among eukaryotic systems tested. Finally, HeLa and LTE show comparable protein yields that are considerably lower than the ones achieved in the E. coli and WGE systems. The protein products produced in the HeLa system display slightly higher integrity, whereas the LTE-produced proteins have the lowest aggregation propensity among the systems analyzed. The high quality of HeLa- and LTE-produced proteins enable their analysis without purification and make them suitable for analysis of multi-domain eukaryotic proteins. © 2015 Wiley Periodicals, Inc.

Insights from the Fungus Fusarium oxysporum Point to High Affinity Glucose Transporters as Targets for Enhancing Ethanol Production from Lignocellulose

PubMed Central

Ali, Shahin S.; Nugent, Brian; Mullins, Ewen; Doohan, Fiona M.

2013-01-01

Ethanol is the most-widely used biofuel in the world today. Lignocellulosic plant biomass derived from agricultural residue can be converted to ethanol via microbial bioprocessing. Fungi such as Fusarium oxysporum can simultaneously saccharify straw to sugars and ferment sugars to ethanol. But there are many bottlenecks that need to be overcome to increase the efficacy of microbial production of ethanol from straw, not least enhancement of the rate of fermentation of both hexose and pentose sugars. This research tested the hypothesis that the rate of sugar uptake by F. oxysporum would enhance the ethanol yields from lignocellulosic straw and that high affinity glucose transporters can enhance ethanol yields from this substrate. We characterized a novel hexose transporter (Hxt) from this fungus. The F. oxysporum Hxt represents a novel transporter with homology to yeast glucose signaling/transporter proteins Rgt2 and Snf3, but it lacks their C-terminal domain which is necessary for glucose signalling. Its expression level decreased with increasing glucose concentration in the medium and in a glucose uptake study the Km(glucose) was 0.9 mM, which indicated that the protein is a high affinity glucose transporter. Post-translational gene silencing or over expression of the Hxt in F. oxysporum directly affected the glucose and xylose transport capacity and ethanol yielded by F. oxysporum from straw, glucose and xylose. Thus we conclude that this Hxt has the capacity to transport both C5 and C6 sugars and to enhance ethanol yields from lignocellulosic material. This study has confirmed that high affinity glucose transporters are ideal candidates for improving ethanol yields from lignocellulose because their activity and level of expression is high in low glucose concentrations, which is very common during the process of consolidated processing. PMID:23382943

Methods for high yield production of terpenes

DOEpatents

Kutchan, Toni; Higashi, Yasuhiro; Feng, Xiaohong

2017-01-03

Provided are enhanced high yield production systems for producing terpenes in plants via the expression of fusion proteins comprising various combinations of geranyl diphosphate synthase large and small subunits and limonene synthases. Also provided are engineered oilseed plants that accumulate monoterpene and sesquiterpene hydrocarbons in their seeds, as well as methods for producing such plants, providing a system for rapidly engineering oilseed crop production platforms for terpene-based biofuels.

Enhancement of recombinant BmK AngM1 production in Pichia pastoris by regulating gene dosage, co-expressing with chaperones and fermenting in fed-batch mode.

PubMed

Wang, Qing-Hua; Liang, Lan; Liu, Wan-Cang; Gong, Ting; Chen, Jing-Jing; Hou, Qi; Yang, Jin-Ling; Zhu, Ping

2017-06-01

The scorpion peptide BmK AngM1 was reported to exhibit evident analgesic effect, but its yield by extraction from scorpion venom limits the research and application. The heterologous expression of BmK AngM1 was achieved in Pichia pastoris in our previous study. In order to realize high-level expression of recombinant BmK AngM1 (rBmK AngM1), the gene dosage of BmK AngM1 was optimized in engineered strains. The yield of rBmK AngM1 in the four-copy strain reached up to 100 mg/L, which was further enhanced to 190 mg/L by co-expressing with chaperones of PDI, BiP, and HAC1. Moreover, the yield of rBmK AngM1 was up to 1200 mg/L by high-density fermentation in 10 L fermenter. Finally, 360 mg rBmK AngM1 was purified from 1 L cultures by a two-step purification method. The efficient and convenient techniques presented in this study could facilitate further scale-up for industrial production of rBmK AngM1.

Differential expression of CURS gene during various growth stages, climatic condition and soil nutrients in turmeric (Curcuma longa): Towards site specific cultivation for high curcumin yield.

PubMed

Sandeep, I Sriram; Das, Suryasnata; Nasim, Noohi; Mishra, Antaryami; Acharya, Laxmikanta; Joshi, Raj Kumar; Nayak, Sanghamitra; Mohanty, Sujata

2017-09-01

Curcuma longa L., accumulates substantial amount of curcumin and essential oil. Little is known about the differential expression of curcumin synthase (CURS) gene and consequent curcumin content variations at different agroclimatic zones. The present study aimed to evaluate the effect of climate, soil and harvesting phase on expression of CURS gene for curcumin yield in two high yielding turmeric cultivars. Expression of CURS gene at different experimental zones as well as at different harvesting phase was studied through transcriptional analysis by qRT-PCR. Curcumin varied from 1.5 to 5% and 1.4-5% in Surama and Roma respectively. The expression of CURS also varied from 0.402 to 5.584 fold in Surama and 0.856-5.217 fold in Roma. Difference in curcumin content at a particular zone varied among different harvesting period from 3.95 to 4.31% in Surama and 3.57-3.83% in Roma. Expression of CURS gene was also effected by harvesting time of the rhizome which varied from 7.389 to 16.882 fold in Surama and 4.41-8.342 fold in Roma. The CURS gene expression was found regardless of variations in curcumin content at different experimental zones. This may be due to the effects of soil and environmental variables. Expression was positively correlated with curcumin content with different harvesting time at a particular zone. This find indicates effect of soil and environment on molecular and biochemical dynamics of curcumin biosynthesis and could be useful in genetic improvement of turmeric. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Soluble Prokaryotic Expression and Purification of Bioactive Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand.

PubMed

Do, Bich Hang; Nguyen, Minh Tan; Song, Jung-A; Park, Sangsu; Yoo, Jiwon; Jang, Jaepyeong; Lee, Sunju; So, Seoungjun; Yoon, Yejin; Kim, Inki; Lee, Kyungjin; Jang, Yeon Jin; Choe, Han

2017-12-28

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered as an antitumor agent owing to its ability to induce apoptosis of cancer cells without imparting toxicity toward most normal cells. TRAIL is produced in poor yield because of its insoluble expression in the cytoplasm of E. coli . In this study, we achieved soluble expression of TRAIL by fusing maltose-binding protein (MBP), b'a' domain of protein disulfide isomerase (PDIb'a'), or protein disulfide isomerase at the N-terminus of TRAIL. The TRAIL was purified using subsequent immobilized metal affinity chromatography and amylose-binding chromatography, with the tag removal using tobacco etch virus protease. Approximately 4.5 mg of pure TRAIL was produced from 125 ml flask culture with a purification yield of 71.6%. The endotoxin level of the final product was 0.4 EU/μg, as measured by the Limulus amebocyte lysate endotoxin assay. The purified TRAIL was validated and shown to cause apoptosis of HeLa cells with an EC₅₀ and Hill coefficient of 0.6 ± 0.03 nM and 2.41 ± 0.15, respectively. The high level of apoptosis in HeLa cells following administration of purified TRAIL indicates the significance and novelty of this method for producing high-grade and high-yield TRAIL.

Soluble FGFR4 extracellular domain inhibits FGF19-induced activation of FGFR4 signaling and prevents nonalcoholic fatty liver disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Qiang; The First Affiliated Hospital of Xiamen University, Xiamen; Jiang, Yuan

2011-06-17

Highlights: {yields} Soluble FGFR4 extracellular domain (FGFR4-ECD) was effectively expressed. {yields} FGFR4-ECD inhibited FGF19-induced activation of FGFR4 signaling. {yields} FGFR4-ECD reduced palmitic acid-induced steatosis of HepG2 cells. {yields} FGFR4-ECD reduced tetracycline-induced fatty liver in mice. {yields} FGFR4-ECD partially restored tetracycline-repressed PPAR{alpha} expression. -- Abstract: Fibroblast growth factor receptor 4 (FGFR4) is a transmembrane tyrosine kinase receptor that plays a crucial role in the regulation of hepatic bile acid and lipid metabolism. FGFR4 underlies high-fat diet-induced hepatic steatosis, suggesting that inhibition of FGFR4 activation may be an effective way to prevent or treat nonalcoholic fatty liver disease (NAFLD). To determine whethermore » neutralization of FGFR4 ligands by soluble FGFR4 extracellular domain (FGFR4-ECD) can inhibit the activation of FGFR4, we constructed FGFR4-ECD expression vector and showed that FGFR4-ECD was effectively expressed in cells and secreted into culture medium. FGFR4-ECD inhibited FGF19-induced activation of FGFR4 signaling and reduced steatosis of HepG2 induced by palmitic acid in vitro. Furthermore, in a tetracycline-induced fatty liver model, expression of FGFR4-ECD in mouse liver reduced the accumulation of hepatic lipids and partially restored the expression of peroxisome proliferator-activated receptor {alpha} (PPAR{alpha}), which promotes the mitochondrial fatty acid beta-oxidation but is repressed by tetracycline. Taken together, these results demonstrate that FGFR4-ECD can block FGFR4 signaling and prevent hepatic steatosis, highlighting the potential value of inhibition of FGFR4 signaling as a method for therapeutic intervention against NAFLD.« less

A phycocyanin·phellandrene synthase fusion enhances recombinant protein expression and β-phellandrene (monoterpene) hydrocarbons production in Synechocystis (cyanobacteria).

PubMed

Formighieri, Cinzia; Melis, Anastasios

2015-11-01

Cyanobacteria can be exploited as photosynthetic platforms for heterologous generation of terpene hydrocarbons with industrial applications. Transformation of Synechocystis and heterologous expression of the β-phellandrene synthase (PHLS) gene alone is necessary and sufficient to confer to Synechocystis the ability to divert intermediate terpenoid metabolites and to generate the monoterpene β-phellandrene during photosynthesis. However, terpene synthases, including the PHLS, have a slow Kcat (low Vmax) necessitating high levels of enzyme concentration to enable meaningful rates and yield of product formation. Here, a novel approach was applied to increase the PHLS protein expression alleviating limitations in the rate and yield of β-phellandrene product generation. Different PHLS fusion constructs were generated with the Synechocystis endogenous cpcB sequence, encoding for the abundant in cyanobacteria phycocyanin β-subunit, expressed under the native cpc operon promoter. In one of these constructs, the CpcB·PHLS fusion protein accumulated to levels approaching 20% of the total cellular protein, i.e., substantially higher than expressing the PHLS protein alone under the same endogenous cpc promoter. The CpcB·PHLS fusion protein retained the activity of the PHLS enzyme and catalyzed β-phellandrene synthesis, yielding an average of 3.2 mg product g(-1) dry cell weight (dcw) versus the 0.03 mg g(-1)dcw measured with low-expressing constructs, i.e., a 100-fold yield improvement. In conclusion, the terpene synthase fusion-protein approach is promising, as, in this case, it substantially increased the amount of the PHLS in cyanobacteria, and commensurately improved rates and yield of β-phellandrene hydrocarbons production in these photosynthetic microorganisms. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Recombinant protein expression for structural biology in HEK 293F suspension cells: a novel and accessible approach.

PubMed

Portolano, Nicola; Watson, Peter J; Fairall, Louise; Millard, Christopher J; Milano, Charles P; Song, Yun; Cowley, Shaun M; Schwabe, John W R

2014-10-16

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two decades, prokaryotic expression systems such as E. coli have dominated the scientific literature over costly and less efficient eukaryotic cell lines. Despite the clear advantage in terms of yields and costs of expressing recombinant proteins in bacteria, the absence of specific co-factors, chaperones and post-translational modifications may cause loss of function, mis-folding and can disrupt protein-protein interactions of certain eukaryotic multi-subunit complexes, surface receptors and secreted proteins. The use of mammalian cell expression systems can address these drawbacks since they provide a eukaryotic expression environment. However, low protein yields and high costs of such methods have until recently limited their use for structural biology. Here we describe a simple and accessible method for expressing and purifying milligram quantities of protein by performing transient transfections of suspension grown HEK (Human Embryonic Kidney) 293 F cells.

Dramatic secretion of recombinant protein expressed in tobacco cells with a designer glycopeptide tag is highly impacted by medium composition.

PubMed

Zhang, Ningning; Dolan, Maureen; Wu, Di; Phillips, Gregory C; Xu, Jianfeng

2016-12-01

Cell growth medium composition has profound impacts on the O -glycosylation of a "designer" arabinogalactan protein-based module; full glycosylation is essential in directing efficient extracellular secretion of the tagged recombinant protein. Expression of recombinant proteins in plant cells as fusion with a de novo designed hydroxyproline (Hyp)-O-glycosylated peptide (HypGP) tag, termed HypGP engineering technology, resulted in dramatically increased secreted protein yields. This is due to the function of the HypGP tag as a molecular carrier in promoting efficient transport of conjoined proteins into culture media. To optimize the cell culture to achieve the best secreted protein yields, the medium effects on the cell growth and protein secretion were investigated using as a model system the tobacco BY-2 cell expressing enhanced green fluorescence protein (EGFP) fused with a (SP) 32 tag (32 tandem repeats of "Ser-Pro" motif). The (SP) 32 tag was found to undergo two-stage Hyp-O-glycosylation in plant cells with the dramatic secretion of the conjoined EGFP correlating with the triggering of the second-stage glycosylation. The BY-2 cell culture in SH medium generated a high secreted protein yield (125 mg/L) with a low cell biomass accumulation (~7.5 gDW/L). In contrast, very low secreted protein yields (~1.5 mg/L) with a high cell biomass accumulation (13.5 gDW/L) were obtained in MS medium. The macronutrients, specifically, the nitrogen supply greatly impacted the glycosylation of the (SP) 32 tag and subsequent protein secretion. Modified MS medium with reduced nitrogen levels boosted the secreted EGFP yields to 168 mg/L. This study demonstrates the profound impacts of medium composition on the secreted yields of a HypGP-tagged protein, and provides a basis for medium design to achieve the highest productivity of the HypGP engineering technology.

Establishing a high yielding streptomyces-based cell-free protein synthesis system.

PubMed

Li, Jian; Wang, He; Kwon, Yong-Chan; Jewett, Michael C

2017-06-01

Cell-free protein synthesis (CFPS) has emerged as a powerful platform for applied biotechnology and synthetic biology, with a range of applications in synthesizing proteins, evolving proteins, and prototyping genetic circuits. To expand the current CFPS repertoire, we report here the development and optimization of a Streptomyces-based CFPS system for the expression of GC-rich genes. By developing a streamlined crude extract preparation protocol and optimizing reaction conditions, we were able to achieve active enhanced green fluorescent protein (EGFP) yields of greater than 50 μg/mL with batch reactions lasting up to 3 h. By adopting a semi-continuous reaction format, the EGFP yield could be increased to 282 ± 8 μg/mL and the reaction time was extended to 48 h. Notably, our extract preparation procedures were robust to multiple Streptomyces lividans and Streptomyces coelicolor strains, although expression yields varied. We show that our optimized Streptomyces lividans system provides benefits when compared to an Escherichia coli-based CFPS system for increasing percent soluble protein expression for four Streptomyces-originated high GC-content genes that are involved in biosynthesis of the nonribosomal peptides tambromycin and valinomycin. Looking forward, we believe that our Streptomyces-based CFPS system will contribute significantly towards efforts to express complex natural product gene clusters (e.g., nonribosomal peptides and polyketides), providing a new avenue for obtaining and studying natural product biosynthesis pathways. Biotechnol. Bioeng. 2017;114: 1343-1353. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

High-level rapid production of full-size monoclonal antibodies in plants by a single-vector DNA replicon system

PubMed Central

Huang, Zhong; Phoolcharoen, Waranyoo; Lai, Huafang; Piensook, Khanrat; Cardineau, Guy; Zeitlin, Larry; Whaley, Kevin J.; Arntzen, Charles J.

2010-01-01

Plant viral vectors have great potential in rapid production of important pharmaceutical proteins. However, high-yield production of heterooligomeric proteins that require the expression and assembly of two or more protein subunits often suffers problems due to the “competing” nature of viral vectors derived from the same virus. Previously we reported that a bean yellow dwarf virus (BeYDV)-derived, three-component DNA replicon system allows rapid production of single recombinant proteins in plants (Huang et al. 2009). In this article, we report further development of this expression system for its application in high-yield production of oligomeric protein complexes including monoclonal antibodies (mAbs) in plants. We showed that the BeYDV replicon system permits simultaneous efficient replication of two DNA replicons and thus, high-level accumulation of two recombinant proteins in the same plant cell. We also demonstrated that a single vector that contains multiple replicon cassettes was as efficient as the three-component system in driving the expression of two distinct proteins. Using either the non-competing, three-vector system or the multi-replicon single vector, we produced both the heavy and light chain subunits of a protective IgG mAb 6D8 against Ebola virus GP1 (Wilson et al. 2000) at 0.5 mg of mAb per gram leaf fresh weight within 4 days post infiltration of Nicotiana benthamiana leaves. We further demonstrated that full-size tetrameric IgG complex containing two heavy and two light chains was efficiently assembled and readily purified, and retained its functionality in specific binding to inactivated Ebola virus. Thus, our single-vector replicon system provides high-yield production capacity for heterooligomeric proteins, yet eliminates the difficult task of identifying non-competing virus and the need for co-infection of multiple expression modules. The multi-replicon vector represents a significant advance in transient expression technology for antibody production in plants. PMID:20047189

High-Yield Expression of M2e Peptide of Avian Influenza Virus H5N1 in Transgenic Duckweed Plants.

PubMed

Firsov, Aleksey; Tarasenko, Irina; Mitiouchkina, Tatiana; Ismailova, Natalya; Shaloiko, Lyubov; Vainstein, Alexander; Dolgov, Sergey

2015-07-01

Avian influenza is a major viral disease in poultry. Antigenic variation of this virus hinders vaccine development. However, the extracellular domain of the virus-encoded M2 protein (peptide M2e) is nearly invariant in all influenza A strains, enabling the development of a broad-range vaccine against them. Antigen expression in transgenic plants is becoming a popular alternative to classical expression methods. Here we expressed M2e from avian influenza virus A/chicken/Kurgan/5/2005(H5N1) in nuclear-transformed duckweed plants for further development of avian influenza vaccine. The N-terminal fragment of M2, including M2e, was selected for expression. The M2e DNA sequence fused in-frame to the 5' end of β-glucuronidase was cloned into pBI121 under the control of CaMV 35S promoter. The resulting plasmid was successfully used for duckweed transformation, and western analysis with anti-β-glucuronidase and anti-M2e antibodies confirmed accumulation of the target protein (M130) in 17 independent transgenic lines. Quantitative ELISA of crude protein extracts from these lines showed M130-β-glucuronidase accumulation ranging from 0.09-0.97 mg/g FW (0.12-1.96 % of total soluble protein), equivalent to yields of up to 40 μg M2e/g plant FW. This relatively high yield holds promise for the development of a duckweed-based expression system to produce an edible vaccine against avian influenza.

Laue Crystal Structure of Shewanella oneidensis Cytochrome c Nitrite Reductase from a High-yield Expression System

PubMed Central

Youngblut, Matthew; Judd, Evan T.; Srajer, Vukica; Sayyed, Bilal; Goelzer, Tyler; Elliott, Sean J.; Schmidt, Marius; Pacheco, A. Andrew

2012-01-01

The high-yield expression and purification of Shewanella oneidensis cytochrome c nitrite reductase (ccNiR), and its characterization by a variety of methods, notably Laue crystallography, is reported. A key component of the expression system is an artificial ccNiR gene in which the N-terminal signal peptide from the highly expressed S. oneidensis protein “Small Tetra-heme c” replaces the wild-type signal peptide. This gene, inserted into the plasmid pHSG298 and expressed in S. oneidensis TSP-1 strain, generated ~20 mg crude ccNiR/L culture, compared with 0.5–1 mg/L for untransformed cells. Purified ccNiR has nitrite and hydroxylamine reductase activities comparable to those previously reported for E. coli ccNiR, and is stable for over two weeks in pH 7 solution at 4° C. UV/Vis spectropotentiometric titrations and protein film voltammetry identified 5 independent 1-electron reduction processes. Global analysis of the spectropotentiometric data also allowed determination of the extinction coefficient spectra for the 5 reduced ccNiR species. The characteristics of the individual extinction coefficient spectra suggest that, within each reduced species, the electrons are distributed amongst the various hemes, rather than being localized on specific heme centers. The purified ccNiR yielded good quality crystals, with which the 2.59 Å resolution structure was solved at room temperature using the Laue diffraction method. The structure is similar to that of E. coli ccNiR, except in the region where the enzyme interacts with its physiological electron donor (CymA in the case of S. oneidensis ccNiR, NrfB in the case of the E. coli protein). PMID:22382353

Kinetics of lipogenic genes expression in milk purified mammary epithelial cells (MEC) across lactation and their correlation with milk and fat yield in buffalo.

PubMed

Yadav, Poonam; Kumar, Parveen; Mukesh, Manishi; Kataria, R S; Yadav, Anita; Mohanty, A K; Mishra, B P

2015-04-01

Expression patterns of lipogenic genes (LPL, ABCG2, ACSS2, ACACA, SCD, BDH, LIPIN1, SREBF1, PPARα and PPARγ) were studied in milk purified MEC across different stages of lactation (15, 30, 45, 60, 90, 120 and 240 days relative to parturition) in buffalo. PPARα was the most abundant gene while ABCG2 and ACSS2 had moderate level of expression; whereas expression of SREBF and PPARγ was very low. The expression patterns of some genes (BDH1, ACSS2, and LIPIN1) across lactation were positively correlated with milk yield while negatively correlated with fat yield. SCD also showed weak correlation with milk yield (p, 0.53) and fat yield (p, -0.47). On the other hand, expression pattern of ACACA was negatively correlated with milk yield (p, -0.88) and positively correlated with fat yield (p, 0.62). Strong correlation was observed between genes involved in de novo milk fat synthesis (BDH1, ACSS2, LIPIN2 and SCD) and milk yield. Copyright © 2015 Elsevier Ltd. All rights reserved.

Coordinated regulation of photosynthesis in rice increases yield and tolerance to environmental stress

PubMed Central

Ambavaram, Madana M. R.; Basu, Supratim; Krishnan, Arjun; Ramegowda, Venkategowda; Batlang, Utlwang; Rahman, Lutfor; Baisakh, Niranjan; Pereira, Andy

2014-01-01

Plants capture solar energy and atmospheric carbon dioxide (CO2) through photosynthesis, which is the primary component of crop yield, and needs to be increased considerably to meet the growing global demand for food. Environmental stresses, which are increasing with climate change, adversely affect photosynthetic carbon metabolism (PCM) and limit yield of cereals such as rice (Oryza sativa) that feeds half the world. To study the regulation of photosynthesis, we developed a rice gene regulatory network and identified a transcription factor HYR (HIGHER YIELD RICE) associated with PCM, which on expression in rice enhances photosynthesis under multiple environmental conditions, determining a morpho-physiological programme leading to higher grain yield under normal, drought and high-temperature stress conditions. We show HYR is a master regulator, directly activating photosynthesis genes, cascades of transcription factors and other downstream genes involved in PCM and yield stability under drought and high-temperature environmental stress conditions. PMID:25358745

Comparative 2D-DIGE Proteomic Analysis of Bovine Mammary Epithelial Cells during Lactation Reveals Protein Signatures for Lactation Persistency and Milk Yield

PubMed Central

Janjanam, Jagadeesh; Singh, Surender; Jena, Manoj K.; Varshney, Nishant; Kola, Srujana; Kumar, Sudarshan; Kaushik, Jai K.; Grover, Sunita; Dang, Ajay K.; Mukesh, Manishi; Prakash, B. S.; Mohanty, Ashok K.

2014-01-01

Mammary gland is made up of a branching network of ducts that end with alveoli which surrounds the lumen. These alveolar mammary epithelial cells (MEC) reflect the milk producing ability of farm animals. In this study, we have used 2D-DIGE and mass spectrometry to identify the protein changes in MEC during immediate early, peak and late stages of lactation and also compared differentially expressed proteins in MEC isolated from milk of high and low milk producing cows. We have identified 41 differentially expressed proteins during lactation stages and 22 proteins in high and low milk yielding cows. Bioinformatics analysis showed that a majority of the differentially expressed proteins are associated in metabolic process, catalytic and binding activity. The differentially expressed proteins were mapped to the available biological pathways and networks involved in lactation. The proteins up-regulated during late stage of lactation are associated with NF-κB stress induced signaling pathways and whereas Akt, PI3K and p38/MAPK signaling pathways are associated with high milk production mediated through insulin hormone signaling. PMID:25111801

Maize ARGOS1 (ZAR1) transgenic alleles increase hybrid maize yield.

PubMed

Guo, Mei; Rupe, Mary A; Wei, Jun; Winkler, Chris; Goncalves-Butruille, Marymar; Weers, Ben P; Cerwick, Sharon F; Dieter, Jo Ann; Duncan, Keith E; Howard, Richard J; Hou, Zhenglin; Löffler, Carlos M; Cooper, Mark; Simmons, Carl R

2014-01-01

Crop improvement for yield and drought tolerance is challenging due to the complex genetic nature of these traits and environmental dependencies. This study reports that transgenic over-expression of Zea mays AR GOS1 (ZAR1) enhanced maize organ growth, grain yield, and drought-stress tolerance. The ZAR1 transgene exhibited environmental interactions, with yield increase under Temperate Dry and yield reduction under Temperate Humid or High Latitude environments. Native ZAR1 allele variation associated with drought-stress tolerance. Two founder alleles identified in the mid-maturity germplasm of North America now predominate in Pioneer's modern breeding programme, and have distinct proteins, promoters and expression patterns. These two major alleles show heterotic group partitioning, with one predominant in Pioneer's female and the other in the male heterotic groups, respectively. These two alleles also associate with favourable crop performance when heterozygous. Allele-specific transgene testing showed that, of the two alleles discussed here, each allele differed in their impact on yield and environmental interactions. Moreover, when transgenically stacked together the allelic pair showed yield and environmental performance advantages over either single allele, resembling heterosis effects. This work demonstrates differences in transgenic efficacy of native alleles and the differences reflect their association with hybrid breeding performance.

Irbesartan increased PPAR{gamma} activity in vivo in white adipose tissue of atherosclerotic mice and improved adipose tissue dysfunction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Iwai, Masaru; Kanno, Harumi; Senba, Izumi

2011-03-04

Research highlights: {yields} Atherosclerotic apolipoprotein E-deficient (ApoEKO) mice were treated with irbesartan. {yields} Irbesartan decreased white adipose tissue weight without affecting body weight. {yields} DNA-binding for PPAR{gamma} was increased in white adipose tissue in vivo by irbesartan. {yields} Irbesartan increased adipocyte number in white adipose tissue. {yields} Irbesatan increased the expression of adiponectin and leptin in white adipose tissue. -- Abstract: The effect of the PPAR{gamma} agonistic action of an AT{sub 1} receptor blocker, irbesartan, on adipose tissue dysfunction was explored using atherosclerotic model mice. Adult male apolipoprotein E-deficient (ApoEKO) mice at 9 weeks of age were treated with amore » high-cholesterol diet (HCD) with or without irbesartan at a dose of 50 mg/kg/day for 4 weeks. The weight of epididymal and retroperitoneal adipose tissue was decreased by irbesartan without changing food intake or body weight. Treatment with irbesartan increased the expression of PPAR{gamma} in white adipose tissue and the DNA-binding activity of PPAR{gamma} in nuclear extract prepared from adipose tissue. The expression of adiponectin, leptin and insulin receptor was also increased by irbesartan. These results suggest that irbesartan induced activation of PPAR{gamma} and improved adipose tissue dysfunction including insulin resistance.« less

Novel Bioengineered Cassava Expressing an Archaeal Starch Degradation System and a Bacterial ADP-Glucose Pyrophosphorylase for Starch Self-Digestibility and Yield Increase

PubMed Central

Ligaba-Osena, Ayalew; Jones, Jenna; Donkor, Emmanuel; Chandrayan, Sanjeev; Pole, Farris; Wu, Chang-Hao; Vieille, Claire; Adams, Michael W. W.; Hankoua, Bertrand B.

2018-01-01

To address national and global low-carbon fuel targets, there is great interest in alternative plant species such as cassava (Manihot esculenta), which are high-yielding, resilient, and are easily converted to fuels using the existing technology. In this study the genes encoding hyperthermophilic archaeal starch-hydrolyzing enzymes, α-amylase and amylopullulanase from Pyrococcus furiosus and glucoamylase from Sulfolobus solfataricus, together with the gene encoding a modified ADP-glucose pyrophosphorylase (glgC) from Escherichia coli, were simultaneously expressed in cassava roots to enhance starch accumulation and its subsequent hydrolysis to sugar. A total of 13 multigene expressing transgenic lines were generated and characterized phenotypically and genotypically. Gene expression analysis using quantitative RT-PCR showed that the microbial genes are expressed in the transgenic roots. Multigene-expressing transgenic lines produced up to 60% more storage root yield than the non-transgenic control, likely due to glgC expression. Total protein extracted from the transgenic roots showed up to 10-fold higher starch-degrading activity in vitro than the protein extracted from the non-transgenic control. Interestingly, transgenic tubers released threefold more glucose than the non-transgenic control when incubated at 85°C for 21-h without exogenous application of thermostable enzymes, suggesting that the archaeal enzymes produced in planta maintain their activity and thermostability. PMID:29541080

Novel Bioengineered Cassava Expressing an Archaeal Starch Degradation System and a Bacterial ADP-Glucose Pyrophosphorylase for Starch Self-Digestibility and Yield Increase.

PubMed

Ligaba-Osena, Ayalew; Jones, Jenna; Donkor, Emmanuel; Chandrayan, Sanjeev; Pole, Farris; Wu, Chang-Hao; Vieille, Claire; Adams, Michael W W; Hankoua, Bertrand B

2018-01-01

To address national and global low-carbon fuel targets, there is great interest in alternative plant species such as cassava ( Manihot esculenta ), which are high-yielding, resilient, and are easily converted to fuels using the existing technology. In this study the genes encoding hyperthermophilic archaeal starch-hydrolyzing enzymes, α-amylase and amylopullulanase from Pyrococcus furiosus and glucoamylase from Sulfolobus solfataricus , together with the gene encoding a modified ADP-glucose pyrophosphorylase ( glgC ) from Escherichia coli , were simultaneously expressed in cassava roots to enhance starch accumulation and its subsequent hydrolysis to sugar. A total of 13 multigene expressing transgenic lines were generated and characterized phenotypically and genotypically. Gene expression analysis using quantitative RT-PCR showed that the microbial genes are expressed in the transgenic roots. Multigene-expressing transgenic lines produced up to 60% more storage root yield than the non-transgenic control, likely due to glgC expression. Total protein extracted from the transgenic roots showed up to 10-fold higher starch-degrading activity in vitro than the protein extracted from the non-transgenic control. Interestingly, transgenic tubers released threefold more glucose than the non-transgenic control when incubated at 85°C for 21-h without exogenous application of thermostable enzymes, suggesting that the archaeal enzymes produced in planta maintain their activity and thermostability.

Improved strategy for recombinant production and purification of antimicrobial peptide tachyplesin I and its analogs with high cell selectivity.

PubMed

Panteleev, Pavel V; Ovchinnikova, Tatiana V

2017-01-01

Here, we report an efficient procedure for recombinant production and purification of tachyplesin I (THI) with a final yield of 17 mg/L of the culture medium. The peptide was expressed in Escherichia coli as a part of the thioredoxin fusion protein. With the use of soluble expression followed by immobilized metal-ion affinity chromatography, the recombinant protein cleavage and reversed-phase high-performance liquid chromatography, a yield of THI did not exceed 6.5 mg/L of the culture medium. Further optimization studies were carried out to improve the protein expression level and simplify purification procedure of the target peptide. To achieve better yield of the peptide, we used high-cell-density bacterial expression. The formed inclusion bodies were highly enriched with the fusion protein, which allowed us to perform direct chemical cleavage of the inclusion bodies solubilized in 6 M guanidine-HCl with subsequent selective precipitation of proteins with trifluoroacetic acid. This enabled us to avoid an extra step of purification by immobilized metal-ion affinity chromatography. The developed procedure has made it possible to obtain biologically active THI and was used for screening a number of its mutant analogs. As a result, several selective and nonhemolytic analogs were developed. Significant reduction in hemolytic activity without losing antimicrobial activity was achieved by substitution of tyrosine or isoleucine residue in the β-turn region of the molecule with hydrophilic serine. The present study affords further insight into molecular mechanism of antimicrobial action of tachyplesin and gains a better understanding of structure-activity relationships in its analogs. This is aimed at searching for novel antibiotics on the basis of antimicrobial peptides with reduced cytotoxicity. © 2015 International Union of Biochemistry and Molecular Biology, Inc.

Cell wall metabolism and hexose allocation contribute to biomass accumulation in high yielding extreme segregants of a Saccharum interspecific F2 population.

PubMed

Wai, Ching Man; Zhang, Jisen; Jones, Tyler C; Nagai, Chifumi; Ming, Ray

2017-10-11

Sugarcane is an emerging dual-purpose biofuel crop for energy and sugar production, owing to its rapid growth rate, high sucrose storage in the stems, and high lignocellulosic yield. It has the highest biomass production reaching 1.9 billion tonnes in 2014 worldwide. To improve sugarcane biomass accumulation, we developed an interspecific cross between Saccharum officinarum 'LA Purple' and Saccharum robustum 'MOL5829'. Selected F1 individuals were self-pollinated to generate a transgressive F2 population with a wide range of biomass yield. Leaf and stem internodes of fourteen high biomass and eight low biomass F2 extreme segregants were used for RNA-seq to decipher the molecular mechanism of rapid plant growth and dry weight accumulation. Gene Ontology terms involved in cell wall metabolism and carbohydrate catabolism were enriched among 3274 differentially expressed genes between high and low biomass groups. Up-regulation of cellulose metabolism, pectin degradation and lignin biosynthesis genes were observed in the high biomass group, in conjunction with higher transcript levels of callose metabolic genes and the cell wall loosening enzyme expansin. Furthermore, UDP-glucose biosynthesis and sucrose conversion genes were differentially expressed between the two groups. A positive correlation between stem glucose, but not sucrose, levels and dry weight was detected. We thus postulated that the high biomass sugarcane plants rapidly convert sucrose to UDP-glucose, which is the building block of cell wall polymers and callose, in order to maintain the rapid plant growth. The gene interaction of cell wall metabolism, hexose allocation and cell division contributes to biomass yield.

The role of class I histone deacetylase (HDAC) on gluconeogenesis in liver

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oiso, Hiroshi; Furukawa, Noboru, E-mail: n-furu@gpo.kumamoto-u.ac.jp; Suefuji, Mihoshi

2011-01-07

Research highlights: {yields} A novel class I HDAC inhibitor decreased hepatic PEPCK mRNA and gluconeogenesis. {yields} Inhibition of HDAC decreased PEPCK by reducing HNF4{alpha} expression and FoxO1 activity. {yields} siRNA knockdown of HDAC1 in HepG2 cells reduced the expression of PEPCK and HNF4{alpha}. {yields} Inhibition of class I HDAC improves glucose homeostasis in HFD mice. -- Abstract: Hepatic gluconeogenesis is crucial for glucose homeostasis. Although sirtuin 1 (Sirt1) is implicated in the regulation of gluconeogenesis in the liver, the effects of other histone deacetylases (HDAC) on gluconeogenesis are unclear. The aim of this study was to identify the role ofmore » class I HDACs in hepatic gluconeogenesis. In HepG2 cells and the liver of mice, the expressions of phosphoenol pyruvate carboxykinase (PEPCK) and hepatocyte nuclear factor 4{alpha} (HNF4{alpha}) were significantly decreased by treatment with a newly designed class I HDAC inhibitor, Ky-2. SiRNA knockdown of HDAC1 expression, but not of HDAC2 or HDAC3, in HepG2 cells decreased PEPCK and HNF4{alpha} expression. In HepG2 cells, insulin-stimulated phosphorylation of Akt and forkhead box O 1 (FoxO1) was increased by Ky-2. Pyruvate tolerance tests in Ky-2-treated high-fat-diet (HFD)-fed mice showed a marked reduction in blood glucose compared with vehicle-treated HFD mice. These data suggest that class I HDACs increase HNF4{alpha} protein expression and the transcriptional activity of FoxO1, followed by the induction of PEPCK mRNA expression and gluconeogenesis in liver.« less

[Expression of human-mouse chimeric antibody directed against Chikungunya virus with site-specific integration system].

PubMed

Li, Jian-min; Chen, Wei; Jia, Xiu-jie; An, Xiao-ping; Li, Bing; Fan, Ying-ru; Tong, Yi-gang

2005-05-01

To obtain CHO/dhfr(-) cells line with integrated FRT sequence in the chromosome transcription active site and to express human-mouse chimeric antibody directed against Chikungunya Virus by using the cell line. The fusion gene of FRT and HBsAg was constructed by PCR and cloned into the MCS of pCI-neo to construct pCI-FRT-HBsAg. The pCI-FRT-HBsAg was transfected into CHO/dhfr(-) cells and cell clones with high expression of HBsAg were screened by detecting the amount of HBsAg with ELISA. A CHO cell clone with the highest expression was chosen and named as CHO/dhfr(-) FRT(+). pAFRT HFLF, a expression plasmid of chimeric antibody with RFT sequence was transfected into CHO/dhfr(-) FRT(+) cells and cell clones with high expression of the chimeric antibody were screened by increasing concentration of MTX. A CHO cell clone with high expression of the chimeric antibody was cultured in large scale and supernatant was collected from which the chimeric antibody was purified. The purified chimeric antibody was analyzed by SDS-PAGE, Western blot and IFA. A CHO/dhfr(-) cells line with integrated FRT sequence in the chromosome transcription active site was obtained successfully. A cell clone with yield of 5 mg/L of chimeric antibody was obtained, as compared with routine CHO cell expression system with a yield of 2 mg/L. A cell line with integrated FRT sequence in the chromosome transcription active site was obtained and with it human-mouse chimeric antibody directed against Chikungunya virus was expressed. This system lays a solid foundation which can be used for expressing antibodies and other proteins.

cAMP-response-element-binding protein positively regulates breast cancer metastasis and subsequent bone destruction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Son, Jieun; Lee, Jong-Ho; Kim, Ha-Neui

2010-07-23

Research highlights: {yields} CREB is highly expressed in advanced breast cancer cells. {yields} Tumor-related factors such as TGF-{beta} further elevate CREB expression. {yields} CREB upregulation stimulates metastatic potential of breast cancer cells. {yields} CREB signaling is required for breast cancer-induced bone destruction. -- Abstract: cAMP-response-element-binding protein (CREB) signaling has been reported to be associated with cancer development and poor clinical outcome in various types of cancer. However, it remains to be elucidated whether CREB is involved in breast cancer development and osteotropism. Here, we found that metastatic MDA-MB-231 breast cancer cells exhibited higher CREB expression than did non-metastatic MCF-7 cellsmore » and that CREB expression was further increased by several soluble factors linked to cancer progression, such as IL-1, IGF-1, and TGF-{beta}. Using wild-type CREB and a dominant-negative form (K-CREB), we found that CREB signaling positively regulated the proliferation, migration, and invasion of MDA-MB-231 cells. In addition, K-CREB prevented MDA-MB-231 cell-induced osteolytic lesions in a mouse model of cancer metastasis. Furthermore, CREB signaling in cancer cells regulated the gene expression of PTHrP, MMPs, and OPG, which are closely involved in cancer metastasis and bone destruction. These results indicate that breast cancer cells acquire CREB overexpression during their development and that this CREB upregulation plays an important role in multiple steps of breast cancer bone metastasis.« less

High-yield secretion of recombinant proteins expressed in tobacco cell culture with a designer glycopeptide tag: Process development.

PubMed

Zhang, Ningning; Gonzalez, Maria; Savary, Brett; Xu, Jianfeng

2016-03-01

Low-yield protein production remains the most significant economic hurdle with plant cell culture technology. Fusions of recombinant proteins with hydroxyproline-O-glycosylated designer glycopeptide tags have consistently boosted secreted protein yields. This prompted us to study the process development of this technology aiming to achieve productivity levels necessary for commercial viability. We used a tobacco BY-2 cell culture expressing EGFP as fusion with a glycopeptide tag comprised of 32 repeat of "Ser-Pro" dipeptide, or (SP)32 , to study cell growth and protein secretion, culture scale-up, and establishment of perfusion cultures for continuous production. The BY-2 cells accumulated low levels of cell biomass (~7.5 g DW/L) in Schenk & Hildebrandt medium, but secreted high yields of (SP)32 -tagged EGFP (125 mg/L). Protein productivity of the cell culture has been stable for 6.0 years. The BY-2 cells cultured in a 5-L bioreactor similarly produced high secreted protein yield at 131 mg/L. Successful operation of a cell perfusion culture for 30 days was achieved under the perfusion rate of 0.25 and 0.5 day(-1) , generating a protein volumetric productivity of 17.6 and 28.9 mg/day/L, respectively. This research demonstrates the great potential of the designer glycopeptide technology for use in commercial production of valuable proteins with plant cell cultures. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Green fluorescent protein as a scaffold for high efficiency production of functional bacteriotoxic proteins in Escherichia coli.

PubMed

Soundrarajan, Nagasundarapandian; Cho, Hye-Sun; Ahn, Byeongyong; Choi, Minkyung; Thong, Le Minh; Choi, Hojun; Cha, Se-Yeoun; Kim, Jin-Hoi; Park, Choi-Kyu; Seo, Kunho; Park, Chankyu

2016-02-11

The availability of simple, robust, and cost-effective methods for the large-scale production of bacteriotoxic peptides such as antimicrobial peptides (AMPs) is essential for basic and pharmaceutical research. However, the production of bacteriotoxic proteins has been difficult due to a high degree of toxicity in bacteria and proteolytic degradation. In this study, we inserted AMPs into the Green fluorescent protein (GFP) in a loop region and expressed them as insoluble proteins in high yield, circumventing the inherent toxicity of AMP production in Escherichia coli. The AMPs inserted were released by cyanogen bromide and purified by chromatography. We showed that highly potent AMPs such as Protegrin-1, PMAP-36, Buforin-2, and Bactridin-1 are produced in high yields and produced AMPs showed similar activities compared to chemically synthesized AMPs. We increased the yield more than two-fold by inserting three copies of Protegrin-1 in the GFP scaffold. The immunogold electron micrographs showed that the expressed Protegrin-1 in the GFP scaffold forms large and small size aggregates in the core region of the inclusion body and become entirely nonfunctional, therefore not influencing the proliferation of E. coli. Our novel method will be applicable for diverse bacteriotoxic peptides which can be exploited in biomedical and pharmaceutical researches.

Green fluorescent protein as a scaffold for high efficiency production of functional bacteriotoxic proteins in Escherichia coli

PubMed Central

Soundrarajan, Nagasundarapandian; Cho, Hye-sun; Ahn, Byeongyong; Choi, Minkyung; Thong, Le Minh; Choi, Hojun; Cha, Se-Yeoun; Kim, Jin-Hoi; Park, Choi-Kyu; Seo, Kunho; Park, Chankyu

2016-01-01

The availability of simple, robust, and cost-effective methods for the large-scale production of bacteriotoxic peptides such as antimicrobial peptides (AMPs) is essential for basic and pharmaceutical research. However, the production of bacteriotoxic proteins has been difficult due to a high degree of toxicity in bacteria and proteolytic degradation. In this study, we inserted AMPs into the Green fluorescent protein (GFP) in a loop region and expressed them as insoluble proteins in high yield, circumventing the inherent toxicity of AMP production in Escherichia coli. The AMPs inserted were released by cyanogen bromide and purified by chromatography. We showed that highly potent AMPs such as Protegrin-1, PMAP-36, Buforin-2, and Bactridin-1 are produced in high yields and produced AMPs showed similar activities compared to chemically synthesized AMPs. We increased the yield more than two-fold by inserting three copies of Protegrin-1 in the GFP scaffold. The immunogold electron micrographs showed that the expressed Protegrin-1 in the GFP scaffold forms large and small size aggregates in the core region of the inclusion body and become entirely nonfunctional, therefore not influencing the proliferation of E. coli. Our novel method will be applicable for diverse bacteriotoxic peptides which can be exploited in biomedical and pharmaceutical researches. PMID:26864123

Characterization and protective efficacy in an animal model of a novel truncated rotavirus VP8 subunit parenteral vaccine candidate.

PubMed

Xue, Miaoge; Yu, Linqi; Che, Yaojian; Lin, Haijun; Zeng, Yuanjun; Fang, Mujin; Li, Tingdong; Ge, Shengxiang; Xia, Ningshao

2015-05-21

The cell-attachment protein VP8* of rotavirus is a potential candidate parenteral vaccine. However, the yield of full-length VP8 protein (VP8*, residues 1-231) expressed in Escherichia coli was low, and a truncated VP8 protein (ΔVP8*, residues 65-231) cannot elicit efficient protective immunity in a mouse model. In this study, tow novel truncated VP8 proteins, VP8-1 (residues 26-231) and VP8-2 (residues 51-231), were expressed in E. coli and evaluated for immunogenicity and protective efficacy, compared with VP8* and ΔVP8*. As well as ΔVP8*, the protein VP8-1 and VP8-2 were successfully expressed in high yield and purified in homogeneous dimeric forms, while the protein VP8* was expressed with lower yield and prone to aggregation and degradation in solution. Although the immunogenicity of the protein VP8*, VP8-1, VP8-2 and ΔVP8* was comparable, immunization of VP8* and VP8-1 elicited significantly higher neutralizing antibody titers than that of VP8-2 and ΔVP8* in mice. Furthermore, when assessed using a mouse maternal antibody model, the efficacy of VP8-1 to protect against rotavirus-induced diarrhea in pups was comparable to that of VP8*, both were dramatically higher than that of VP8-2 and ΔVP8*. Taken together, the novel truncated protein VP8-1, with increased yield, improved homogeneity and high protective efficacy, is a viable candidate for further development of a parenterally administrated prophylactic vaccine against rotavirus infection. Copyright © 2015 Elsevier Ltd. All rights reserved.

Glutamate co-transmission from developing medial nucleus of the trapezoid body - Lateral superior olive synapses is cochlear dependent in kanamycin-treated rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Jae Ho; Pradhan, Jonu; Maskey, Dhiraj

Research highlights: {yields} Glutamate co-transmission is enhanced in kanamycin-treated rats. {yields} VGLUT3 expression is increased in kanamycin-treated rats. {yields} GlyR expression is decreased in kanamycin-treated rats. {yields} GlyR, VGLUT3 expression patterns are asymmetric in unilaterally cochlear ablated rat. -- Abstract: Cochlear dependency of glutamate co-transmission at the medial nucleus of the trapezoid body (MNTB) - the lateral superior olive (LSO) synapses was investigated using developing rats treated with high dose kanamycin. Rats were treated with kanamycin from postnatal day (P) 3 to P8. A scanning electron microscopic study on P9 demonstrated partial cochlear hair cell damage. A whole cell voltagemore » clamp experiment demonstrated the increased glutamatergic portion of postsynaptic currents (PSCs) elicited by MNTB stimulation in P9-P11 kanamycin-treated rats. The enhanced VGLUT3 immunoreactivities (IRs) in kanamycin-treated rats and asymmetric VGLUT3 IRs in the LSO of unilaterally cochlear ablated rats supported the electrophysiologic data. Taken together, it is concluded that glutamate co-transmission is cochlear-dependent and enhanced glutamate co-transmission in kanamycin-treated rats is induced by partial cochlear damage.« less

High yield recombinant production of a self-assembling polycationic peptide for silica biomineralization.

PubMed

Zerfaß, Christian; Braukmann, Sandra; Nietzsche, Sandor; Hobe, Stephan; Paulsen, Harald

2015-04-01

We report the recombinant bacterial expression and purification at high yields of a polycationic oligopeptide, P5S3. The sequence of P5S3 was inspired by a diatom silaffin, a silica precipitating peptide. Like its native model, P5S3 exhibits silica biomineralizing activity, but furthermore has unusual self-assembling properties. P5S3 is efficiently expressed in Escherichia coli as fusion with ketosteroid isomerase (KSI), which causes deposition in inclusion bodies. After breaking the fusion by cyanogen bromide reaction, P5S3 was purified by cation exchange chromatography, taking advantage of the exceptionally high content of basic amino acids. The numerous cationic charges do not prevent, but may even promote counterion-independent self-assembly which in turn leads to silica precipitation. Enzymatic phosphorylation, a common modification in native silica biomineralizing peptides, can be used to modify the precipitation activity. Copyright © 2015 Elsevier Inc. All rights reserved.

High-Yield Secretion of Multiple Client Proteins in Aspergillus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Segato, F.; Damasio, A. R. L.; Goncalves, T. A.

2012-07-15

Production of pure and high-yield client proteins is an important technology that addresses the need for industrial applications of enzymes as well as scientific experiments in protein chemistry and crystallization. Fungi are utilized in industrial protein production because of their ability to secrete large quantities of proteins. In this study, we engineered a high-expression-secretion vector, pEXPYR that directs proteins towards the extracellular medium in two Aspergillii host strains, examine the effect of maltose-induced over-expression and protein secretion as well as time and pH-dependent protein stability in the medium. We describe five client proteins representing a core set of hemicellulose degradingmore » enzymes that accumulated up to 50-100 mg/L of protein. Using a recyclable genetic marker that allows serial insertion of multiple genes, simultaneous hyper-secretion of three client proteins in a single host strain was accomplished.« less

The heterologous expression strategies of antimicrobial peptides in microbial systems.

PubMed

Deng, Ting; Ge, Haoran; He, Huahua; Liu, Yao; Zhai, Chao; Feng, Liang; Yi, Li

2017-12-01

Antimicrobial peptides (AMPs) consist of molecules acting on the defense systems of numerous organisms toward tumor and multiple pathogens, such as bacteria, fungi, viruses, and parasites. Compared to traditional antibiotics, AMPs are more stable and have lower propensity for developing resistance through functioning in the innate immune system, thus having important applications in the fields of medicine, food and so on. However, despite of their high economic values, the low yield and the cumbersome extraction process in AMPs production are problems that limit their industrial application and scientific research. To conquer these obstacles, optimized heterologous expression technologies were developed that could provide effective ways to increase the yield of AMPs. In this review, the research progress on heterologous expression of AMPs using Escherichia coli, Bacillus subtilis, Pichia pastoris and Saccharomyces cerevisiae as host cells was mainly summarized, which might guide the expression strategies of AMPs in these cells. Copyright © 2017 Elsevier Inc. All rights reserved.

Homodimeric β-Galactosidase from Lactobacillus delbrueckii subsp. bulgaricus DSM 20081: Expression in Lactobacillus plantarum and Biochemical Characterization

PubMed Central

2012-01-01

The lacZ gene from Lactobacillus delbrueckii subsp. bulgaricus DSM 20081, encoding a β-galactosidase of the glycoside hydrolase family GH2, was cloned into different inducible lactobacillal expression vectors for overexpression in the host strain Lactobacillus plantarum WCFS1. High expression levels were obtained in laboratory cultivations with yields of approximately 53000 U of β-galactosidase activity per liter of medium, which corresponds to ∼170 mg of recombinant protein per liter and β-galactosidase levels amounting to 63% of the total intracellular protein of the host organism. The wild-type (nontagged) and histidine-tagged recombinant enzymes were purified to electrophoretic homogeneity and further characterized. β-Galactosidase from L. bulgaricus was used for lactose conversion and showed very high transgalactosylation activity. The maximum yield of galacto-oligosaccharides (GalOS) was approximately 50% when using an initial concentration of 600 mM lactose, indicating that the enzyme can be of interest for the production of GalOS. PMID:22283494

Homodimeric β-galactosidase from Lactobacillus delbrueckii subsp. bulgaricus DSM 20081: expression in Lactobacillus plantarum and biochemical characterization.

PubMed

Nguyen, Tien-Thanh; Nguyen, Hoang Anh; Arreola, Sheryl Lozel; Mlynek, Georg; Djinović-Carugo, Kristina; Mathiesen, Geir; Nguyen, Thu-Ha; Haltrich, Dietmar

2012-02-22

The lacZ gene from Lactobacillus delbrueckii subsp. bulgaricus DSM 20081, encoding a β-galactosidase of the glycoside hydrolase family GH2, was cloned into different inducible lactobacillal expression vectors for overexpression in the host strain Lactobacillus plantarum WCFS1. High expression levels were obtained in laboratory cultivations with yields of approximately 53000 U of β-galactosidase activity per liter of medium, which corresponds to ~170 mg of recombinant protein per liter and β-galactosidase levels amounting to 63% of the total intracellular protein of the host organism. The wild-type (nontagged) and histidine-tagged recombinant enzymes were purified to electrophoretic homogeneity and further characterized. β-Galactosidase from L. bulgaricus was used for lactose conversion and showed very high transgalactosylation activity. The maximum yield of galacto-oligosaccharides (GalOS) was approximately 50% when using an initial concentration of 600 mM lactose, indicating that the enzyme can be of interest for the production of GalOS.

Targeted impairment of thymidine kinase 2 expression in cells induces mitochondrial DNA depletion and reveals molecular mechanisms of compensation of mitochondrial respiratory activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Villarroya, Joan, E-mail: joanvillarroya@gmail.com; Institut de Recerca l'Hospital de la Santa Creu i Sant Pau, Barcelona; Lara, Mari-Carmen

Highlights: {yields} We impaired TK2 expression in Ost TK1{sup -} cells via siRNA-mediated interference (TK2{sup -}). {yields} TK2 impairment caused severe mitochondrial DNA (mtDNA) depletion in quiescent cells. {yields} Despite mtDNA depletion, TK2{sup -} cells show high cytochrome oxidase activity. {yields} Depletion of mtDNA occurs without imbalance in the mitochondrial dNTP pool. {yields} Nuclear-encoded ENT1, DNA-pol {gamma}, TFAM and TP gene expression is lowered in TK2{sup -} cells. -- Abstract: The mitochondrial DNA (mtDNA) depletion syndrome comprises a clinically heterogeneous group of diseases characterized by reductions of the mtDNA abundance, without associated point mutations or rearrangements. We have developed themore » first in vitro model to study of mtDNA depletion due to reduced mitochondrial thymidine kinase 2 gene (TK2) expression in order to understand the molecular mechanisms involved in mtDNA depletion syndrome due to TK2 mutations. Small interfering RNA targeting TK2 mRNA was used to decrease TK2 expression in Ost TK1{sup -} cells, a cell line devoid of endogenous thymidine kinase 1 (TK1). Stable TK2-deficient cell lines showed a reduction of TK2 levels close to 80%. In quiescent conditions, TK2-deficient cells showed severe mtDNA depletion, also close to 80% the control levels. However, TK2-deficient clones showed increased cytochrome c oxidase activity, higher cytochrome c oxidase subunit I transcript levels and higher subunit II protein expression respect to control cells. No alterations of the deoxynucleotide pools were found, whereas a reduction in the expression of genes involved in nucleoside/nucleotide homeostasis (human equilibrative nucleoside transporter 1, thymidine phosphorylase) and mtDNA maintenance (DNA-polymerase {gamma}, mitochondrial transcription factor A) was observed. Our findings highlight the importance of cellular compensatory mechanisms that enhance the expression of respiratory components to ensure respiratory activity despite profound depletion in mtDNA levels.« less

Enhancement of solubility, purification and inclusion-bodies-refolding of an active pectin lyase from Penicillium occitanis expressed in Escherichia coli.

PubMed

Hadj Sassi, Azza; Trigui-Lahiani, Hèla; Abdeljalil, Salma; Gargouri, Ali

2017-02-01

Pectin lyase (pnl) is the only pectinase able to hydrolyze directly the highly methylated pectin without liberating the toxic methanol and without disturbing ester content responsible for specific aroma of juices. The cDNA of Penicillium occitanis pnl (mature form) was cloned into pET-21a as expression vector and over-expressed into Esherichia coli. Most of recombinant pnl was expressed as inclusion bodies. Pnl activity was confirmed by colorimetric assay. To enhance the solubility yield of the expressed pnl, the effects of induction temperature, host strain and expression level were optimized. Maximal production of functional pnl was obtained after induction by 0.4mM IPTG at 30°C and 150rpm for 16h. Interestingly, the use of Origami host strain, having an oxidized cytoplasm favoring disulfide bonds formation required for the active conformation of the enzyme, has significantly improved the yield of the soluble active form of recombinant pnl. This pnl was successfully purified through a single step purification using His-Trap affinity column chromatography. This work is the first to report pnl expression into Origami strain. Alternatively, the inclusion bodies were isolated, denatured by high concentration of urea and gradually refolded by successive dialysis, leading to their transformation into soluble and active form. Copyright © 2016 Elsevier B.V. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Forward, Nicholas A.; Conrad, David M.; Power Coombs, Melanie R.

Highlights: {yields} Curcumin inhibits CD4{sup +} T-lymphocyte proliferation. {yields} Curcumin inhibits interleukin-2 (IL-2) synthesis and CD25 expression by CD4{sup +} T-lymphocytes. {yields} Curcumin interferes with IL-2 receptor signaling by inhibiting JAK3 and STAT5 phosphorylation. {yields} IL-2-dependent regulatory T-lymphocyte function and Foxp3 expression is downregulated by curcumin. -- Abstract: Curcumin (diferulomethane) is the principal curcuminoid in the spice tumeric and a potent inhibitor of activation-induced T-lymphocyte proliferation; however, the molecular basis of this immunosuppressive effect has not been well studied. Here we show that micromolar concentrations of curcumin inhibited DNA synthesis by mouse CD4{sup +} T-lymphocytes, as well as interleukin-2 (IL-2)more » and CD25 ({alpha} chain of the high affinity IL-2 receptor) expression in response to antibody-mediated cross-linking of CD3 and CD28. Curcumin acted downstream of protein kinase C activation and intracellular Ca{sup 2+} release to inhibit I{kappa}B phosphorylation, which is required for nuclear translocation of the transcription factor NF{kappa}B. In addition, IL-2-dependent DNA synthesis by mouse CTLL-2 cells, but not constitutive CD25 expression, was impaired in the presence of curcumin, which demonstrated an inhibitory effect on IL-2 receptor (IL-2R) signaling. IL-2-induced phosphorylation of STAT5A and JAK3, but not JAK1, was diminished in the presence of curcumin, indicating inhibition of critical proximal events in IL-2R signaling. In line with the inhibitory action of curcumin on IL-2R signaling, pretreatment of CD4{sup +}CD25{sup +} regulatory T-cells with curcumin downregulated suppressor function, as well as forkhead box p3 (Foxp3) expression. We conclude that curcumin inhibits IL-2 signaling by reducing available IL-2 and high affinity IL-2R, as well as interfering with IL-2R signaling.« less

Expression and purification of recombinant apolipoprotein A-I Zaragoza (L144R) and formation of reconstituted HDL particles.

PubMed

Fiddyment, Sarah; Barceló-Batllori, Sílvia; Pocoví, Miguel; García-Otín, Angel-Luis

2011-11-01

Apolipoprotein A-I Zaragoza (L144R) (apo A-I Z), has been associated with severe hypoalphalipoproteinemia and an enhanced effect of high density lipoprotein (HDL) reverse cholesterol transport. In order to perform further studies with this protein we have optimized an expression and purification method of recombinant wild-type apo A-I and apo A-I Z and produced mimetic HDL particles with each protein. An pET-45 expression system was used to produce N-terminal His-tagged apo A-I, wild-type or mutant, in Escherichia coli BL21 (DE3) which was subsequently purified by affinity chromatography in non-denaturing conditions. HDL particles were generated via a modified sodium cholate method. Expression and purification of both proteins was verified by SDS-PAGE, MALDI-TOF MS and immunochemical procedures. Yield was 30mg of purified protein (94% purity) per liter of culture. The reconstituted HDL particles checked via non-denaturing PAGE showed high homogeneity in their size when reconstituted both with wild-type apo A-I and apo A-I Z. An optimized system for the expression and purification of wild-type apo A-I and apo A-I Z with high yield and purity grade has been achieved, in addition to their use in reconstituted HDL particles, as a basis for further studies. Copyright © 2011 Elsevier Inc. All rights reserved.

Efficient production of antibody Fab fragment by transient gene expression in insect cells.

PubMed

Mori, Keita; Hamada, Hirotsugu; Ogawa, Takafumi; Ohmuro-Matsuyama, Yuki; Katsuda, Tomohisa; Yamaji, Hideki

2017-08-01

Transient gene expression allows a rapid production of diverse recombinant proteins in early-stage preclinical and clinical developments of biologics. Insect cells have proven to be an excellent platform for the production of functional recombinant proteins. In the present study, the production of an antibody Fab fragment by transient gene expression in lepidopteran insect cells was investigated. The DNA fragments encoding heavy-chain (Hc; Fd fragment) and light-chain (Lc) genes of an Fab fragment were individually cloned into the plasmid vector pIHAneo, which contained the Bombyx mori actin promoter downstream of the B. mori nucleopolyhedrovirus (BmNPV) IE-1 transactivator and the BmNPV HR3 enhancer for high-level expression. Trichoplusia ni BTI-TN-5B1-4 (High Five) cells were co-transfected with the resultant plasmid vectors using linear polyethyleneimine. When the transfection efficiency was evaluated, a plasmid vector encoding an enhanced green fluorescent protein (EGFP) gene was also co-transfected. Transfection and culture conditions were optimized based on both the flow cytometry of the EGFP expression in transfected cells and the yield of the secreted Fab fragments determined by enzyme-linked immunosorbent assay (ELISA). Under optimal conditions, a yield of approximately 120 mg/L of Fab fragments was achieved in 5 days in a shake-flask culture. Transient gene expression in insect cells may offer a promising approach to the high-throughput production of recombinant proteins. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Expression and bioactivity of human α-fetoprotein in a Bac-to-Bac system

PubMed Central

Lin, Bo; Liu, Kun; Wang, Wenting; Li, Wei; Dong, Xu; Chen, Yi; Lu, Yan; Guo, Junli; Li, Mengsen

2016-01-01

α-fetoprotein (AFP) is an early serum growth factor in foetal embryonic development and hepatic oncogenesis. A growing number of investigations of AFP as a tumour-specific biomarker have concluded that AFP is an important target for cancer treatment. AFP also plays an immunomodulatory role in the treatment of several autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis, myasthenia gravis and thyroiditis. In an effort to support biochemical screening and drug design and discovery, we attempted to express and purify human AFP in a Bac-to-Bac system. Two key factors affecting the expression of recombinant human AFP (R-AFP), namely the infectious baculovirus inoculum volume and the culturing time post-infection, were optimized to maximize the yield. We achieved a high yield of approximately 1.5 mg/l of harvested medium with a 72–96 h incubation period after infection and an inoculum volume ratio of 1:100. We also assessed the role of R-AFP in the proliferation of the human liver cancer cell line Bel 7402, and the results indicated that R-AFP promoted the growth of hepatoma cells. We concluded that this method can produce high yields of R-AFP, which can be used for studies related to AFP. PMID:27913752

Expression and bioactivity of human α-fetoprotein in a Bac-to-Bac system.

PubMed

Lin, Bo; Liu, Kun; Wang, Wenting; Li, Wei; Dong, Xu; Chen, Yi; Lu, Yan; Guo, Junli; Zhu, Mingyue; Li, Mengsen

2017-02-28

α-fetoprotein (AFP) is an early serum growth factor in foetal embryonic development and hepatic oncogenesis. A growing number of investigations of AFP as a tumour-specific biomarker have concluded that AFP is an important target for cancer treatment. AFP also plays an immunomodulatory role in the treatment of several autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis, myasthenia gravis and thyroiditis. In an effort to support biochemical screening and drug design and discovery, we attempted to express and purify human AFP in a Bac-to-Bac system. Two key factors affecting the expression of recombinant human AFP (R-AFP), namely the infectious baculovirus inoculum volume and the culturing time post-infection, were optimized to maximize the yield. We achieved a high yield of approximately 1.5 mg/l of harvested medium with a 72-96 h incubation period after infection and an inoculum volume ratio of 1:100. We also assessed the role of R-AFP in the proliferation of the human liver cancer cell line Bel 7402, and the results indicated that R-AFP promoted the growth of hepatoma cells. We concluded that this method can produce high yields of R-AFP, which can be used for studies related to AFP. © 2017 The Author(s).

ACC oxidase and miRNA 159a, and their involvement in fresh fruit bunch yield (FFB) via sex ratio determination in oil palm.

PubMed

Somyong, Suthasinee; Poopear, Supannee; Sunner, Supreet Kaur; Wanlayaporn, Kitti; Jomchai, Nukoon; Yoocha, Thippawan; Ukoskit, Kittipat; Tangphatsornruang, Sithichoke; Tragoonrung, Somvong

2016-06-01

Oil palm (Elaeis guineesis Jacq.) is the most productive oil-bearing crop, yielding more oil per area than any other oil-bearing crops. However, there are still efforts to improve oil palm yield, in order to serve consumer and manufacturer demand. Oil palm produces female and male inflorescences in an alternating cycle. So, high sex ratio (SR), the ratio of female inflorescences to the total inflorescences, is a favorable trait in term of increasing yields in oil palm. This study aims to understand the genetic control for SR related traits, such as fresh fruit bunch yield (FFB), by characterizing genes at FFB quantitative trait loci (QTLs) on linkage 10 (chromosome 6) and linkage 15 (chromosome 10). Published oil palm sequences at the FFB QTLs were used to develop gene-based and simple sequence repeat (SSR) markers. We used the multiple QTL analysis model (MQM) to characterize the relationship of new markers with the SR traits in the oil palm population. The RNA expression of the most linked QTL genes was also evaluated in various tissues of oil palm. We identified EgACCO1 (encoding aminocyclopropane carboxylate (ACC) oxidase) at chromosome 10 and EgmiR159a (microRNA 159a) at chromosome 6 to be the most linked QTL genes or determinants for FFB yield and/or female inflorescence number with a phenotype variance explained (PVE) from 10.4 to 15 % and suggest that these play the important roles in sex determination and differentiation in oil palm. The strongest expression of EgACCO1 and the predicted precursor of EgmiR159a was found in ovaries and, to a lesser extent, fruit development. In addition, highly normalized expression of EgmiR159a was found in female flowers. In summary, the QTL analysis and the RNA expression reveal that EgACCO1 and EgmiR159a are the potential genetic factors involved in female flower determination and hence would affect yield in oil palm. However, to clarify how these genetic factors regulate female flower determination, more investigation of their down regulation or target may be essential. Additionally, if more sex determination genes controlled by plant hormones are identified, it may possible to reveal a crosstalk of sex determination genes with hormones and environment factors.

Engineering Halomonas spp. as A Low-Cost Production Host for Production of Bio-surfactant Protein PhaP.

PubMed

Lan, Lu-Hong; Zhao, Han; Chen, Jin-Chun; Chen, Guo-Qiang

2016-12-01

Halomonas spp. have been studied as a low cost production host for producing bulk materials such as polyhydroxyalkanoates (PHA) bioplastics, since they are able to grow at high pH and high NaCl concentration under unsterile and continuous conditions without microbial contamination. In this paper, Halomonas strain TD is used as a host to produce a protein named PHA phasin or PhaP which has a potential to be developed into a bio-surfactant. Four Halomonas TD expression strains are constructed based on a strong T7-family expression system. Of these, the strain with phaC deletion and chromosomal expression system resulted in the highest production of PhaP in soluble form, reaching 19% of total cellular soluble proteins and with a yield of 1.86 g/L in an open fed-batch fermentation process. A simple "heat lysis and salt precipitation" method is applied to allow rapid PhaP purification from a mixture of cellular proteins with a PhaP recovery rate of 63%. It clearly demonstrated that Halomonas TD could be used for high yield expression of a bio-surfactant protein PhaP for industrial application in an economical way. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Hematopoietic Cancer Cell Lines Can Support Replication of Sabin Poliovirus Type 1

PubMed Central

van Eikenhorst, Gerco; de Gruijl, Tanja D.; van der Pol, Leo A.; Bakker, Wilfried A. M.

2015-01-01

Viral vaccines can be produced in adherent or in suspension cells. The objective of this work was to screen human suspension cell lines for the capacity to support viral replication. As the first step, it was investigated whether poliovirus can replicate in such cell lines. Sabin poliovirus type 1 was serially passaged on five human cell lines, HL60, K562, KG1, THP-1, and U937. Sabin type 1 was capable of efficiently replicating in three cell lines (K562, KG1, and U937), yielding high viral titers after replication. Expression of CD155, the poliovirus receptor, did not explain susceptibility to replication, since all cell lines expressed CD155. Furthermore, we showed that passaged virus replicated more efficiently than parental virus in KG1 cells, yielding higher virus titers in the supernatant early after infection. Infection of cell lines at an MOI of 0.01 resulted in high viral titers in the supernatant at day 4. Infection of K562 with passaged Sabin type 1 in a bioreactor system yielded high viral titers in the supernatant. Altogether, these data suggest that K562, KG1, and U937 cell lines are useful for propagation of poliovirus. PMID:25815312

Maize ARGOS1 (ZAR1) transgenic alleles increase hybrid maize yield

PubMed Central

Guo, Mei

2014-01-01

Crop improvement for yield and drought tolerance is challenging due to the complex genetic nature of these traits and environmental dependencies. This study reports that transgenic over-expression of Zea mays ARGOS1 (ZAR1) enhanced maize organ growth, grain yield, and drought-stress tolerance. The ZAR1 transgene exhibited environmental interactions, with yield increase under Temperate Dry and yield reduction under Temperate Humid or High Latitude environments. Native ZAR1 allele variation associated with drought-stress tolerance. Two founder alleles identified in the mid-maturity germplasm of North America now predominate in Pioneer’s modern breeding programme, and have distinct proteins, promoters and expression patterns. These two major alleles show heterotic group partitioning, with one predominant in Pioneer’s female and the other in the male heterotic groups, respectively. These two alleles also associate with favourable crop performance when heterozygous. Allele-specific transgene testing showed that, of the two alleles discussed here, each allele differed in their impact on yield and environmental interactions. Moreover, when transgenically stacked together the allelic pair showed yield and environmental performance advantages over either single allele, resembling heterosis effects. This work demonstrates differences in transgenic efficacy of native alleles and the differences reflect their association with hybrid breeding performance. PMID:24218327

Involvement of viral envelope GP2 in Ebola virus entry into cells expressing the macrophage galactose-type C-type lectin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Usami, Katsuaki; Matsuno, Keita; Igarashi, Manabu

2011-04-01

Highlights: {yields} Ebola virus infection is mediated by binding to and fusion with the target cells. {yields} Structural feature of the viral glycoprotein determines the infectivity. {yields} Surface C-type lectin, MGL, of macrophages and dendritic cells mediate the infection. {yields} GP2, one of glycoprotein subunits, plays an essential role in MGL-mediated infection. {yields} There is a critical amino acid residue involved in high infectivity. -- Abstract: Ebola virus (EBOV) infection is initiated by the interaction of the viral surface envelope glycoprotein (GP) with the binding sites on target cells. Differences in the mortality among different species of the Ebola viruses,more » i.e., Zaire ebolavirus (ZEBOV) and Reston ebolavirus (REBOV), correspond to the in vitro infectivity of the pseudo-typed virus constructed with the GPs in cells expressing macrophage galactose-type calcium-type lectin (MGL/CD301). Through mutagenesis of GP2, the transmembrane-anchored subunit of GP, we found that residues 502-527 of the GP2 sequence determined the different infectivity between VSV-ZEBOV GP and -REBOV GP in MGL/CD301-expressing cells and a histidine residue at position 516 of ZEBOV GP2 appeared essential in the differential infectivity. These findings may provide a clue to clarify a molecular basis of different pathogenicity among EBOV species.« less

Enhanced xylose fermentation by engineered yeast expressing NADH oxidase through high cell density inoculums.

PubMed

Zhang, Guo-Chang; Turner, Timothy L; Jin, Yong-Su

2017-03-01

Accumulation of reduced byproducts such as glycerol and xylitol during xylose fermentation by engineered Saccharomyces cerevisiae hampers the economic production of biofuels and chemicals from cellulosic hydrolysates. In particular, engineered S. cerevisiae expressing NADPH-linked xylose reductase (XR) and NAD + -linked xylitol dehydrogenase (XDH) produces substantial amounts of the reduced byproducts under anaerobic conditions due to the cofactor difference of XR and XDH. While the additional expression of a water-forming NADH oxidase (NoxE) from Lactococcus lactis in engineered S. cerevisiae with the XR/XDH pathway led to reduced glycerol and xylitol production and increased ethanol yields from xylose, volumetric ethanol productivities by the engineered yeast decreased because of growth defects from the overexpression of noxE. In this study, we introduced noxE into an engineered yeast strain (SR8) exhibiting near-optimal xylose fermentation capacity. To overcome the growth defect caused by the overexpression of noxE, we used a high cell density inoculum for xylose fermentation by the SR8 expressing noxE. The resulting strain, SR8N, not only showed a higher ethanol yield and lower byproduct yields, but also exhibited a high ethanol productivity during xylose fermentation. As noxE overexpression elicits a negligible growth defect on glucose conditions, the beneficial effects of noxE overexpression were substantial when a mixture of glucose and xylose was used. Consumption of glucose led to rapid cell growth and therefore enhanced the subsequent xylose fermentation. As a result, the SR8N strain produced more ethanol and fewer byproducts from a mixture of glucose and xylose than the parental SR8 strain without noxE overexpression. Our results suggest that the growth defects from noxE overexpression can be overcome in the case of fermenting lignocellulose-derived sugars such as glucose and xylose.

Independent and high-level dual-gene expression in adult stem-progenitor cells from a single lentiviral vector.

PubMed

Tian, J; Andreadis, S T

2009-07-01

Expression of multiple genes from the same target cell is required in several technological and therapeutic applications such as quantitative measurements of promoter activity or in vivo tracking of stem cells. In spite of such need, reaching independent and high-level dual-gene expression cannot be reliably accomplished by current gene transfer vehicles. To address this issue, we designed a lentiviral vector carrying two transcriptional units separated by polyadenylation, terminator and insulator sequences. With this design, the expression level of both genes was as high as that yielded from lentiviral vectors containing only a single transcriptional unit. Similar results were observed with several promoters and cell types including epidermal keratinocytes, bone marrow mesenchymal stem cells and hair follicle stem cells. Notably, we demonstrated quantitative dynamic monitoring of gene expression in primary cells with no need for selection protocols suggesting that this optimized lentivirus may be useful in high-throughput gene expression profiling studies.

The metabolic costs of improving ethanol yield by reducing glycerol formation capacity under anaerobic conditions in Saccharomyces cerevisiae

PubMed Central

2013-01-01

Background Finely regulating the carbon flux through the glycerol pathway by regulating the expression of the rate controlling enzyme, glycerol-3-phosphate dehydrogenase (GPDH), has been a promising approach to redirect carbon from glycerol to ethanol and thereby increasing the ethanol yield in ethanol production. Here, strains engineered in the promoter of GPD1 and deleted in GPD2 were used to investigate the possibility of reducing glycerol production of Saccharomyces cerevisiae without jeopardising its ability to cope with process stress during ethanol production. For this purpose, the mutant strains TEFmut7 and TEFmut2 with different GPD1 residual expression were studied in Very High Ethanol Performance (VHEP) fed-batch process under anaerobic conditions. Results Both strains showed a drastic reduction of the glycerol yield by 44 and 61% while the ethanol yield improved by 2 and 7% respectively. TEFmut2 strain showing the highest ethanol yield was accompanied by a 28% reduction of the biomass yield. The modulation of the glycerol formation led to profound redox and energetic changes resulting in a reduction of the ATP yield (YATP) and a modulation of the production of organic acids (acetate, pyruvate and succinate). Those metabolic rearrangements resulted in a loss of ethanol and stress tolerance of the mutants, contrarily to what was previously observed under aerobiosis. Conclusions This work demonstrates the potential of fine-tuned pathway engineering, particularly when a compromise has to be found between high product yield on one hand and acceptable growth, productivity and stress resistance on the other hand. Previous study showed that, contrarily to anaerobiosis, the resulting gain in ethanol yield was accompanied with no loss of ethanol tolerance under aerobiosis. Moreover those mutants were still able to produce up to 90 gl-1 ethanol in an anaerobic SSF process. Fine tuning metabolic strategy may then open encouraging possibilities for further developing robust strains with improved ethanol yield. PMID:23537043

The metabolic costs of improving ethanol yield by reducing glycerol formation capacity under anaerobic conditions in Saccharomyces cerevisiae.

PubMed

Pagliardini, Julien; Hubmann, Georg; Alfenore, Sandrine; Nevoigt, Elke; Bideaux, Carine; Guillouet, Stephane E

2013-03-28

Finely regulating the carbon flux through the glycerol pathway by regulating the expression of the rate controlling enzyme, glycerol-3-phosphate dehydrogenase (GPDH), has been a promising approach to redirect carbon from glycerol to ethanol and thereby increasing the ethanol yield in ethanol production. Here, strains engineered in the promoter of GPD1 and deleted in GPD2 were used to investigate the possibility of reducing glycerol production of Saccharomyces cerevisiae without jeopardising its ability to cope with process stress during ethanol production. For this purpose, the mutant strains TEFmut7 and TEFmut2 with different GPD1 residual expression were studied in Very High Ethanol Performance (VHEP) fed-batch process under anaerobic conditions. Both strains showed a drastic reduction of the glycerol yield by 44 and 61% while the ethanol yield improved by 2 and 7% respectively. TEFmut2 strain showing the highest ethanol yield was accompanied by a 28% reduction of the biomass yield. The modulation of the glycerol formation led to profound redox and energetic changes resulting in a reduction of the ATP yield (YATP) and a modulation of the production of organic acids (acetate, pyruvate and succinate). Those metabolic rearrangements resulted in a loss of ethanol and stress tolerance of the mutants, contrarily to what was previously observed under aerobiosis. This work demonstrates the potential of fine-tuned pathway engineering, particularly when a compromise has to be found between high product yield on one hand and acceptable growth, productivity and stress resistance on the other hand. Previous study showed that, contrarily to anaerobiosis, the resulting gain in ethanol yield was accompanied with no loss of ethanol tolerance under aerobiosis. Moreover those mutants were still able to produce up to 90 gl-1 ethanol in an anaerobic SSF process. Fine tuning metabolic strategy may then open encouraging possibilities for further developing robust strains with improved ethanol yield.

Stress inducible overexpression of AtHDG11 leads to improved drought and salt stress tolerance in peanut (Arachis hypogaea L.)

NASA Astrophysics Data System (ADS)

Banavath, Jayanna N.; Chakradhar, Thammineni; Pandit, Varakumar; Konduru, Sravani; Guduru, Krishna K.; Akila, Chandra S.; Podha, Sudhakar; Puli, Chandra O. R.

2018-03-01

Peanut is an important oilseed and food legume cultivated as a rain-fed crop in semi-arid tropics. Drought and high salinity are the major abiotic stresses limiting the peanut productivity in this region. Development of drought and salt tolerant peanut varieties with improved yield potential using biotechnological approach is highly desirable to improve the peanut productivity in marginal geographies. As abiotic stress tolerance and yield represent complex traits, engineering of regulatory genes to produce abiotic stress-resilient transgenic crops appears to be a viable approach. In the present study, we developed transgenic peanut plants expressing an Arabidopsis homeodomain-leucine zipper transcription factor (AtHDG11) under stress inducible rd29Apromoter. A stress-inducible expression of AtHDG11 in three independent homozygous transgenic peanut lines resulted in improved drought and salt tolerance through up-regulation of known stress responsive genes(LEA, HSP70, Cu/Zn SOD, APX, P5CS, NCED1, RRS5, ERF1, NAC4, MIPS, Aquaporin, TIP, ELIP ) in the stress gene network , antioxidative enzymes, free proline along with improved water use efficiency traits such as longer root system, reduced stomatal density, higher chlorophyll content, increased specific leaf area, improved photosynthetic rates and increased intrinsic instantaneous WUE. Transgenic peanut plants displayed high yield compared to non-transgenic plants under both drought and salt stress conditions. Holistically, our study demonstrates the potentiality of stress-induced expression of AtHDG11 to improve the drought, salt tolerance in peanut.

Transient Expression and Purification of Horseradish Peroxidase C in Nicotiana benthamiana.

PubMed

Huddy, Suzanne M; Hitzeroth, Inga I; Meyers, Ann E; Weber, Brandon; Rybicki, Edward P

2018-01-01

Horseradish peroxidase (HRP) is a commercially important reagent enzyme used in molecular biology and in the diagnostic product industry. It is typically purified from the roots of the horseradish ( Armoracia rusticana ); however, this crop is only available seasonally, yields are variable and often low, and the product is a mixture of isoenzymes. Engineering high-level expression in transiently transformed tobacco may offer a solution to these problems. In this study, a synthetic Nicotiana benthamiana codon-adapted full-length HRP isoenzyme gene as well as C-terminally truncated and both N- and C-terminally truncated versions of the HRP C gene were synthesized, and their expression in N. benthamiana was evaluated using an Agrobacterium tumefaciens -mediated transient expression system. The influence on HRP C expression levels of co-infiltration with a silencing suppressor (NSs) construct was also evaluated. Highest HRP C levels were consistently obtained using either the full length or C-terminally truncated HRP C constructs. HRP C purification by ion exchange chromatography gave an overall yield of 54% with a Reinheitszahl value of >3 and a specific activity of 458 U/mg. The high level of HRP C production in N. benthamiana in just five days offers an alternative, viable, and scalable system for production of this commercially significant enzyme.

Transient Expression and Purification of Horseradish Peroxidase C in Nicotiana benthamiana

PubMed Central

Huddy, Suzanne M.; Hitzeroth, Inga I.; Weber, Brandon; Rybicki, Edward P.

2018-01-01

Horseradish peroxidase (HRP) is a commercially important reagent enzyme used in molecular biology and in the diagnostic product industry. It is typically purified from the roots of the horseradish (Armoracia rusticana); however, this crop is only available seasonally, yields are variable and often low, and the product is a mixture of isoenzymes. Engineering high-level expression in transiently transformed tobacco may offer a solution to these problems. In this study, a synthetic Nicotiana benthamiana codon-adapted full-length HRP isoenzyme gene as well as C-terminally truncated and both N- and C-terminally truncated versions of the HRP C gene were synthesized, and their expression in N. benthamiana was evaluated using an Agrobacterium tumefaciens-mediated transient expression system. The influence on HRP C expression levels of co-infiltration with a silencing suppressor (NSs) construct was also evaluated. Highest HRP C levels were consistently obtained using either the full length or C-terminally truncated HRP C constructs. HRP C purification by ion exchange chromatography gave an overall yield of 54% with a Reinheitszahl value of >3 and a specific activity of 458 U/mg. The high level of HRP C production in N. benthamiana in just five days offers an alternative, viable, and scalable system for production of this commercially significant enzyme. PMID:29301255

Increasing plant growth by modulating omega-amidase expression in plants

DOEpatents

Unkefer, Pat J.; Anderson, Penelope S.; Knight, Thomas J.

2015-06-30

The present disclosure relates to compositions and methods for increasing the leaf-to-root ratio of the signal metabolite 2-oxoglutaramate and related proline molecules in plants by modulating levels of .omega.-amidase to increase nitrogen use efficiency, resulting in enhanced growth, faster growth rates, greater seed and fruit/pod yields, earlier and more productive flowering, increased tolerance to high salt conditions, and increased biomass yields.

The Processing and Mechanical Properties of High Temperature/High Performance Composites. Book 3. Constituent Properties and Macroscopic Performance: MMCs

DTIC Science & Technology

1993-04-01

re - expressed as, v .= hCSw (C3) Combining Eqns. (C2) and C3) yields, Se = - ’. S (C4...of vi( s ) or v ’( s ). Substituting eq. (B10) into eq. (25), one finds the finite element method expression for functional Ud [ v ] which is U, d [] = v , K...Measurements 1- D 2- D S ,_DL 4 Constitutive W Constitutive Laws Laws Matrix Cracking Labor Models Models Stress Redistribution Numerical Calculations

Breeding maize for resistance to ear rot caused by Fusarium moniliforme.

PubMed

Hefny, M; Attaa, S; Bayoumi, T; Ammar, S; El-Bramawy, M

2012-01-15

Maize ear rots are among the most important impediments to increased maize production in Egypt. The present research was conducted to estimate combining abilities, heterosis and correlation coefficients for resistance to ear rot disease in seven corn inbred lines and their 21 crosses under field conditions. Results demonstrated that both additive and non-additive gene actions were responsible for the genetic expression of all characters with the preponderance of non-additive actions for days to 50% silking. The parental line L51 was the best combiner for earliness, low infection severity %, high phenols content, short plants and reasonable grain yield, while L101 was good combiner for low ear rot infection only. The cross: L122 x L84, L122 x L101, L51 x L101, L76 x L36, L76 x L84, L36 x L84, L36 x L81 and L36 x L101 which involved one or both parents with good General Combining Ability (GCA) effects expressed useful significant heterosis and Specific Combining Ability (SCA) effects for low infection severity %, high phenol contents, early silking, tall plants and high grain yield. Phenotypic and genotypic correlation coefficients suggest that selection for resistance to ear rot should identify lines with high yielding ability, early silking, tall plants, high phenols content and chitinase activity.

Assessment of diesel-contaminated domestic wastewater treated by constructed wetlands for irrigation of chillies grown in a greenhouse.

PubMed

Al-Isawi, Rawaa H K; Scholz, Miklas; Al-Faraj, Furat A M

2016-12-01

In order to avoid environmental pollution and eliminate the need for using fertiliser, this study assessed for the first time the optimum performance of mature (in operation since 2011) vertical flow constructed wetlands in treating domestic wastewater (with and without hydrocarbon) and the subsequent recycling of the outflow to irrigate chillies (De Cayenne; Capsicum annuum (Linnaeus) Longum Group 'De Cayenne') grown in a greenhouse. Various variables were investigated to assess the treatment performance. Concerning chilli fruit numbers, findings showed that the highest fruit yields for all wetland filters were associated with those that received inflow wastewater with a high loading rate, reflecting the high nutrient availability in treated wastewater, which is of obvious importance for yield production. Findings also indicated that wetlands without hydrocarbon, small aggregate size, low contact time and low inflow loading rate provided high marketable yields (expressed in economic return). In comparison, chillies irrigated by filters with hydrocarbon contamination, small aggregate size, high contact time and high loading rate also resulted in high marketable yields of chillies, which pointed out the role of high contact time and high inflow load for better diesel degradation rates.

Alternative preparation of inclusion bodies excludes interfering non-protein contaminants and improves the yield of recombinant proinsulin.

PubMed

Mackin, Robert B

2014-01-01

The goal of simple, high-yield expression and purification of recombinant human proinsulin has proven to be a considerable challenge. First, proinsulin forms inclusion bodies during bacterial expression. While this phenomenon can be exploited as a capture step, conventionally prepared inclusion bodies contain significant amounts of non-protein contaminants that interfere with subsequent chromatographic purification. Second, the proinsulin molecules within the inclusion bodies are incorrectly folded, and likely cross-linked to one another, making it difficult to quantify the amount of expressed proinsulin. Third, proinsulin is an intermediate between the initial product of ribosomal translation (preproinsulin) and the final product secreted by pancreatic beta cells (insulin). Therefore, to be efficiently produced in bacteria, it must be produced as an N-terminally extended fusion protein, which has to be converted to authentic proinsulin during the purification scheme. To address all three of these problems, while simultaneously streamlining the procedure and increasing the yield of recombinant proinsulin, we have made three substantive modifications to our previous method for producing proinsulin:.•Conditions for the preparation of inclusion bodies have been altered so contaminants that interfere with semi-preparative reversed-phase chromatography are excluded while the proinsulin fusion protein is retained at high yield.•Aliquots are taken following important steps in the procedure and the quantity of proinsulin-related polypeptide in the sample is compared to the amount present prior to that step.•Final purification is performed using a silica-based reversed-phase matrix in place of a polystyrene-divinylbenzene-based matrix.

Alternative preparation of inclusion bodies excludes interfering non-protein contaminants and improves the yield of recombinant proinsulin

PubMed Central

Mackin, Robert B.

2014-01-01

The goal of simple, high-yield expression and purification of recombinant human proinsulin has proven to be a considerable challenge. First, proinsulin forms inclusion bodies during bacterial expression. While this phenomenon can be exploited as a capture step, conventionally prepared inclusion bodies contain significant amounts of non-protein contaminants that interfere with subsequent chromatographic purification. Second, the proinsulin molecules within the inclusion bodies are incorrectly folded, and likely cross-linked to one another, making it difficult to quantify the amount of expressed proinsulin. Third, proinsulin is an intermediate between the initial product of ribosomal translation (preproinsulin) and the final product secreted by pancreatic beta cells (insulin). Therefore, to be efficiently produced in bacteria, it must be produced as an N-terminally extended fusion protein, which has to be converted to authentic proinsulin during the purification scheme. To address all three of these problems, while simultaneously streamlining the procedure and increasing the yield of recombinant proinsulin, we have made three substantive modifications to our previous method for producing proinsulin:.•Conditions for the preparation of inclusion bodies have been altered so contaminants that interfere with semi-preparative reversed-phase chromatography are excluded while the proinsulin fusion protein is retained at high yield.•Aliquots are taken following important steps in the procedure and the quantity of proinsulin-related polypeptide in the sample is compared to the amount present prior to that step.•Final purification is performed using a silica-based reversed-phase matrix in place of a polystyrene-divinylbenzene-based matrix. PMID:26150942

The combination of plant translational enhancers and terminator increase the expression of human glucocerebrosidase in Nicotiana benthamiana plants.

PubMed

Limkul, Juthamard; Misaki, Ryo; Kato, Ko; Fujiyama, Kazuhito

2015-11-01

Gaucher's disease is a lysosomal storage disorder caused by mutations in the gene encoding glucocerebrosidase (GCase). It is currently treated by enzyme replacement therapy using recombinant GCase expressed in mammalian cells. Plant production systems are among the most attractive alternatives for pharmaceutical protein production due to such advantages as low-cost, high-scalability, and safety from human pathogen contamination. Because of its high biomass yield, Nicotiana benthamiana could be an economical recombinant GCase production system. In this study, a translational enhancer and suitable terminator were utilized to obtain a powerful expression system for GCase production in N. benthamiana plants. Six plasmid constructs were used. The highest activity of 44.5units/mg protein (after subtraction of endogenous glucosidase activity of the wild-type plant) was observed in transgenic plants transformed with pAt-GC-HSP combined with a 5' untranslated region of the Arabidopsis alcohol dehydrogenase gene with the Arabidopsis heat shock protein terminator. These transgenic plant lines could pave the way to a stable plant-production system for low-cost, high-yield human GCase production. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Strategies for analysing and improving the expression and quality of recombinant proteins made in mammalian cells.

PubMed

Jenkins, Nigel; Meleady, Paula; Tyther, Raymond; Murphy, Lisa

2009-05-06

The production of monoclonal antibodies and other recombinant proteins is one of the highest growth areas in the pharmaceutical industry. Mammalian cells are used to manufacture the majority of biotherapeutics, largely due to their ability to perform complex post-translational modifications. Although significant progress has been made recently in improving product yields and protein quality, many challenges still lie ahead to achieve consistently high yields while avoiding potentially damaging protein modifications. The present review first considers the strategies used to analyse and improve recombinant protein expression of industrial cell lines, with an emphasis on proteomic technologies. Next, cellular and environmental influences on protein production and quality are examined, and strategies for improvements in product yield and quality are reviewed. The analytical techniques required to detect these protein changes are also described, together with prospects for assay improvements.

Expression of Arabidopsis glycine-rich RNA-binding protein AtGRP2 or AtGRP7 improves grain yield of rice (Oryza sativa) under drought stress conditions.

PubMed

Yang, Deok Hee; Kwak, Kyung Jin; Kim, Min Kyung; Park, Su Jung; Yang, Kwang-Yeol; Kang, Hunseung

2014-01-01

Although posttranscriptional regulation of RNA metabolism is increasingly recognized as a key regulatory process in plant response to environmental stresses, reports demonstrating the importance of RNA metabolism control in crop improvement under adverse environmental stresses are severely limited. To investigate the potential use of RNA-binding proteins (RBPs) in developing stress-tolerant transgenic crops, we generated transgenic rice plants (Oryza sativa) that express Arabidopsis thaliana glycine-rich RBP (AtGRP) 2 or 7, which have been determined to harbor RNA chaperone activity and confer stress tolerance in Arabidopsis, and analyzed the response of the transgenic rice plants to abiotic stresses. AtGRP2- or AtGRP7-expressing transgenic rice plants displayed similar phenotypes comparable with the wild-type plants under high salt or cold stress conditions. By contrast, AtGRP2- or AtGRP7-expressing transgenic rice plants showed much higher recovery rates and grain yields compared with the wild-type plants under drought stress conditions. The higher grain yield of the transgenic rice plants was due to the increases in filled grain numbers per panicle. Collectively, the present results show the importance of posttranscriptional regulation of RNA metabolism in plant response to environmental stress and suggest that GRPs can be utilized to improve the yield potential of crops under stress conditions. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Overexpressing of OsAMT1-3, a High Affinity Ammonium Transporter Gene, Modifies Rice Growth and Carbon-Nitrogen Metabolic Status

PubMed Central

Bao, Aili; Liang, Zhijun; Zhao, Zhuqing; Cai, Hongmei

2015-01-01

AMT1-3 encodes the high affinity NH4+ transporter in rice roots and is predominantly expressed under nitrogen starvation. In order to evaluate the effect of AMT1-3 gene on rice growth, nitrogen absorption and metabolism, we generated AMT1-3-overexpressing plants and analyzed the growth phenotype, yield, carbon and nitrogen metabolic status, and gene expression profiles. Although AMT1-3 mRNA accumulated in transgenic plants, these plants displayed significant decreases in growth when compared to the wild-type plants. The nitrogen uptake assay using a 15N tracer revealed poor nitrogen uptake ability in AMT1-3-overexpressing plants. We found significant decreases in AMT1-3-overexpressing plant leaf carbon and nitrogen content accompanied with a higher leaf C/N ratio. Significant changes in soluble proteins and carbohydrates were also observed in AMT1-3-overexpressing plants. In addition, metabolite profile analysis demonstrated significant changes in individual sugars, organic acids and free amino acids. Gene expression analysis revealed distinct expression patterns of genes that participate in carbon and nitrogen metabolism. Additionally, the correlation between the metabolites and gene expression patterns was consistent in AMT1-3-overexpressing plants under both low and high nitrogen growth conditions. Therefore, we hypothesized that the carbon and nitrogen metabolic imbalance caused by AMT1-3 overexpressing attributed to the poor growth and yield of transgenic plants. PMID:25915023

One-Pot Production of l-threo-3-Hydroxyaspartic Acid Using Asparaginase-Deficient Escherichia coli Expressing Asparagine Hydroxylase of Streptomyces coelicolor A3(2)

PubMed Central

Nakano, Masashi; Kino, Kuniki

2015-01-01

We developed a novel process for efficient synthesis of l-threo-3-hydroxyaspartic acid (l-THA) using microbial hydroxylase and hydrolase. A well-characterized mutant of asparagine hydroxylase (AsnO-D241N) and its homologous enzyme (SCO2693-D246N) were adaptable to the direct hydroxylation of l-aspartic acid; however, the yields were strictly low. Therefore, the highly stable and efficient wild-type asparagine hydroxylases AsnO and SCO2693 were employed to synthesize l-THA. By using these recombinant enzymes, l-THA was obtained by l-asparagine hydroxylation by AsnO followed by amide hydrolysis by asparaginase via 3-hydroxyasparagine. Subsequently, the two-step reaction was adapted to one-pot bioconversion in a test tube. l-THA was obtained in a small amount with a molar yield of 0.076% by using intact Escherichia coli expressing the asnO gene, and thus, two asparaginase-deficient mutants of E. coli were investigated. A remarkably increased l-THA yield of 8.2% was obtained with the asparaginase I-deficient mutant. When the expression level of the asnO gene was enhanced by using the T7 promoter in E. coli instead of the lac promoter, the l-THA yield was significantly increased to 92%. By using a combination of the E. coli asparaginase I-deficient mutant and the T7 expression system, a whole-cell reaction in a jar fermentor was conducted, and consequently, l-THA was successfully obtained from l-asparagine with a maximum yield of 96% in less time than with test tube-scale production. These results indicate that asparagine hydroxylation followed by hydrolysis would be applicable to the efficient production of l-THA. PMID:25795668

Estimating the potential refolding yield of recombinant proteins expressed as inclusion bodies.

PubMed

Ho, Jason G S; Middelberg, Anton P J

2004-09-05

Recombinant protein production in bacteria is efficient except that insoluble inclusion bodies form when some gene sequences are expressed. Such proteins must undergo renaturation, which is an inefficient process due to protein aggregation on dilution from concentrated denaturant. In this study, the protein-protein interactions of eight distinct inclusion-body proteins are quantified, in different solution conditions, by measurement of protein second virial coefficients (SVCs). Protein solubility is shown to decrease as the SVC is reduced (i.e., as protein interactions become more attractive). Plots of SVC versus denaturant concentration demonstrate two clear groupings of proteins: a more aggregative group and a group having higher SVC and better solubility. A correlation of the measured SVC with protein molecular weight and hydropathicity, that is able to predict which group each of the eight proteins falls into, is presented. The inclusion of additives known to inhibit aggregation during renaturation improves solubility and increases the SVC of both protein groups. Furthermore, an estimate of maximum refolding yield (or solubility) using high-performance liquid chromatography was obtained for each protein tested, under different environmental conditions, enabling a relationship between "yield" and SVC to be demonstrated. Combined, the results enable an approximate estimation of the maximum refolding yield that is attainable for each of the eight proteins examined, under a selected chemical environment. Although the correlations must be tested with a far larger set of protein sequences, this work represents a significant move beyond empirical approaches for optimizing renaturation conditions. The approach moves toward the ideal of predicting maximum refolding yield using simple bioinformatic metrics that can be estimated from the gene sequence. Such a capability could potentially "screen," in silico, those sequences suitable for expression in bacteria from those that must be expressed in more complex hosts.

Sensitivity of malignant rhabdoid tumor cell lines to PD 0332991 is inversely correlated with p16 expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Katsumi, Yoshiki; Iehara, Tomoko; Miyachi, Mitsuru

Highlights: {yields} PD 0332991 (PD) could suppress four of five malignant rhabdoid tumor (MRT) cell lines. {yields} The sensitivity of the MRT cell lines to PD was inversely correlated with p16 expression (r = 0.951). {yields} p16 expression in MRT could be used to predict its sensitivity to PD. {yields} PD may be an attractive agent for patients with MRT whose tumors express low levels of p16. -- Abstract: Malignant rhabdoid tumor (MRT) is a rare and highly aggressive neoplasm of young children. MRT is characterized by inactivation of integrase interactor 1 (INI1). Cyclin-dependent kinase 4 (CDK4), which acts downstreammore » of INI1, is required for the proliferation of MRT cells. Here we investigated the effects of PD 0332991 (PD), a potent inhibitor of CDK4, against five human MRT cell lines (MP-MRT-AN, KP-MRT-RY, G401, KP-MRT-NS, KP-MRT-YM). In all of the cell lines except KP-MRT-YM, PD inhibited cell proliferation >50%, (IC{sub 50} values 0.01 to 0.6 {mu}M) by WST-8 assay, and induced G1-phase cell cycle arrest, as shown by flow cytometry and BrdU incorporation assay. The sensitivity of the MRT cell lines to PD was inversely correlated with p16 expression (r = 0.951). KP-MRT-YM cells overexpress p16 and were resistant to the growth inhibitory effect of PD. Small interfering RNA against p16 significantly increased the sensitivity of KP-MRT-YM cells to PD (p < 0.05). These results suggest that p16 expression in MRT could be used to predict its sensitivity to PD. PD may be an attractive agent for patients with MRT whose tumors express low levels of p16.« less

Combined strategies for improving expression of Citrobacter amalonaticus phytase in Pichia pastoris.

PubMed

Li, Cheng; Lin, Ying; Zheng, Xueyun; Pang, Nuo; Liao, Xihao; Liu, Xiaoxiao; Huang, Yuanyuan; Liang, Shuli

2015-09-26

Phytase is used as an animal feed additive that degrades phytic acid and reduces feeding costs and pollution caused by fecal excretion of phosphorus. Some phytases have been expressed in Pichia pastoris, among which the phytase from Citrobacter amalonaticus CGMCC 1696 had high specific activity (3548 U/mg). Improvement of the phytase expression level will contribute to facilitate its industrial applications. To improve the phytase expression, we use modification of P AOX1 and the α-factor signal peptide, increasing the gene copy number, and overexpressing HAC1 (i) to enhance folding and secretion of the protein in the endoplasmic reticulum. The genetic stability and fermentation in 10-L scaled-up fed-batch fermenter was performed to prepare for the industrial production. The phytase gene from C. amalonaticus CGMCC 1696 was cloned under the control of the AOX1 promoter (P AOX1 ) and expressed in P. pastoris. The phytase activity achieved was 414 U/mL. Modifications of P AOX1 and the α-factor signal peptide increased the phytase yield by 35 and 12%, respectively. Next, on increasing the copy number of the Phy gene to six, the phytase yield was 141% higher than in the strain containing only a single gene copy. Furthermore, on overexpression of HAC1 (i) (i indicating induced), a gene encoding Hac1p that regulates the unfolded protein response, the phytase yield achieved was 0.75 g/L with an activity of 2119 U/mL, 412% higher than for the original strain. The plasmids in this high-phytase expression strain were stable during incubation at 30 °C in Yeast Extract Peptone Dextrose (YPD) Medium. In a 10-L scaled-up fed-batch fermenter, the phytase yield achieved was 9.58 g/L with an activity of 35,032 U/mL. The production of a secreted protein will reach its limit at a specific gene copy number where further increases in transcription and translation due to the higher abundance of gene copies will not enhance the secretion process any further. Enhancement of protein folding in the ER can alleviate bottlenecks in the folding and secretion pathways during the overexpression of heterologous proteins in P. pastoris. Using modification of P AOX1 and the α-factor signal peptide, increasing the gene copy number, and overexpressing HAC1 (i) to enhance folding and secretion of the protein in the endoplasmic reticulum, we have successfully increased the phytase yield 412% relative to the original strain. In a 10-L fed-batch fermenter, the phytase yield achieved was 9.58 g/L with an activity of 35,032 U/mL. Large-scale production of phytase can be applied towards different biocatalytic and feed additive applications.

Efficient expression systems for cysteine proteases of malaria parasites

PubMed Central

Sarduy, Emir Salas; de los A. Chávez Planes, María

2013-01-01

Papain-like cysteine proteases of malaria parasites are considered important chemotherapeutic targets or valuable models for the evaluation of drug candidates. Consequently, many of these enzymes have been cloned and expressed in Escherichia coli for their biochemical characterization. However, their expression has been problematic, showing low yield and leading to the formation of insoluble aggregates. Given that highly-productive expression systems are required for the high-throughput evaluation of inhibitors, we analyzed the existing expression systems to identify the causes of such apparent issues. We found that significant divergences in codon and nucleotide composition from host genes are the most probable cause of expression failure, and propose several strategies to overcome these limitations. Finally we predict that yeast hosts Saccharomyces cerevisiae and Pichia pastoris may be better suited than E. coli for the efficient expression of plasmodial genes, presumably leading to soluble and active products reproducing structural and functional characteristics of the natural enzymes. PMID:23018863

The high producing dairy cow and its reproductive performance

PubMed Central

Dobson, H; Smith, RF; Royal, MD; Knight, CH; Sheldon, IM

2009-01-01

Contents: Intensive genetic selection has resulted in modern dairy cow with very high milk yields but reduced fertility, due mainly to an increase in postpartum clinical problems, poor expression of oestrus, defective oocytes/embryos and uterine infections. It is a challenge to get enough food into these cows to meet the high demands of peak milk yields in early lactation and the animals require considerable veterinary attention in the early period after calving. Both genetic and management changes to increase the persistency of lactations would reduce the number and intensity of clinical risk periods throughout a cow's life without compromising milk output. PMID:17688598

Genome shuffling of Saccharomyces cerevisiae for enhanced glutathione yield and relative gene expression analysis using fluorescent quantitation reverse transcription polymerase chain reaction.

PubMed

Yin, Hua; Ma, Yanlin; Deng, Yang; Xu, Zhenbo; Liu, Junyan; Zhao, Junfeng; Dong, Jianjun; Yu, Junhong; Chang, Zongming

2016-08-01

Genome shuffling is an efficient and promising approach for the rapid improvement of microbial phenotypes. In this study, genome shuffling was applied to enhance the yield of glutathione produced by Saccharomyces cerevisiae YS86. Six isolates with subtle improvements in glutathione yield were obtained from populations generated by ultraviolet (UV) irradiation and nitrosoguanidine (NTG) mutagenesis. These yeast strains were then subjected to recursive pool-wise protoplast fusion. A strain library that was likely to yield positive colonies was created by fusing the lethal protoplasts obtained from both UV irradiation and heat treatments. After two rounds of genome shuffling, a high-yield recombinant YSF2-19 strain that exhibited 3.2- and 3.3-fold increases in glutathione production in shake flask and fermenter respectively was obtained. Comparative analysis of synthetase gene expression was conducted between the initial and shuffled strains using FQ (fluorescent quantitation) RT-PCR (reverse transcription polymerase chain reaction). Delta CT (threshold cycle) relative quantitation analysis revealed that glutathione synthetase gene (GSH-I) expression at the transcriptional level in the YSF2-19 strain was 9.9-fold greater than in the initial YS86. The shuffled yeast strain has a potential application in brewing, other food, and pharmaceutical industries. Simultaneously, the analysis of improved phenotypes will provide more valuable data for inverse metabolic engineering. Copyright © 2016 Elsevier B.V. All rights reserved.

Respiratory glycerol metabolism of Actinobacillus succinogenes 130Z for succinate production.

PubMed

Schindler, Bryan D; Joshi, Rajasi V; Vieille, Claire

2014-09-01

Actinobacillus succinogenes 130Z naturally produces among the highest levels of succinate from a variety of inexpensive carbon substrates. A few studies have demonstrated that A. succinogenes can anaerobically metabolize glycerol, a waste product of biodiesel manufacture and an inexpensive feedstock, to produce high yields of succinate. However, all these studies were performed in the presence of yeast extract, which largely removes the redox constraints associated with fermenting glycerol, a highly reduced molecule. We demonstrated that A. succinogenes cannot ferment glycerol in minimal medium, but that it can metabolize glycerol by aerobic or anaerobic respiration. These results were expected based on the A. succinogenes genome, which encodes respiratory enzymes, but no pathway for 1,3-propanediol production. We investigated A. succinogenes's glycerol metabolism in minimal medium in a variety of respiratory conditions by comparing growth, metabolite production, and in vitro activity of terminal oxidoreductases. Nitrate inhibited succinate production by inhibiting fumarate reductase expression. In contrast, growth in the presence of dimethylsulfoxide and in microaerobic conditions allowed high succinate yields. The highest succinate yield was 0.75 mol/mol glycerol (75 % of the maximum theoretical yield) in continuous microaerobic cultures. A. succinogenes could also grow and produce succinate on partially refined glycerols obtained directly from biodiesel manufacture. Finally, by expressing a heterologous 1,3-propanediol synthesis pathway in A. succinogenes, we provide the first proof of concept that A. succinogenes can be engineered to grow fermentatively on glycerol.

Proteomic analysis of high yield rice variety mutated from spaceflight

NASA Astrophysics Data System (ADS)

Ma, Y.; Cheng, Z.; Wang, W.; Sun, Y.

Seeds of pure rice varieties were flown on Chinese recoverable satellite, JB-1, for a 15-day flight in 1996. Many mutant rice varieties with various phenotypes were generated after continuous selection and breeding. Among the mutants, a variety 971-5 showed a significant increase in grain yield compared to its control (971ck). In this study, proteomic analysis of both mutant variety 971-5 and control variety 971ck were carried out to investigate the changes of protein expression level in their leaves at three different growth stages (early and middle stage of tillering, and booting stage). Results showed that (1) almost all differentially expressed proteins were down-regulated in 971-5 with only one exception, (2) the percentages of differentially expressed proteins were 3.1%, 2.1% and 3.1% at the three stages, respectively, and (3) one protein showed a significant alteration in its molecular weight (MW). These data demonstrated that the space environment can alter the expression level of rice proteins both quantitatively and qualitatively.

Production of functional bacteriorhodopsin by an Escherichia coli cell-free protein synthesis system supplemented with steroid detergent and lipid.

PubMed

Shimono, Kazumi; Goto, Mie; Kikukawa, Takashi; Miyauchi, Seiji; Shirouzu, Mikako; Kamo, Naoki; Yokoyama, Shigeyuki

2009-10-01

Cell-free expression has become a highly promising tool for the efficient production of membrane proteins. In this study, we used a dialysis-based Escherichia coli cell-free system for the production of a membrane protein actively integrated into liposomes. The membrane protein was the light-driven proton pump bacteriorhodopsin, consisting of seven transmembrane alpha-helices. The cell-free expression system in the dialysis mode was supplemented with a combination of a detergent and a natural lipid, phosphatidylcholine from egg yolk, in only the reaction mixture. By examining a variety of detergents, we found that the combination of a steroid detergent (digitonin, cholate, or CHAPS) and egg phosphatidylcholine yielded a large amount (0.3-0.7 mg/mL reaction mixture) of the fully functional bacteriorhodopsin. We also analyzed the process of functional expression in our system. The synthesized polypeptide was well protected from aggregation by the detergent-lipid mixed micelles and/or lipid disks, and was integrated into liposomes upon detergent removal by dialysis. This approach might be useful for the high yield production of functional membrane proteins.

A single major QTL controls expression of larval Cry1F resistance trait in Ostrinia nubilalis (Lepidoptera: Crambidae) and is independent of midgut receptor genes

USDA-ARS?s Scientific Manuscript database

European corn borer, Ostrinia nubilalis (Lepidoptera: Crambidae), is an introduced crop pest in North America that causes major damage to corn, and reduces yield of food, feed, and biofuel materials. Transgenic hybrid corn expressing the Bacillus thuringiensis (Bt) toxin Cry1F is highly lethal to O...

Enantioconvergent hydrolysis of racemic styrene oxide at high concentration by a pair of novel epoxide hydrolases into (R)-phenyl-1,2-ethanediol.

PubMed

Wang, Rui; Hu, Die; Zong, Xuncheng; Li, Jinping; Ding, Lei; Wu, Minchen; Li, Jianfang

2017-12-01

To prepare (R)-phenyl-1,2-ethanediol ((R)-PED) with high enantiomeric excess (ee p ) and yield from racemic styrene oxide (rac-SO) at high concentration by bi-enzymatic catalysis. The bi-enzymatic catalysis was designed for enantioconvergent hydrolysis of rac-SO by a pair of novel epoxide hydrolases (EHs), a Vigna radiata EH3 (VrEH3) and a variant (AuEH2 A250I ) of Aspergillus usamii EH2. The simultaneous addition mode of VrEH3 and AuEH2 A250I , exhibiting the highest average turnover frequency (aTOF) of 0.12 g h -1 g -1 , was selected, by which rac-SO (10 mM) was converted into (R)-PED with 92.6% ee p and 96.3% yield. Under the optimized reaction conditions: dry weight ratio 14:1 of VrEH3-expressing E. coli/vreh3 to AuEH2 A250I -expressing E. coli/Aueh2 A250I and reaction at 20 °C, rac-SO (10 mM) was completely hydrolyzed in 2.3 h, affording (R)-PED with 98% ee p . At the weight ratio 0.8:1 of rac-SO to two mixed dry cells, (R)-PED with 97.4% ee p and 98.7% yield was produced from 200 mM (24 mg/ml) rac-SO in 10.5 h. Enantioconvergent hydrolysis of rac-SO at high concentration catalyzed by both VrEH3 and AuEH2 A250I is an effective method for preparing (R)-PED with high ee p and yield.

Draft Assembly of Elite Inbred Line PH207 Provides Insights into Genomic and Transcriptome Diversity in Maize

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hirsch, Candice N.; Hirsch, Cory D.; Brohammer, Alex B.

Intense artificial selection over the last 100 years has produced elite maize (Zea mays) inbred lines that combine to produce high-yielding hybrids. To further our understanding of how genome and transcriptome variation contribute to the production of high-yielding hybrids, we generated a draft genome assembly of the inbred line PH207 to complement and compare with the existing B73 reference sequence. B73 is a founder of the Stiff Stalk germplasm pool, while PH207 is a founder of Iodent germplasm, both of which have contributed substantially to the production of temperate commercial maize and are combined to make heterotic hybrids. Comparison ofmore » these two assemblies revealed over 2500 genes present in only one of the two genotypes and 136 gene families that have undergone extensive expansion or contraction. Transcriptome profiling revealed extensive expression variation, with as many as 10,564 differentially expressed transcripts and 7128 transcripts expressed in only one of the two genotypes in a single tissue. Genotype-specific genes were more likely to have tissue/condition-specific expression and lower transcript abundance. The availability of a high-quality genome assembly for the elite maize inbred PH207 expands our knowledge of the breadth of natural genome and transcriptome variation in elite maize inbred lines across heterotic pools.« less

Draft Assembly of Elite Inbred Line PH207 Provides Insights into Genomic and Transcriptome Diversity in Maize

DOE PAGES

Hirsch, Candice N.; Hirsch, Cory D.; Brohammer, Alex B.; ...

2016-11-01

Intense artificial selection over the last 100 years has produced elite maize (Zea mays) inbred lines that combine to produce high-yielding hybrids. To further our understanding of how genome and transcriptome variation contribute to the production of high-yielding hybrids, we generated a draft genome assembly of the inbred line PH207 to complement and compare with the existing B73 reference sequence. B73 is a founder of the Stiff Stalk germplasm pool, while PH207 is a founder of Iodent germplasm, both of which have contributed substantially to the production of temperate commercial maize and are combined to make heterotic hybrids. Comparison ofmore » these two assemblies revealed over 2500 genes present in only one of the two genotypes and 136 gene families that have undergone extensive expansion or contraction. Transcriptome profiling revealed extensive expression variation, with as many as 10,564 differentially expressed transcripts and 7128 transcripts expressed in only one of the two genotypes in a single tissue. Genotype-specific genes were more likely to have tissue/condition-specific expression and lower transcript abundance. The availability of a high-quality genome assembly for the elite maize inbred PH207 expands our knowledge of the breadth of natural genome and transcriptome variation in elite maize inbred lines across heterotic pools.« less

Draft Assembly of Elite Inbred Line PH207 Provides Insights into Genomic and Transcriptome Diversity in Maize[OPEN

PubMed Central

Soifer, Ilya; Barad, Omer; Shem-Tov, Doron; Baruch, Kobi; Lu, Fei; Hernandez, Alvaro G.; Wright, Chris L.; Koehler, Klaus; Buell, C. Robin; de Leon, Natalia

2016-01-01

Intense artificial selection over the last 100 years has produced elite maize (Zea mays) inbred lines that combine to produce high-yielding hybrids. To further our understanding of how genome and transcriptome variation contribute to the production of high-yielding hybrids, we generated a draft genome assembly of the inbred line PH207 to complement and compare with the existing B73 reference sequence. B73 is a founder of the Stiff Stalk germplasm pool, while PH207 is a founder of Iodent germplasm, both of which have contributed substantially to the production of temperate commercial maize and are combined to make heterotic hybrids. Comparison of these two assemblies revealed over 2500 genes present in only one of the two genotypes and 136 gene families that have undergone extensive expansion or contraction. Transcriptome profiling revealed extensive expression variation, with as many as 10,564 differentially expressed transcripts and 7128 transcripts expressed in only one of the two genotypes in a single tissue. Genotype-specific genes were more likely to have tissue/condition-specific expression and lower transcript abundance. The availability of a high-quality genome assembly for the elite maize inbred PH207 expands our knowledge of the breadth of natural genome and transcriptome variation in elite maize inbred lines across heterotic pools. PMID:27803309

Enhanced expression and purification of camelid single domain VHH antibodies from classical inclusion bodies.

PubMed

Maggi, Maristella; Scotti, Claudia

2017-08-01

Single domain antibodies (sdAbs) are small antigen-binding domains derived from naturally occurring, heavy chain-only immunoglobulins isolated from camelid and sharks. They maintain the same binding capability of full-length IgGs but with improved thermal stability and permeability, which justifies their scientific, medical and industrial interest. Several described recombinant forms of sdAbs have been produced in different hosts and with different strategies. Here we present an optimized method for a time-saving, high yield production and extraction of a poly-histidine-tagged sdAb from Escherichia coli classical inclusion bodies. Protein expression and extraction were attempted using 4 different methods (e.g. autoinducing or IPTG-induced soluble expression, non-classical and classical inclusion bodies). The best method resulted to be expression in classical inclusion bodies and urea-mediated protein extraction which yielded 60-70 mg/l bacterial culture. The method we here describe can be of general interest for an enhanced and efficient heterologous expression of sdAbs for research and industrial purposes. Copyright © 2017 Elsevier Inc. All rights reserved.

Transgenic Cotton Plants Expressing the HaHR3 Gene Conferred Enhanced Resistance to Helicoverpa armigera and Improved Cotton Yield

PubMed Central

Han, Qiang; Wang, Zhenzhen; He, Yunxin; Xiong, Yehui; Lv, Shun; Li, Shupeng; Zhang, Zhigang; Qiu, Dewen; Zeng, Hongmei

2017-01-01

RNA interference (RNAi) has been developed as an efficient technology. RNAi insect-resistant transgenic plants expressing double-stranded RNA (dsRNA) that is ingested into insects to silence target genes can affect the viability of these pests or even lead to their death. HaHR3, a molt-regulating transcription factor gene, was previously selected as a target expressed in bacteria and tobacco plants to control Helicoverpa armigera by RNAi technology. In this work, we selected the dsRNA-HaHR3 fragment to silence HaHR3 in cotton bollworm for plant mediated-RNAi research. A total of 19 transgenic cotton lines expressing HaHR3 were successfully cultivated, and seven generated lines were used to perform feeding bioassays. Transgenic cotton plants expressing dsHaHR3 were shown to induce high larval mortality and deformities of pupation and adult eclosion when used to feed the newly hatched larvae, and 3rd and 5th instar larvae of H. armigera. Moreover, HaHR3 transgenic cotton also demonstrated an improved cotton yield when compared with controls. PMID:28867769

Identification of genes related to high royal jelly production in the honey bee (Apis mellifera) using microarray analysis

PubMed Central

Nie, Hongyi; Liu, Xiaoyan; Pan, Jiao; Li, Wenfeng; Li, Zhiguo; Zhang, Shaowu; Chen, Shenglu; Miao, Xiaoqing; Zheng, Nenggan; Su, Songkun

2017-01-01

Abstract China is the largest royal jelly producer and exporter in the world, and high royal jelly-yielding strains have been bred in the country for approximately three decades. However, information on the molecular mechanism underlying high royal jelly production is scarce. Here, a cDNA microarray was used to screen and identify differentially expressed genes (DEGs) to obtain an overview on the changes in gene expression levels between high and low royal jelly producing bees. We developed a honey bee gene chip that covered 11,689 genes, and this chip was hybridised with cDNA generated from RNA isolated from heads of nursing bees. A total of 369 DEGs were identified between high and low royal jelly producing bees. Amongst these DEGs, 201 (54.47%) genes were up-regulated, whereas 168 (45.53%) were down-regulated in high royal jelly-yielding bees. Gene ontology (GO) analyses showed that they are mainly involved in four key biological processes, and pathway analyses revealed that they belong to a total of 46 biological pathways. These results provide a genetic basis for further studies on the molecular mechanisms involved in high royal jelly production. PMID:28981563

Identification of genes related to high royal jelly production in the honey bee (Apis mellifera) using microarray analysis.

PubMed

Nie, Hongyi; Liu, Xiaoyan; Pan, Jiao; Li, Wenfeng; Li, Zhiguo; Zhang, Shaowu; Chen, Shenglu; Miao, Xiaoqing; Zheng, Nenggan; Su, Songkun

2017-01-01

China is the largest royal jelly producer and exporter in the world, and high royal jelly-yielding strains have been bred in the country for approximately three decades. However, information on the molecular mechanism underlying high royal jelly production is scarce. Here, a cDNA microarray was used to screen and identify differentially expressed genes (DEGs) to obtain an overview on the changes in gene expression levels between high and low royal jelly producing bees. We developed a honey bee gene chip that covered 11,689 genes, and this chip was hybridised with cDNA generated from RNA isolated from heads of nursing bees. A total of 369 DEGs were identified between high and low royal jelly producing bees. Amongst these DEGs, 201 (54.47%) genes were up-regulated, whereas 168 (45.53%) were down-regulated in high royal jelly-yielding bees. Gene ontology (GO) analyses showed that they are mainly involved in four key biological processes, and pathway analyses revealed that they belong to a total of 46 biological pathways. These results provide a genetic basis for further studies on the molecular mechanisms involved in high royal jelly production.

MDM4 overexpression contributes to synoviocyte proliferation in patients with rheumatoid arthritis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Nanwei; Wang, Yuji, E-mail: yujiwang@sohu.com; State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200433

Research highlights: {yields} Elevated MDM4 mRNA and protein levels in FLS from patients with RA and OA. {yields} Strong MDM4 staining in synovial cells of inflammatory synovium. {yields} MDM4 knockdown increased p53 and p21 levels, and inhibited the proliferation of RA FLS. {yields} MDM4 overexpression increased p53 while decreased p21 levels, and promoted the growth of RA FLS. -- Abstract: Rheumatoid arthritis (RA) is a chronic autoimmune disease with features of inflammatory cell infiltration, synovial cell invasive proliferation, and ultimately, irreversible joint destruction. It has been reported that the p53 pathway is involved in RA pathogenesis. MDM4/MDMX is a majormore » negative regulator of p53. To determine whether MDM4 contributes to RA pathogenesis, MDM4 mRNA and protein expression were assessed in fibroblast-like synoviocytes (FLS) by real-time PCR, western blotting, and in synovial tissues by immunohistochemistry. Furthermore, MDM4 was knocked down and overexpressed by lentivirus-mediated expression, and the proliferative capacity of FLS was determined by MTS assay. We found that cultured FLS from RA and osteoarthritis (OA) patients exhibited higher levels of MDM4 mRNA and protein expression than those from trauma controls. MDM4 protein was highly expressed in the synovial lining and sublining cells from both types of arthritis. Finally, MDM4 knockdown inhibited the proliferation of RA FLS by enhancing functional p53 levels while MDM4 overexpression promoted the growth of RA FLS by inhibiting p53 effects. Taken together, our results suggest that the abundant expression of MDM4 in FLS may contribute to the hyperplasia phenotype of RA synovial tissues.« less

Genetic variations in ARE1 mediate grain yield by modulating nitrogen utilization in rice.

PubMed

Wang, Qing; Nian, Jinqiang; Xie, Xianzhi; Yu, Hong; Zhang, Jian; Bai, Jiaoteng; Dong, Guojun; Hu, Jiang; Bai, Bo; Chen, Lichao; Xie, Qingjun; Feng, Jian; Yang, Xiaolu; Peng, Juli; Chen, Fan; Qian, Qian; Li, Jiayang; Zuo, Jianru

2018-02-21

In crops, nitrogen directly determines productivity and biomass. However, the improvement of nitrogen utilization efficiency (NUE) is still a major challenge in modern agriculture. Here, we report the characterization of are1, a genetic suppressor of a rice fd-gogat mutant defective in nitrogen assimilation. ARE1 is a highly conserved gene, encoding a chloroplast-localized protein. Loss-of-function mutations in ARE1 cause delayed senescence and result in 10-20% grain yield increases, hence enhance NUE under nitrogen-limiting conditions. Analysis of a panel of 2155 rice varieties reveals that 18% indica and 48% aus accessions carry small insertions in the ARE1 promoter, which result in a reduction in ARE1 expression and an increase in grain yield under nitrogen-limiting conditions. We propose that ARE1 is a key mediator of NUE and represents a promising target for breeding high-yield cultivars under nitrogen-limiting condition.

Genistein promotes DNA demethylation of the steroidogenic factor 1 (SF-1) promoter in endometrial stromal cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Matsukura, Hiroshi, E-mail: hmatsukura.epi@mri.tmd.ac.jp; Aisaki, Ken-ichi; Igarashi, Katsuhide

2011-08-26

Highlights: {yields} Genistein (GEN) is a phytoestrogen found in soy products. {yields} GEN demethylated/unsilenced the steroidogenic factor 1 gene in endometrial tissue. {yields} GEN thus altered mRNA expression in uteri of ovariectomized (OVX) mice. {yields} A high-resolution melting assay was used to screen for epigenetic change. {yields} We isolated an endometrial cell clone that was epigenetically modulated by GEN. -- Abstract: It has recently been demonstrated that genistein (GEN), a phytoestrogen in soy products, is an epigenetic modulator in various types of cells; but its effect on endometrium has not yet been determined. We investigated the effects of GEN onmore » mouse uterine cells, in vivo and in vitro. Oral administration of GEN for 1 week induced mild proliferation of the endometrium in ovariectomized (OVX) mice, which was accompanied by the induction of steroidogenic factor 1 (SF-1) gene expression. GEN administration induced demethylation of multiple CpG sites in the SF-1 promoter; these sites are extensively methylated and thus silenced in normal endometrium. The GEN-mediated promoter demethylation occurred predominantly on the luminal side, as opposed to myometrium side, indicating that the epigenetic change was mainly shown in regenerated cells. Primary cultures of endometrial stromal cell colonies were screened for GEN-mediated alterations of DNA methylation by a high-resolution melting (HRM) method. One out of 20 colony-forming cell clones showed GEN-induced demethylation of SF-1. This clone exhibited a high proliferation capacity with continuous colony formation activity through multiple serial clonings. We propose that only a portion of endometrial cells are capable of receiving epigenetic modulation by GEN.« less

A Rare Allele of GS2 Enhances Grain Size and Grain Yield in Rice.

PubMed

Hu, Jiang; Wang, Yuexing; Fang, Yunxia; Zeng, Longjun; Xu, Jie; Yu, Haiping; Shi, Zhenyuan; Pan, Jiangjie; Zhang, Dong; Kang, Shujing; Zhu, Li; Dong, Guojun; Guo, Longbiao; Zeng, Dali; Zhang, Guangheng; Xie, Lihong; Xiong, Guosheng; Li, Jiayang; Qian, Qian

2015-10-05

Grain size determines grain weight and affects grain quality. Several major quantitative trait loci (QTLs) regulating grain size have been cloned; however, our understanding of the underlying mechanism that regulates the size of rice grains remains fragmentary. Here, we report the cloning and characterization of a dominant QTL, grain size on chromosome 2 (GS2), which encodes Growth-Regulating Factor 4 (OsGRF4), a transcriptional regulator. GS2 localizes to the nucleus and may act as a transcription activator. A rare mutation of GS2 affecting the binding site of a microRNA, OsmiR396c, causes elevated expression of GS2/OsGRF4. The increase in GS2 expression leads to larger cells and increased numbers of cells, which thus enhances grain weight and yield. The introduction of this rare allele of GS2/OsGRF4 into rice cultivars could significantly enhance grain weight and increase grain yield, with possible applications in breeding high-yield rice varieties. Copyright © 2015 The Author. Published by Elsevier Inc. All rights reserved.

Putative carotenoid genes expressed under the regulation of Shine-Dalgarno regions in Escherichia coli for efficient lycopene production.

PubMed

Jin, Weiyue; Xu, Xian; Jiang, Ling; Zhang, Zhidong; Li, Shuang; Huang, He

2015-11-01

Putative genes crtE, crtB, and crtI from Deinococcus wulumiqiensis R12, a novel species, were identified by genome mining and were co-expressed using the optimized Shine-Dalgarno (SD) regions to improve lycopene yield. A lycopene biosynthesis pathway was constructed by co-expressing these three genes in Escherichia coli. After optimizing the upstream SD regions and the culture medium, the recombinant strain EDW11 produced 88 mg lycopene g(-1) dry cell wt (780 mg lycopene l(-1)) after 40 h fermentation without IPTG induction, while the strain EDW without optimized SD regions only produced 49 mg lycopene g(-1) dry cell wt (417 mg lycopene l(-1)). Based on the optimization of the upstream SD regions and culture medium, the yield of the strain EDW11 reached a high level during microbial lycopene production until now.

High Level Expression and Purification of the Clinically Active Antimicrobial Peptide P-113 in Escherichia coli.

PubMed

Cheng, Kuang-Ting; Wu, Chih-Lung; Yip, Bak-Sau; Yu, Hui-Yuan; Cheng, Hsi-Tsung; Chih, Ya-Han; Cheng, Jya-Wei

2018-03-30

P-113, which was originally derived from the human saliva protein histatin 5, is a histidine-rich antimicrobial peptide with the sequence AKRHHGYKRKFH. P-113 is currently undergoing phase II clinical trial as a pharmaceutical agent to fight against fungal infections in HIV patients with oral candidiasis. Previously, we developed a new procedure for the high-yield expression and purification of hG31P, an analogue and antagonist of human CXCL8. Moreover, we have successfully removed lipopolysaccharide (LPS, endotoxin) associated with hG31P in the expression with Escherichia coli . In this paper, we have used hG31P as a novel fusion protein for the expression and purification of P-113. The purity of the expressed P-113 is more than 95% and the yield is 4 mg P-113 per liter of E. coli cell culture in Luria-Bertani (LB) medium. The antimicrobial activity of the purified P-113 was tested. Furthermore, we used circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy to study the structural properties of P-113. Our results indicate that using hG31P as a fusion protein to obtain large quantities of P-113 is feasible and is easy to scale up for commercial production. An effective way of producing enough P-113 for future clinical studies is evident in this study.

Elevation of the Yields of Very Long Chain Polyunsaturated Fatty Acids via Minimal Codon Optimization of Two Key Biosynthetic Enzymes

PubMed Central

Zheng, Desong; Sun, Quanxi; Liu, Jiang; Li, Yaxiao; Hua, Jinping

2016-01-01

Eicosapentaenoic acid (EPA, 20:5Δ5,8,11,14,17) and Docosahexaenoic acid (DHA, 22:6Δ4,7,10,13,16,19) are nutritionally beneficial to human health. Transgenic production of EPA and DHA in oilseed crops by transferring genes originating from lower eukaryotes, such as microalgae and fungi, has been attempted in recent years. However, the low yield of EPA and DHA produced in these transgenic crops is a major hurdle for the commercialization of these transgenics. Many factors can negatively affect transgene expression, leading to a low level of converted fatty acid products. Among these the codon bias between the transgene donor and the host crop is one of the major contributing factors. Therefore, we carried out codon optimization of a fatty acid delta-6 desaturase gene PinD6 from the fungus Phytophthora infestans, and a delta-9 elongase gene, IgASE1 from the microalga Isochrysis galbana for expression in Saccharomyces cerevisiae and Arabidopsis respectively. These are the two key genes encoding enzymes for driving the first catalytic steps in the Δ6 desaturation/Δ6 elongation and the Δ9 elongation/Δ8 desaturation pathways for EPA/DHA biosynthesis. Hence expression levels of these two genes are important in determining the final yield of EPA/DHA. Via PCR-based mutagenesis we optimized the least preferred codons within the first 16 codons at their N-termini, as well as the most biased CGC codons (coding for arginine) within the entire sequences of both genes. An expression study showed that transgenic Arabidopsis plants harbouring the codon-optimized IgASE1 contained 64% more elongated fatty acid products than plants expressing the native IgASE1 sequence, whilst Saccharomyces cerevisiae expressing the codon optimized PinD6 yielded 20 times more desaturated products than yeast expressing wild-type (WT) PinD6. Thus the codon optimization strategy we developed here offers a simple, effective and low-cost alternative to whole gene synthesis for high expression of foreign genes in yeast and Arabidopsis. PMID:27433934

Ectopic expression of H2AX protein promotes TrkA-induced cell death via modulation of TrkA tyrosine-490 phosphorylation and JNK activity upon DNA damage

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jung, Eun Joo; Kim, Deok Ryong, E-mail: drkim@gnu.ac.kr

2011-01-21

Research highlights: {yields} We established TrkA-inducible U2OS cells stably expressing GFP-H2AX proteins. {yields} GFP-H2AX was colocalized with TrkA in the cytoplasm. {yields} {gamma}H2AX production was significantly increased upon activation of TrkA and suppressed by TrkA inhibitor or JNK inhibitor. {yields} Ectopic expression of H2AX promoted TrkA-mediated cell death through the modulation of TrkA tyrosine-490 phosphorylation and JNK activity upon DNA damage. -- Abstract: We previously reported that TrkA overexpression causes accumulation of {gamma}H2AX proteins in the cytoplasm, subsequently leading to massive cell death in U2OS cells. To further investigate how cytoplasmic H2AX is associated with TrkA-induced cell death, we establishedmore » TrkA-inducible cells stably expressing GFP-tagged H2AX. We found that TrkA co-localizes with ectopically expressed GFP-H2AX proteins in the cytoplasm, especially at the juxta-nuclear membranes, which supports our previous results about a functional connection between TrkA and {gamma}H2AX in TrkA-induced cell death. {gamma}H2AX production from GFP-H2AX proteins was significantly increased when TrkA was overexpressed. Moreover, ectopic expression of H2AX activated TrkA-mediated signal pathways via up-regulation of TrkA tyrosine-490 phosphorylation. In addition, suppression of TrkA tyrosine-490 phosphorylation under a certain condition was removed by ectopic expression of H2AX, indicating a functional role of H2AX in the maintenance of TrkA activity. Indeed, TrkA-induced cell death was highly elevated by ectopic H2AX expression, and it was further accelerated by DNA damage via JNK activation. These all results suggest that cytoplasmic H2AX could play an important role in TrkA-mediated cell death by modulating TrkA upon DNA damage.« less

Liver regeneration after partial hepatectomy in rat is more impaired in a steatotic liver induced by dietary fructose compared to dietary fat

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tanoue, Shirou; Uto, Hirofumi, E-mail: hirouto@m2.kufm.kagoshima-u.ac.jp; Kumamoto, Ryo

Highlights: {yields} Hepatic steatosis in rats fed a high fructose diet was less severe than that in rats fed a high fat diet. {yields} Liver regeneration was more impaired in rats fed a high fructose diet than in rats fed a high fat diet. {yields} Dysregulation of genes associated with metabolism may contribute to impairment of liver regeneration. {yields} Regulation of the TGF-{beta}1 level after partial hepatectomy may be impaired in rats fed a high fructose diet. -- Abstract: Hepatic steatosis (HS) has a negative effect on liver regeneration, but different pathophysiologies of HS may lead to different outcomes. Malemore » Sprague-Dawley rats were fed a high fructose (66% fructose; H-fruc), high fat (54% fat; H-fat), or control chow diet for 4 weeks. Based on hepatic triglyceride content and oil red O staining, HS developed in the H-fruc group, but was less severe compared to the H-fat group. Hepatic mRNA expression levels of fatty acid synthase and fructokinase were increased and those of carnitine palmitoyltransferase-1 and peroxisome proliferator-activated receptor-{alpha} were decreased in the H-fruc group compared to the H-fat group. Liver regeneration after 70% partial hepatectomy (PHx) was evaluated by measuring the increase in postoperative liver mass and PCNA-positive hepatocytes, and was impaired in the H-fruc group compared to the H-fat and control groups on days 3 and 7. Serum levels of tumor necrosis factor-{alpha}, interleukin-6 and hepatocyte growth factor did not change significantly after PHx. In contrast, serum TGF-{beta}1 levels were slightly but significantly lower in the control group on day 1 and in the H-fat group on day 3 compared to the level in each group on day 0, and then gradually increased. However, the serum TGF-{beta}1 level did not change after PHx in the H-fruc group. These results indicate that impairment of liver regeneration after PHx in HS is related to the cause, rather than the degree, of steatosis. This difference may result from altered metabolic gene expression profiles and potential dysregulation of TGF-{beta}1 expression.« less

Accelerated high-yield generation of limb-innervating motor neurons from human stem cells

PubMed Central

Amoroso, Mackenzie W.; Croft, Gist F.; Williams, Damian J.; O’Keeffe, Sean; Carrasco, Monica A.; Davis, Anne R.; Roybon, Laurent; Oakley, Derek H.; Maniatis, Tom; Henderson, Christopher E.; Wichterle, Hynek

2013-01-01

Human pluripotent stem cells are a promising source of differentiated cells for developmental studies, cell transplantation, disease modeling, and drug testing. However, their widespread use even for intensely studied cell types like spinal motor neurons is hindered by the long duration and low yields of existing protocols for in vitro differentiation and by the molecular heterogeneity of the populations generated. We report a combination of small molecules that within 3 weeks induce motor neurons at up to 50% abundance and with defined subtype identities of relevance to neurodegenerative disease. Despite their accelerated differentiation, motor neurons expressed combinations of HB9, ISL1 and column-specific markers that mirror those observed in vivo in human fetal spinal cord. They also exhibited spontaneous and induced activity, and projected axons towards muscles when grafted into developing chick spinal cord. Strikingly, this novel protocol preferentially generates motor neurons expressing markers of limb-innervating lateral motor column motor neurons (FOXP1+/LHX3−). Access to high-yield cultures of human limb-innervating motor neuron subtypes will facilitate in-depth study of motor neuron subtype-specific properties, disease modeling, and development of large-scale cell-based screening assays. PMID:23303937

Transcriptional transitions in Nicotiana benthamiana leaves upon induction of oil synthesis by WRINKLED1 homologs from diverse species and tissues.

PubMed

Grimberg, Åsa; Carlsson, Anders S; Marttila, Salla; Bhalerao, Rishikesh; Hofvander, Per

2015-08-08

Carbon accumulation and remobilization are essential mechanisms in plants to ensure energy transfer between plant tissues with different functions or metabolic needs and to support new generations. Knowledge about the regulation of carbon allocation into oil (triacylglycerol) in plant storage tissue can be of great economic and environmental importance for developing new high-yielding oil crops. Here, the effect on global gene expression as well as on physiological changes in leaves transiently expressing five homologs of the transcription factor WRINKLED1 (WRI1) originating from diverse species and tissues; Arabidopsis thaliana and potato (Solanum tuberosum) seed embryo, poplar (Populus trichocarpa) stem cambium, oat (Avena sativa) grain endosperm, and nutsedge (Cyperus esculentus) tuber parenchyma, were studied by agroinfiltration in Nicotiana benthamiana. All WRI1 homologs induced oil accumulation when expressed in leaf tissue. Transcriptome sequencing revealed that all homologs induced the same general patterns with a drastic shift in gene expression profiles of leaves from that of a typical source tissue to a source-limited sink-like tissue: Transcripts encoding enzymes for plastid uptake and metabolism of phosphoenolpyruvate, fatty acid and oil biosynthesis were up-regulated, as were also transcripts encoding starch degradation. Transcripts encoding enzymes in photosynthesis and starch synthesis were instead down-regulated. Moreover, transcripts representing fatty acid degradation were up-regulated indicating that fatty acids might be degraded to feed the increased need to channel carbons into fatty acid synthesis creating a futile cycle. RT-qPCR analysis of leaves expressing Arabidopsis WRI1 showed the temporal trends of transcripts selected as 'markers' for key metabolic pathways one to five days after agroinfiltration. Chlorophyll fluorescence measurements of leaves expressing Arabidopsis WRI1 showed a significant decrease in photosynthesis, even though effect on starch content could not be observed. This data gives for the first time a general view on the transcriptional transitions in leaf tissue upon induction of oil synthesis by WRI1. This yields important information about what effects WRI1 may exert on global gene expression during seed and embryo development. The results suggest why high oil content in leaf tissue cannot be achieved by solely transcriptional activation by WRI1, which can be essential knowledge in the development of new high-yielding oil crops.

Efficient secretory expression of the sweet-tasting protein brazzein in the yeast Kluyveromyces lactis.

PubMed

Jo, Hyun-Joo; Noh, Jin-Seok; Kong, Kwang-Hoon

2013-08-01

Brazzein is an intensely sweet-tasting protein with high water solubility, heat stability, and taste properties resembling those of carbohydrate sweeteners. In the present study, we describe the expression of the synthetic gene encoding brazzein, a sweet protein in the yeast Kluyveromyces lactis. The synthetic brazzein gene was designed based on the biased codons of the yeast, so as to optimize its expression, as well as on the extracellular secretion for expression in an active, soluble form. The synthesized brazzein gene was cloned into the secretion vector pKLAC2, which contains the yeast prepropeptide signal from the Saccharomycescerevisiae α-mating factor. The constructed plasmid pKLAC2-des-pE1M-brazzein was introduced into the yeast K. lactis GG799. The yeast transformants were cultured for high-yield secretion of the recombinant des-pE1M-brazzein in YPGal medium for 96 h at 30°C. The expressed recombinant des-pE1M-brazzein was purified by CM-Sepharose column chromatography and approximately 104 mg/L was obtained. The purity and conformational state of the recombinant des-pE1M-brazzein were confirmed using SDS-PAGE, HPLC, and circular dichroism. The identity of the recombinant protein was also confirmed by N-terminal amino acid analysis and taste testing. The purified recombinant des-pE1M-brazzein had an intrinsic sweetness in its minor form, approximately 2130 times sweeter than sucrose on a weight basis. These results demonstrate that the K. lactis expression system is useful for producing the recombinant brazzein in active form at a high yield with attributes useful in the food industry. Copyright © 2013 Elsevier Inc. All rights reserved.

A chloride tolerant laccase from the plant pathogen ascomycete Botrytis aclada expressed at high levels in Pichia pastoris.

PubMed

Kittl, Roman; Mueangtoom, Kitti; Gonaus, Christoph; Khazaneh, Shima Tahvilda; Sygmund, Christoph; Haltrich, Dietmar; Ludwig, Roland

2012-01-20

Fungal laccases from basidiomycetous fungi are thoroughly investigated in respect of catalytic mechanism and industrial applications, but the number of reported and well characterized ascomycetous laccases is much smaller although they exhibit interesting catalytic properties. We report on a highly chloride tolerant laccase produced by the plant pathogen ascomycete Botrytis aclada, which was recombinantly expressed in Pichia pastoris with an extremely high yield and purified to homogeneity. In a fed-batch fermentation, 495 mg L(-1) of laccase was measured in the medium, which is the highest concentration obtained for a laccase by a yeast expression system. The recombinant B. aclada laccase has a typical molecular mass of 61,565 Da for the amino acid chain. The pI is approximately 2.4, a very low value for a laccase. Glycosyl residues attached to the recombinant protein make up for approximately 27% of the total protein mass. B. aclada laccase exhibits very low K(M) values and high substrate turnover numbers for phenolic and non-phenolic substrates at acidic and near neutral pH. The enzyme's stability increases in the presence of chloride ions and, even more important, its substrate turnover is only weakly inhibited by chloride ions (I(50)=1.4M), which is in sharp contrast to most other described laccases. This high chloride tolerance is mandatory for some applications such as implantable biofuel cells and laccase catalyzed reactions, which suffer from the presence of chloride ions. The high expression yield permits fast and easy production for further basic and applied research. Copyright © 2011 Elsevier B.V. All rights reserved.

Engineering Extracellular Expression Systems in Escherichia coli Based on Transcriptome Analysis and Cell Growth State.

PubMed

Gao, Wen; Yin, Jun; Bao, Lichen; Wang, Qun; Hou, Shan; Yue, Yali; Yao, Wenbing; Gao, Xiangdong

2018-05-18

Escherichia coli extracellular expression systems have a number of advantages over other systems, such as lower pyrogen levels and a simple purification process. Various approaches, such as the generation of leaky mutants via chromosomal engineering, have been explored for this expression system. However, extracellular protein yields in leaky mutants are relatively low compared to that in intracellular expression systems and therefore need to be improved. In this work, we describe the construction, characterization, and mechanism of enhanced extracellular expression in Escherichia coli. On the basis of the localizations, functions, and transcription levels of cell envelope proteins, we systematically elucidated the effects of multiple gene deletions on cell growth and extracellular expression using modified CRISPR/Cas9-based genome editing and a FlAsH labeling assay. High extracellular yields of heterologous proteins of different sizes were obtained by screening multiple gene mutations. The enhancement of extracellular secretion was associated with the derepression of translation and translocation. This work utilized universal methods in the design of extracellular expression systems for genes not directly associated with protein synthesis that were used to generate strains with higher protein expression capability. We anticipate that extracellular expression systems may help to shed light on the poorly understood aspects of these secretion processes as well as to further assist in the construction of engineered prokaryotic cells for efficient extracellular production of heterologous proteins.

Meta-transcriptomics indicates biotic cross-tolerance in willow trees cultivated on petroleum hydrocarbon contaminated soil.

PubMed

Gonzalez, Emmanuel; Brereton, Nicholas J B; Marleau, Julie; Guidi Nissim, Werther; Labrecque, Michel; Pitre, Frederic E; Joly, Simon

2015-10-12

High concentrations of petroleum hydrocarbon (PHC) pollution can be hazardous to human health and leave soils incapable of supporting agricultural crops. A cheap solution, which can help restore biodiversity and bring land back to productivity, is cultivation of high biomass yielding willow trees. However, the genetic mechanisms which allow these fast-growing trees to tolerate PHCs are as yet unclear. Salix purpurea 'Fish Creek' trees were pot-grown in soil from a former petroleum refinery, either lacking or enriched with C10-C50 PHCs. De novo assembled transcriptomes were compared between tree organs and impartially annotated without a priori constraint to any organism. Over 45% of differentially expressed genes originated from foreign organisms, the majority from the two-spotted spidermite, Tetranychus urticae. Over 99% of T. urticae transcripts were differentially expressed with greater abundance in non-contaminated trees. Plant transcripts involved in the polypropanoid pathway, including phenylalanine ammonia-lyase (PAL), had greater expression in contaminated trees whereas most resistance genes showed higher expression in non-contaminated trees. The impartial approach to annotation of the de novo transcriptomes, allowing for the possibility for multiple species identification, was essential for interpretation of the crop's response treatment. The meta-transcriptomic pattern of expression suggests a cross-tolerance mechanism whereby abiotic stress resistance systems provide improved biotic resistance. These findings highlight a valuable but complex biotic and abiotic stress response to real-world, multidimensional contamination which could, in part, help explain why crops such as willow can produce uniquely high biomass yields on challenging marginal land.

High-yield production of canine parvovirus virus-like particles in a baculovirus expression system.

PubMed

Jin, Hongli; Xia, Xiaohong; Liu, Bing; Fu, Yu; Chen, Xianping; Wang, Huihui; Xia, Zhenqiang

2016-03-01

An optimized VP2 gene from the current prevalent CPV strain (new CPV-2a) in China was expressed in a baculovirus expression system. It was found that the VP2 proteins assembled into virus-like particles (VLPs) with antigenic properties similar to those of natural CPV and with an especially high hemagglutination (HA) titer (1:2(20)). Dogs intramuscularly or orally immunized with VLPs produced antibodies against CPV with >1:80 hemagglutination inhibition (HI) units for at least 3 months. The CPV VLPs could be considered for use as a vaccine against CPV or as a platform for research on chimeric VLP vaccines against other diseases.

High-level expression of a synthetic gene encoding a sweet protein, monellin, in Escherichia coli.

PubMed

Chen, Zhongjun; Cai, Heng; Lu, Fuping; Du, Lianxiang

2005-11-01

The expression of a synthetic gene encoding monellin, a sweet protein, in E. coli under the control of T7 promoter from phage is described. The single-chain monellin gene was designed based on the biased codons of E. coli so as to optimize its expression. Monellin was produced and accounted for 45% of total soluble proteins. It was purified to yield 43 mg protein per g dry cell wt. The purity of the recombinant protein was confirmed by SDS-PAGE.

Efficient soluble expression of disulfide bonded proteins in the cytoplasm of Escherichia coli in fed-batch fermentations on chemically defined minimal media.

PubMed

Gąciarz, Anna; Khatri, Narendar Kumar; Velez-Suberbie, M Lourdes; Saaranen, Mirva J; Uchida, Yuko; Keshavarz-Moore, Eli; Ruddock, Lloyd W

2017-06-15

The production of recombinant proteins containing disulfide bonds in Escherichia coli is challenging. In most cases the protein of interest needs to be either targeted to the oxidizing periplasm or expressed in the cytoplasm in the form of inclusion bodies, then solubilized and re-folded in vitro. Both of these approaches have limitations. Previously we showed that soluble expression of disulfide bonded proteins in the cytoplasm of E. coli is possible at shake flask scale with a system, known as CyDisCo, which is based on co-expression of a protein of interest along with a sulfhydryl oxidase and a disulfide bond isomerase. With CyDisCo it is possible to produce disulfide bonded proteins in the presence of intact reducing pathways in the cytoplasm. Here we scaled up production of four disulfide bonded proteins to stirred tank bioreactors and achieved high cell densities and protein yields in glucose fed-batch fermentations, using an E. coli strain (BW25113) with the cytoplasmic reducing pathways intact. Even without process optimization production of purified human single chain IgA 1 antibody fragment reached 139 mg/L and hen avidin 71 mg/L, while purified yields of human growth hormone 1 and interleukin 6 were around 1 g/L. Preliminary results show that human growth hormone 1 was also efficiently produced in fermentations of W3110 strain and when glucose was replaced with glycerol as the carbon source. Our results show for the first time that efficient production of high yields of soluble disulfide bonded proteins in the cytoplasm of E. coli with the reducing pathways intact is feasible to scale-up to bioreactor cultivations on chemically defined minimal media.

Development of a high yielding E. coli periplasmic expression system for the production of humanized Fab' fragments.

PubMed

Ellis, Mark; Patel, Pareshkumar; Edon, Marjory; Ramage, Walter; Dickinson, Robert; Humphreys, David P

2017-01-01

Humanized Fab' fragments may be produced in the periplasm of Escherichia coli but can be subject to degradation by host cell proteases. In order to increase Fab' yield and reduce proteolysis we developed periplasmic protease deficient strains of E. coli. These strains lacked the protease activity of Tsp, protease III and DegP. High cell density fermentations indicated Tsp deficient strains increased productivity two fold but this increase was accompanied by premature cell lysis soon after the induction of Fab' expression. To overcome the reduction in cell viability we introduced suppressor mutations into the spr gene. The mutations partially restored the wild type phenotype of the cells. Furthermore, we coexpressed a range of periplasmic chaperone proteins with the Fab', DsbC had the most significant impact, increasing humanized Fab' production during high cell density fermentation. When DsbC coexpression was combined with a Tsp deficient spr strain we observed an increase in yield and essentially restored "wild type" cell viability. We achieved a final periplasmic yield of over 2.4g/L (final cell density OD 600 105), 40 h post Fab' induction with minimal cell lysis.The data suggests that proteolysis, periplasm integrity, protein folding and disulphide bond formation are all potential limiting steps in the production of Fab' fragments in the periplasm of E. coli. In this body of work, we have addressed these limiting steps by utilizing stabilized protease deficient strains and chaperone coexpression. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:212-220, 2017. © 2016 American Institute of Chemical Engineers.

A Pseudomonas putida efflux pump acts on short-chain alcohols.

PubMed

Basler, Georg; Thompson, Mitchell; Tullman-Ercek, Danielle; Keasling, Jay

2018-01-01

The microbial production of biofuels is complicated by a tradeoff between yield and toxicity of many fuels. Efflux pumps enable bacteria to tolerate toxic substances by their removal from the cells while bypassing the periplasm. Their use for the microbial production of biofuels can help to improve cell survival, product recovery, and productivity. However, no native efflux pump is known to act on the class of short-chain alcohols, important next-generation biofuels, and it was considered unlikely that such an efflux pump exists. We report that controlled expression of the RND-type efflux pump TtgABC from Pseudomonas putida DOT-T1E strongly improved cell survival in highly toxic levels of the next-generation biofuels n -butanol, isobutanol, isoprenol, and isopentanol. GC-FID measurements indicated active efflux of n -butanol when the pump is expressed. Conversely, pump expression did not lead to faster growth in media supplemented with low concentrations of n -butanol and isopentanol. TtgABC is the first native efflux pump shown to act on multiple short-chain alcohols. Its controlled expression can be used to improve cell survival and increase production of biofuels as an orthogonal approach to metabolic engineering. Together with the increased interest in P. putida for metabolic engineering due to its flexible metabolism, high native tolerance to toxic substances, and various applications of engineering its metabolism, our findings endorse the strain as an excellent biocatalyst for the high-yield production of next-generation biofuels.

Material and methods to increase plant growth and yield

DOEpatents

Kirst, Matias

2015-09-15

The present invention relates to materials and methods for modulating growth rates, yield, and/or resistance to drought conditions in plants. In one embodiment, a method of the invention comprises increasing expression of an hc1 gene (or a homolog thereof that provides for substantially the same activity), or increasing expression or activity of the protein encoded by an hc1 gene thereof, in a plant, wherein expression of the hc1 gene or expression or activity of the protein encoded by an hc1 gene results in increased growth rate, yield, and/or drought resistance in the plant.

Materials and methods to increase plant growth and yield

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kirst, Matias

The present invention relates to materials and methods for modulating growth rates, yield, and/or resistance to drought conditions in plants. In one embodiment, a method of the invention comprises increasing expression of an hc1 gene (or a homolog thereof that provides for substantially the same activity), or increasing expression or activity of the protein encoded by an hc1 gene thereof, in a plant, wherein expression of the hc1 gene or expression or activity of the protein encoded by an hc1 gene results in increased growth rate, yield, and/or drought resistance in the plant.

Characterization of the 4,6-α-glucanotransferase GTFB enzyme of Lactobacillus reuteri 121 isolated from inclusion bodies.

PubMed

Bai, Yuxiang; van der Kaaij, Rachel Maria; Woortman, Albert Jan Jacob; Jin, Zhengyu; Dijkhuizen, Lubbert

2015-06-09

The GTFB enzyme of the probiotic bacterium Lactobacillus reuteri 121 is a 4,6-α-glucanotransferase of glycoside hydrolase family 70 (GH70; http://www.cazy.org ). Contrary to the glucansucrases in GH70, GTFB is unable to use sucrose as substrate, but instead converts malto-oligosaccharides and starch into isomalto-/malto- polymers that may find application as prebiotics and dietary fibers. The GTFB enzyme expresses well in Escherichia coli BL21 Star (DE3), but mostly accumulates in inclusion bodies (IBs) which generally contain wrongly folded protein and inactive enzyme. Denaturation followed by refolding, as well as ncIB preparation were used for isolation of active GTFB protein from inclusion bodies. Soluble, refolded and ncIB GTFB were compared using activity assays, secondary structure analysis by FT-IR, and product analyses by NMR, HPAEC and SEC. Expression of GTFB in E. coli yielded > 100 mg/l relatively pure and active but mostly insoluble GTFB protein in IBs, regardless of the expression conditions used. Following denaturing, refolding of GTFB protein was most efficient in double distilled H2O. Also, GTFB ncIBs were active, with approx. 10 % of hydrolysis activity compared to the soluble protein. When expressed as units of activity obtained per liter E. coli culture, the total amount of ncIB GTFB expressed possessed around 180 % hydrolysis activity and 100 % transferase activity compared to the amount of soluble GTFB enzyme obtained from one liter culture. The product profiles obtained for the three GTFB enzyme preparations were similar when analyzed by HPAEC and NMR. SEC investigation also showed that these 3 enzyme preparations yielded products with similar size distributions. FT-IR analysis revealed extended β-sheet formation in ncIB GTFB providing an explanation at the molecular level for reduced GTFB activity in ncIBs. The thermostability of ncIB GTFB was relatively high compared to the soluble and refolded GTFB. In view of their relatively high yield, activity and high thermostability, both refolded and ncIB GTFB derived from IBs in E. coli may find industrial application in the synthesis of modified starches.

Camelid-derived heavy-chain nanobody against Clostridium botulinum neurotoxin E in Pichia pastoris.

PubMed

Baghban, Roghayyeh; Gargari, Seyed Latif Mousavi; Rajabibazl, Masoumeh; Nazarian, Shahram; Bakherad, Hamid

2016-01-01

Botulinum neurotoxins (BoNTs) result in severe and often fatal disease, botulism. Common remedial measures such as equine antitoxin and human botulism immunoglobulin in turn are problematic and time-consuming. Therefore, diagnosis and therapy of BoNTs are vital. The variable domain of heavy-chain antibodies (VHH) has unique features, such as the ability to identify and bind specifically to target epitopes and ease of production in bacteria and yeast. The Pichia pastoris is suitable for expression of recombinant antibody fragments. Disulfide bond formation and correct folds of protein with a high yield are some of the advantages of this eukaryotic host. In this study, we have expressed and purified the camelid VHH against BoNT/E in P. pastoris. The final yield of P. pastoris-expressed antibody was estimated to be 16 mg/l, which is higher than that expressed by Escherichia coli. The nanobody expressed in P. pastoris neutralized 4LD50 of the BoNT/E upon i.p. injection in 25% of mice. The nanobody expressed in E. coli extended the mice's survival to 1.5-fold compared to the control. This experiment indicated that the quality of expressed protein in the yeast is superior to that of the bacterial expression. Favorable protein folding by P. pastoris seems to play a role in its better toxin-binding property. © 2014 International Union of Biochemistry and Molecular Biology, Inc.

Highly active promoters and native secretion signals for protein production during extremely low growth rates in Aspergillus niger.

PubMed

Wanka, Franziska; Arentshorst, Mark; Cairns, Timothy C; Jørgensen, Thomas; Ram, Arthur F J; Meyer, Vera

2016-08-20

The filamentous ascomycete Aspergillus niger is used in many industrial processes for the production of enzymes and organic acids by batch and fed-batch cultivation. An alternative technique is continuous cultivation, which promises improved yield and optimized pipeline efficiency. In this work, we have used perfusion (retentostat) cultivation to validate two promoters that are suitable for A. niger continuous cultivation of industrially relevant products. Firstly, promoters of genes encoding either an antifungal protein (Panafp) or putative hydrophobin (PhfbD) were confirmed as active throughout retentostat culture by assessing mRNA and protein levels using a luciferase (mluc) reporter system. This demonstrated the anafp promoter mediates a high but temporally variable expression profile, whereas the hfbD promoter mediates a semi-constant, moderate-to-high protein expression during retentostat culture. In order to assess whether these promoters were suitable to produce heterologous proteins during retentostat cultivation, the secreted antifungal protein (AFP) from Aspergillus giganteus, which has many potential biotechnological applications, was expressed in A. niger during retentostat cultivation. Additionally, this assay was used to concomitantly validate that native secretion signals encoded in anafp and hfbD genes can be harnessed for secretion of heterologous proteins. Afp mRNA and protein abundance were comparable to luciferase measurements throughout retentostat cultivation, validating the use of Panafp and PhfbD for perfusion cultivation. Finally, a gene encoding the highly commercially relevant thermal hysteresis protein (THP) was expressed in this system, which did not yield detectable protein. Both hfbD and anafp promoters are suitable for production of useful products in A. niger during perfusion cultivation. These findings provide a platform for further optimisations for high production of heterologous proteins with industrial relevance.

High-efficiency production of bioactive recombinant human fibroblast growth factor 18 in Escherichia coli and its effects on hair follicle growth.

PubMed

Song, Lintao; Huang, Zhifeng; Chen, Yu; Li, Haiyan; Jiang, Chao; Li, Xiaokun

2014-01-01

Using fusion tags, expression of recombinant human fibroblast growth factor 18 (rhFGF18) in mammalian cells and Escherichia coli has been extensively used for fundamental research and clinical applications, including chondrogenesis and osteogenesis, hair growth, and neuroprotection. However, high-level rhFGF18 expression is difficult and the products are often not homogeneous. Furthermore, fusion-tagged protein has higher immunogenicity and lower bioactivity, and the removal of the fused tag is expensive. To overcome the limitations of fusion-tagged expression of protein and to prepare soluble highly bioactive rhFGF18, we have developed a rapid and efficient expression strategy. Optimized hFGF18 gene was amplified by polymerase chain reaction and cloned into pET22b and pET3c vectors, then transformed into E. coli strains Origima (DE3) and BL21 (DE3)PlysS. The best combination of plasmid and host strain was selected, and only Origima (DE3)/pET3c-rhFGF18 was screened for high-level expressed rhFGF18. Under optimal conditions in a 30-L fermentor, the average bacterial yield and expression level of rhFGF18 of three batches were more than 652 g and 30 % respectively, after treatment with 1 mM isopropyl-thio-β-galactopyranoside for 10 h at 25 °C. The target protein was purified by CM Sepharose FF and heparin affinity chromatography. The purity of rhFGF18 was shown by HPLC to be higher than 95 %, and the yield was 155 mg/L. In vitro MTT assays demonstrated that the purified rhFGF18 could stimulate significant proliferation of NIH3T3 cells, and animal experiments showed that rhFGF18 could effectively regulate hair growth. In conclusion, this may be a better method of producing rhFGF18 to meet the increasing demand in its pharmacological application.

Using RNA-seq to Profile Gene Expression of Spikelet Development in Response to Temperature and Nitrogen during Meiosis in Rice (Oryza sativa L.).

PubMed

Yang, Jun; Chen, Xiaorong; Zhu, Changlan; Peng, Xiaosong; He, Xiaopeng; Fu, Junru; Ouyang, Linjuan; Bian, Jianmin; Hu, Lifang; Sun, Xiaotang; Xu, Jie; He, Haohua

2015-01-01

Rice reproductive development is sensitive to high temperature and soil nitrogen supply, both of which are predicted to be increased threats to rice crop yield. Rice spikelet development is a critical process that determines yield, yet little is known about the transcriptional regulation of rice spikelet development in response to the combination of heat stress and low nitrogen availability. Here, we profiled gene expression of rice spikelet development during meiosis under heat stress and different nitrogen levels using RNA-seq. We subjected plants to four treatments: 1) NN: normal nitrogen level (165 kg ha-1) with normal temperature (30°C); 2) HH: high nitrogen level (264 kg ha-1) with high temperature (37°C); 3) NH: normal nitrogen level and high temperature; and 4) HN: high nitrogen level and normal temperature. The de novo transcriptome assembly resulted in 52,250,482 clean reads aligned with 76,103 unigenes, which were then used to compare differentially expressed genes (DEGs) in the different treatments. Comparing gene expression in samples with the same nitrogen levels but different temperatures, we identified 70 temperature-responsive DEGs in normal nitrogen levels (NN vs NH) and 135 DEGs in high nitrogen levels (HN vs HH), with 27 overlapping DEGs. We identified 17 and seven nitrogen-responsive DEGs by comparing changes in nitrogen levels in lower temperature (NN vs HN) and higher temperature (NH vs HH), with one common DEG. The temperature-responsive genes were principally associated with cytochrome, heat shock protein, peroxidase, and ubiquitin, while the nitrogen-responsive genes were mainly involved in glutamine synthetase, amino acid transporter, pollen development, and plant hormone. Rice spikelet fertility was significantly reduced under high temperature, but less reduced under high-nitrogen treatment. In the high temperature treatments, we observed downregulation of genes involved in spikelet development, such as pollen tube growth, pollen maturation, especially sporopollenin biosynthetic process, and pollen exine formation. Moreover, we observed higher expression levels of the co-expressed DEGs in HN vs HH compared to NN vs NH. These included the six downregulated genes (one pollen maturation and five pollen exine formation genes), as well as the four upregulated DEGs in response to heat. This suggests that high-nitrogen treatment may enhance the gene expression levels to mitigate aspects of heat-stress. The spikelet genes identified in this study may play important roles in response to the combined effects of high temperature and high nitrogen, and may serve as candidates for crop improvement.

Climatically driven yield variability of major crops in Khakassia (South Siberia)

NASA Astrophysics Data System (ADS)

Babushkina, Elena A.; Belokopytova, Liliana V.; Zhirnova, Dina F.; Shah, Santosh K.; Kostyakova, Tatiana V.

2018-06-01

We investigated the variability of yield of the three main crop cultures in the Khakassia Republic: spring wheat, spring barley, and oats. In terms of yield values, variability characteristics, and climatic response, the agricultural territory of Khakassia can be divided into three zones: (1) the Northern Zone, where crops yield has a high positive response to the amount of precipitation, May-July, and a moderately negative one to the temperatures of the same period; (2) the Central Zone, where crops yield depends mainly on temperatures; and (3) the Southern Zone, where climate has the least expressed impact on yield. The dominant pattern in the crops yield is caused by water stress during periods of high temperatures and low moisture supply with heat stress as additional reason. Differences between zones are due to combinations of temperature latitudinal gradient, precipitation altitudinal gradient, and the presence of a well-developed hydrological network and the irrigational system as moisture sources in the Central Zone. More detailed analysis shows differences in the climatic sensitivity of crops during phases of their vegetative growth and grain development and, to a lesser extent, during harvesting period. Multifactor linear regression models were constructed to estimate climate- and autocorrelation-induced variability of the crops yield. These models allowed prediction of the possibility of yield decreasing by at least 2-11% in the next decade due to increasing of the regional summer temperatures.

Climatically driven yield variability of major crops in Khakassia (South Siberia)

NASA Astrophysics Data System (ADS)

Babushkina, Elena A.; Belokopytova, Liliana V.; Zhirnova, Dina F.; Shah, Santosh K.; Kostyakova, Tatiana V.

2017-12-01

We investigated the variability of yield of the three main crop cultures in the Khakassia Republic: spring wheat, spring barley, and oats. In terms of yield values, variability characteristics, and climatic response, the agricultural territory of Khakassia can be divided into three zones: (1) the Northern Zone, where crops yield has a high positive response to the amount of precipitation, May-July, and a moderately negative one to the temperatures of the same period; (2) the Central Zone, where crops yield depends mainly on temperatures; and (3) the Southern Zone, where climate has the least expressed impact on yield. The dominant pattern in the crops yield is caused by water stress during periods of high temperatures and low moisture supply with heat stress as additional reason. Differences between zones are due to combinations of temperature latitudinal gradient, precipitation altitudinal gradient, and the presence of a well-developed hydrological network and the irrigational system as moisture sources in the Central Zone. More detailed analysis shows differences in the climatic sensitivity of crops during phases of their vegetative growth and grain development and, to a lesser extent, during harvesting period. Multifactor linear regression models were constructed to estimate climate- and autocorrelation-induced variability of the crops yield. These models allowed prediction of the possibility of yield decreasing by at least 2-11% in the next decade due to increasing of the regional summer temperatures.

Human blood RNA stabilization in samples collected and transported for a large biobank

PubMed Central

2012-01-01

Background The Norwegian Mother and Child Cohort Study (MoBa) is a nation-wide population-based pregnancy cohort initiated in 1999, comprising more than 108.000 pregnancies recruited between 1999 and 2008. In this study we evaluated the feasibility of integrating RNA analyses into existing MoBa protocols. We compared two different blood RNA collection tube systems – the PAXgene™ Blood RNA system and the Tempus™ Blood RNA system - and assessed the effects of suboptimal blood volumes in collection tubes and of transportation of blood samples by standard mail. Endpoints to characterize the samples were RNA quality and yield, and the RNA transcript stability of selected genes. Findings High-quality RNA could be extracted from blood samples stabilized with both PAXgene and Tempus tubes. The RNA yields obtained from the blood samples collected in Tempus tubes were consistently higher than from PAXgene tubes. Higher RNA yields were obtained from cord blood (3 – 4 times) compared to adult blood with both types of tubes. Transportation of samples by standard mail had moderate effects on RNA quality and RNA transcript stability; the overall RNA quality of the transported samples was high. Some unexplained changes in gene expression were noted, which seemed to correlate with suboptimal blood volumes collected in the tubes. Temperature variations during transportation may also be of some importance. Conclusions Our results strongly suggest that special collection tubes are necessary for RNA stabilization and they should be used for establishing new biobanks. We also show that the 50,000 samples collected in the MoBa biobank provide RNA of high quality and in sufficient amounts to allow gene expression analyses for studying the association of disease with altered patterns of gene expression. PMID:22988904

Significantly improving the yield of recombinant proteins in Bacillus subtilis by a novel powerful mutagenesis tool (ARTP): Alkaline α-amylase as a case study.

PubMed

Ma, Yingfang; Yang, Haiquan; Chen, Xianzhong; Sun, Bo; Du, Guocheng; Zhou, Zhemin; Song, Jiangning; Fan, You; Shen, Wei

2015-10-01

In this study, atmospheric and room temperature plasma (ARTP), a promising mutation breeding technique, was successfully applied to generate Bacillus subtilis mutants that yielded large quantities of recombinant protein. The high throughput screening platform was implemented to select those mutants with the highest yield of recombinant alkaline α-amylase (AMY), including the preferred mutant B. subtilis WB600 mut-12#. The yield and productivity of recombinant AMY in B. subtilis WB600 mut-12# increased 35.0% and 8.8%, respectively, the extracellular protein concentration of which increased 37.9%. B. subtilis WB600 mut-12# exhibited good genetic stability. Cells from B. subtilis WB600 mut-12# became shorter and wider than those from the wild-type. This study is the first to report a novel powerful mutagenesis tool (ARTP) that significantly improves the yield of recombinant proteins in B. subtilis and may therefore play an important role in the high expression level of proteins in recombinant microbial hosts. Copyright © 2015 Elsevier Inc. All rights reserved.

Efficient Degradation of Malathion in the Presence of Detergents Using an Engineered Organophosphorus Hydrolase Highly Expressed by Pichia pastoris without Methanol Induction.

PubMed

Bai, Yun-Peng; Luo, Xiao-Jing; Zhao, Yu-Lian; Li, Chun-Xiu; Xu, Dian-Sheng; Xu, Jian-He

2017-10-18

The biodegradation of pesticides by organophosphorus hydrolases (OPHs) requires an efficient enzyme production technology in industry. Herein, a Pichia pastoris strain was constructed for the extracellular expression of PoOPH M9 , an engineered malathion-degrading enzyme. After optimization, the maximum titer and yield of fermentation reached 50.8 kU/L and 4.1 g protein /L after 3 days, with the highest space-time yield (STY) reported so far, 640 U L -1 h -1 . PoOPH M9 displayed its high activity and stability in the presence of 0.1% (w/w) plant-derived detergent. Only 0.04 mg/mL enzyme could completely remove 0.15 mM malathion in aqueous solution within 20 min. Furthermore, 12 μmol malathion on apples and cucumbers surfaces was completely removed by 0.05 mg/mL PoOPH M9 in tap water after 35 min washing. The efficient production of the highly active PoOPH M9 has cleared a major barrier to biodegradation of pesticide residues in food industry.

TaER Expression Is Associated with Transpiration Efficiency Traits and Yield in Bread Wheat

PubMed Central

Zheng, Jiacheng; Yang, Zhiyuan; Madgwick, Pippa J.; Carmo-Silva, Elizabete; Parry, Martin A. J.; Hu, Yin-Gang

2015-01-01

ERECTA encodes a receptor-like kinase and is proposed as a candidate for determining transpiration efficiency of plants. Two genes homologous to ERECTA in Arabidopsis were identified on chromosomes 6 (TaER2) and 7 (TaER1) of bread wheat (Triticum aestivum L.), with copies of each gene on the A, B and D genomes of wheat. Similar expression patterns were observed for TaER1 and TaER2 with relatively higher expression of TaER1 in flag leaves of wheat at heading (Z55) and grain-filling (Z73) stages. Significant variations were found in the expression levels of both TaER1 and TaER2 in the flag leaves at both growth stages among 48 diverse bread wheat varieties. Based on the expression of TaER1 and TaER2, the 48 wheat varieties could be classified into three groups having high (5 varieties), medium (27 varieties) and low (16 varieties) levels of TaER expression. Significant differences were also observed between the three groups varying for TaER expression for several transpiration efficiency (TE)- related traits, including stomatal density (SD), transpiration rate, photosynthetic rate (A), instant water use efficiency (WUEi) and carbon isotope discrimination (CID), and yield traits of biomass production plant-1 (BYPP) and grain yield plant-1 (GYPP). Correlation analysis revealed that the expression of TaER1 and TaER2 at the two growth stages was significantly and negatively associated with SD (P<0.01), transpiration rate (P<0.05) and CID (P<0.01), while significantly and positively correlated with flag leaf area (FLA, P<0.01), A (P<0.05), WUEi (P<0.05), BYPP (P<0.01) and GYPP (P<0.01), with stronger correlations for TaER1 than TaER2 and at grain-filling stage than at heading stage. These combined results suggested that TaER involved in development of transpiration efficiency -related traits and yield in bread wheat, implying a function for TaER in regulating leaf development of bread wheat and contributing to expression of these traits. Moreover, the results indicate that TaER could be exploitable for manipulating important agronomical traits in wheat improvement. PMID:26047019

TaER Expression Is Associated with Transpiration Efficiency Traits and Yield in Bread Wheat.

PubMed

Zheng, Jiacheng; Yang, Zhiyuan; Madgwick, Pippa J; Carmo-Silva, Elizabete; Parry, Martin A J; Hu, Yin-Gang

2015-01-01

ERECTA encodes a receptor-like kinase and is proposed as a candidate for determining transpiration efficiency of plants. Two genes homologous to ERECTA in Arabidopsis were identified on chromosomes 6 (TaER2) and 7 (TaER1) of bread wheat (Triticum aestivum L.), with copies of each gene on the A, B and D genomes of wheat. Similar expression patterns were observed for TaER1 and TaER2 with relatively higher expression of TaER1 in flag leaves of wheat at heading (Z55) and grain-filling (Z73) stages. Significant variations were found in the expression levels of both TaER1 and TaER2 in the flag leaves at both growth stages among 48 diverse bread wheat varieties. Based on the expression of TaER1 and TaER2, the 48 wheat varieties could be classified into three groups having high (5 varieties), medium (27 varieties) and low (16 varieties) levels of TaER expression. Significant differences were also observed between the three groups varying for TaER expression for several transpiration efficiency (TE)- related traits, including stomatal density (SD), transpiration rate, photosynthetic rate (A), instant water use efficiency (WUEi) and carbon isotope discrimination (CID), and yield traits of biomass production plant-1 (BYPP) and grain yield plant-1 (GYPP). Correlation analysis revealed that the expression of TaER1 and TaER2 at the two growth stages was significantly and negatively associated with SD (P<0.01), transpiration rate (P<0.05) and CID (P<0.01), while significantly and positively correlated with flag leaf area (FLA, P<0.01), A (P<0.05), WUEi (P<0.05), BYPP (P<0.01) and GYPP (P<0.01), with stronger correlations for TaER1 than TaER2 and at grain-filling stage than at heading stage. These combined results suggested that TaER involved in development of transpiration efficiency -related traits and yield in bread wheat, implying a function for TaER in regulating leaf development of bread wheat and contributing to expression of these traits. Moreover, the results indicate that TaER could be exploitable for manipulating important agronomical traits in wheat improvement.

Purification of proteins from baculovirus-infected insect cells.

PubMed

O'Shaughnessy, Luke; Doyle, Sean

2011-01-01

Expression of recombinant proteins in the baculovirus/insect cell expression system is employed because it enables post-translational protein modification and high yields of recombinant protein. The system is capable of facilitating the functional expression of many proteins - either secreted or intracellularly located within infected insect cells. Strategies for the isolation and extraction of soluble proteins are presented in this chapter and involve selective cell lysis, precipitation and chromatography. Protein insolubility, following recombinant expression in insect cells, can occur. However, using the methods described herein, it is possible to extract and purify insoluble protein using affinity, ion-exchange and gel filtration chromatography. Indeed, protein insolubility often aids protein purification.

Expression of the hemagglutinin HA1 subunit of the equine influenza virus using a baculovirus expression system.

PubMed

Sguazza, Guillermo H; Fuentealba, Nadia A; Tizzano, Marco A; Galosi, Cecilia M; Pecoraro, Marcelo R

2013-01-01

Equine influenza virus is a leading cause of respiratory disease in horses worldwide. Disease prevention is by vaccination with inactivated whole virus vaccines. Most current influenza vaccines are generated in embryonated hens' eggs. Virions are harvested from allantoic fluid and chemically inactivated. Although this system has served well over the years, the use of eggs as the substrate for vaccine production has several well-recognized disadvantages (cost, egg supply, waste disposal and yield in eggs). The aim of this study was to evaluate a baculovirus system as a potential method for producing recombinant equine influenza hemagglutinin to be used as a vaccine. The hemagglutinin ectodomain (HA1 subunit) was cloned and expressed using a baculovirus expression vector. The expression was determined by SDS-PAGE and immunoblotting. A high yield, 20μg/ml of viral protein, was obtained from recombinant baculovirus-infected cells. The immune response in BALB/c mice was examined following rHA1 inoculation. Preliminary results show that recombinant hemagglutinin expressed from baculovirus elicits a strong antibody response in mice; therefore it could be used as an antigen for subunit vaccines and diagnostic tests. Copyright © 2013 Asociación Argentina de Microbiología. Publicado por Elsevier España. All rights reserved.

Pulsed Dilution Method for the Recovery of Aggregated Mouse TNF-α.

PubMed

Mahmoodi, Merat; Ghodsi, Maryam; Moghadam, Malihe; Sankian, Mojtaba

2017-04-01

The expression of mouse tumor necrosis factor alpha (TNF-α) in Escherichia coli is a favorable way to get high yield of protein; however, the formation of cytoplasmic inclusion bodies, which is the consequence of insoluble accumulated proteins, is a major obstacle in this system. To overcome this obstacle, we used a pulsed dilution method to convert the product to its native conformation. Reducing agent and guanidine hydrochloride were used to solubilize inclusion bodies formed after TNF-(α) expression. Then, the refolding procedure was performed by pulsed dilution of the denatured protein into a refolding buffer. The properly-folded protein was purified by metal affinity chromatography. SDS-PAGE showed a 19.9 kDa band related to the mature TNF-(α) protein. The protein was recognized by anti-mouse TNF-(α) on western blots. The final concentration of the purified recombinant TNF-(α) was 62.5 µg/mL. Our study demonstrates the efficiency of this method to produce a high yield of folded mature TNF- (α).

Circadian Clock genes Per2 and clock regulate steroid production, cell proliferation, and luteinizing hormone receptor transcription in ovarian granulosa cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shimizu, Takashi, E-mail: shimizut@obihiro.ac.jp; Hirai, Yuko; Murayama, Chiaki

2011-08-19

Highlights: {yields} Treatment with Per2 and Clock siRNAs decreased the number of granulosa cells and LHr expression. {yields}Per2 siRNA treatment did not stimulate the production of estradiol and expression of P450arom. {yields} Clock siRNA treatment inhibited the production of estradiol and expression of P450arom mRNA. {yields}Per2 and Clock siRNA treatment increased and unchanged, respectively, progesterone production in FSH-treated granulosa cells. {yields} The expression of StAR mRNA was increased by Per2 siRNA and unchanged by Clock siRNA. -- Abstract: Circadian Clock genes are associated with the estrous cycle in female animals. Treatment with Per2 and Clock siRNAs decreased the number ofmore » granulosa cells and LHr expression in follicle-stimulating hormone FSH-treated granulosa cells. Per2 siRNA treatment did not stimulate the production of estradiol and expression of P450arom, whereas Clock siRNA treatment inhibited the production of estradiol and expression of P450arom mRNA. Per2 and Clock siRNA treatment increased and unchanged, respectively, progesterone production in FSH-treated granulosa cells. Similarly, expression of StAR mRNA was increased by Per2 siRNA and unchanged by Clock siRNA. Our data provide a new insight that Per2 and Clock have different action on ovarian granulosa cell functions.« less

miRNA engineering of CHO cells facilitates production of difficult-to-express proteins and increases success in cell line development.

PubMed

Fischer, Simon; Marquart, Kim F; Pieper, Lisa A; Fieder, Juergen; Gamer, Martin; Gorr, Ingo; Schulz, Patrick; Bradl, Harald

2017-07-01

In recent years, coherent with growing biologics portfolios also the number of complex and thus difficult-to-express (DTE) therapeutic proteins has increased considerably. DTE proteins challenge bioprocess development and can include various therapeutic protein formats such as monoclonal antibodies (mAbs), multi-specific affinity scaffolds (e.g., bispecific antibodies), cytokines, or fusion proteins. Hence, the availability of robust and versatile Chinese hamster ovary (CHO) host cell factories is fundamental for high-yielding bioprocesses. MicroRNAs (miRNAs) have emerged as potent cell engineering tools to improve process performance of CHO manufacturing cell lines. However, there has not been any report demonstrating the impact of beneficial miRNAs on industrial cell line development (CLD) yet. To address this question, we established novel CHO host cells constitutively expressing a pro-productive miRNA: miR-557. Novel host cells were tested in two independent CLD campaigns using two different mAb candidates including a normal as well as a DTE antibody. Presence of miR-557 significantly enhanced each process step during CLD in a product independent manner. Stable expression of miR-557 increased the probability to identify high-producing cell clones. Furthermore, production cell lines derived from miR-557 expressing host cells exhibited significantly increased final product yields in fed-batch cultivation processes without compromising product quality. Strikingly, cells co-expressing miR-557 and a DTE antibody achieved a twofold increase in product titer compared to clones co-expressing a negative control miRNA. Thus, host cell engineering using miRNAs represents a promising tool to overcome limitations in industrial CLD especially with regard to DTE proteins. Biotechnol. Bioeng. 2017;114: 1495-1510. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

High-level expression of human immunodeficiency virus antigens from the tobacco and tomato plastid genomes.

PubMed

Zhou, Fei; Badillo-Corona, Jesus A; Karcher, Daniel; Gonzalez-Rabade, Nuria; Piepenburg, Katrin; Borchers, A-M Inka; Maloney, Alan P; Kavanagh, Tony A; Gray, John C; Bock, Ralph

2008-12-01

Transgene expression from the plant's plastid genome represents a promising strategy in molecular farming because of the plastid's potential to accumulate foreign proteins to high levels and the increased biosafety provided by the maternal mode of organelle inheritance. In this article, we explore the potential of transplastomic plants to produce human immunodeficiency virus (HIV) antigens as potential components of an acquired immunodeficiency syndrome (AIDS) vaccine. It is shown that the HIV antigens p24 (the major target of T-cell-mediated immune responses in HIV-positive individuals) and Nef can be expressed to high levels in plastids of tobacco, a non-food crop, and tomato, a food crop with an edible fruit. Optimized p24-Nef fusion gene cassettes trigger antigen protein accumulation to up to approximately 40% of the plant's total protein, demonstrating the great potential of transgenic plastids to produce AIDS vaccine components at low cost and high yield.

A human parvovirus, adeno-associated virus, as a eucaryotic vector: Transient expression and encapsidation of the procaryotic gene for chloramphenicol acetyltransferase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tratschin, J.D.; West, M.H.P.; Sandbank, T.

1984-10-01

The authors have used the defective human parvovirus adeno-associated virus (AAV) as a novel eurocaryotic vector (parvector) for the expression of a foreign gene in human cells. The recombinant, pAV2, contains the AAV genome in a pBR322-derived bacterial plasmid. When pAV2 is transfected into human cells together with helper adenovirus particles, the AAV genome is rescued from the recombinant plasmid and replicated to produce infectious AAV particles at high efficiency. To create a vector, we inserted a procaryotic sequence coding for chloramphenicol acetyltransferase (CAT) into derivatives of pAV2 following either of the AAV promoters p/sub 40/ (pAVHiCAT) and p/sub 19/more » (pAVBcCAT). When transfected into human 293 cells or HeLa cells, pAVHiCAT expressed CAT activity in the absence of adenovirus. In the presence of adenovirus, this vector produced increased amounts of CAT activity and the recombinant AAV-CAT genome was replicated. In 293 cells, pAVBcCAT expressed a similar amount of CAT activity in the absence or presence of adenovirus and the recombinant AAV-CAT genome was not replicated. In HeLa cells, pAVBcCAT expressed low levels of CAT activity, but this level was elevated by coinfection with adenovirus particles or by cotransfection with a plasmid which expressed the adenovirus early region 1A (E1A) product. The E1A product is a transcriptional activator and is expressed in 293 cells. Thus, expression from two AAV promoters is differentially regulated: expression from p/sub 19/ is increased by E1A, whereas p/sub 40/ yields high levels of constitutive expression in the absence of E1A. Both AAV vectors were packaged into AAV particles by complementation with wild-type AAV and yielded CAT activity when subsequently infected into cells in the presence of adenovirus.« less

The melatonin action on stromal stem cells within pericryptal area in colon cancer model under constant light

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kannen, Vinicius, E-mail: kannen71@yahoo.com.br; Marini, Tassiana; Zanette, Dalila L.

Research highlights: {yields} We investigated melatonin against the malignant effects of constant light. {yields} Melatonin supplementation increased its serum levels and its receptor expression. {yields} Melatonin decreased cancer stem cells and dysplastic injuries in colon tissue. {yields} Melatonin controlled proliferative process and apoptosis induction. -- Abstract: Constant light (LL) is associated with high incidence of colon cancer. MLT supplementation was related to the significant control of preneoplastic patterns. We sought to analyze preneoplastic patterns in colon tissue from animals exposed to LL environment (14 days; 300 lx), MLT-supplementation (10 mg/kg/day) and DMH-treatment (1,2 dimethylhydrazine; 125 mg/kg). Rodents were sacrificed andmore » MLT serum levels were measured by radioimmunoassay. Our results indicated that LL induced ACF development (p < 0.001) with a great potential to increase the number of CD133(+) and CD68(+) cells (p < 0.05 and p < 0.001). LL also increased the proliferative process (PCNA-Li; p < 0.001) as well as decreased caspase-3 protein (p < 0.001), related to higher COX-2 protein expression (p < 0.001) within pericryptal colonic stroma (PCCS). However, MLT-supplementation controlled the development of dysplastic ACF (p < 0.001) diminishing preneoplastic patterns into PCCS as CD133 and CD68 (p < 0.05 and p < 0.001). These events were relative to decreased PCNA-Li index and higher expression of caspase-3 protein. Thus, MLT showed a great potential to control the preneoplastic patterns induced by LL.« less

An efficient Foxtail mosaic virus vector system with reduced environmental risk

PubMed Central

2010-01-01

Background Plant viral vectors offer high-yield expression of pharmaceutical and commercially important proteins with a minimum of cost and preparation time. The use of Agrobacterium tumefaciens has been introduced to deliver the viral vector as a transgene to each plant cell via a simple, nonsterile infiltration technique called "agroinoculation". With agroinoculation, a full length, systemically moving virus is no longer necessary for excellent protein yield, since the viral transgene is transcribed and replicates in every infiltrated cell. Viral genes may therefore be deleted to decrease the potential for accidental spread and persistence of the viral vector in the environment. Results In this study, both the coat protein (CP) and triple gene block (TGB) genetic segments were eliminated from Foxtail mosaic virus to create the "FECT" vector series, comprising a deletion of 29% of the genome. This viral vector is highly crippled and expresses little or no marker gene within the inoculated leaf. However, when co-agroinoculated with a silencing suppressor (p19 or HcPro), FECT expressed GFP at 40% total soluble protein in the tobacco host, Nicotiana benthamiana. The modified FoMV vector retained the full-length replicase ORF, the TGB1 subgenomic RNA leader sequence and either 0, 22 or 40 bases of TGB1 ORF (in vectors FECT0, FECT22 and FECT40, respectively). As well as N. benthamiana, infection of legumes was demonstrated. Despite many attempts, expression of GFP via syringe agroinoculation of various grass species was very low, reflecting the low Agrobacterium-mediated transformation rate of monocots. Conclusions The FECT/40 vector expresses foreign genes at a very high level, and yet has a greatly reduced biohazard potential. It can form no virions and can effectively replicate only in a plant with suppressed silencing. PMID:21162736

Enhancement of welan gum production in Sphingomonas sp. HT-1 via heterologous expression of Vitreoscilla hemoglobin gene.

PubMed

Liu, Xiaoliu; Zhu, Ping; Jiang, Ruifan; Wu, Lingtian; Feng, Xiaohai; Li, Sha; Xu, Hong

2017-01-20

Welan gum is a microbial polysaccharide produced by Sphingomonas sp. Its production is limited by the dissolved oxygen levels in the highly viscous fermentation. A strategy of heterologous expression of the Vitreoscilla hemoglobin gene in Sphingomonas sp. HT-1 was investigated to alleviate oxygen limitation and improve the yield of welan gum. Ultimately, the welan gum production increased from 25.3g/L to 34.6g/L, whereas the rheological behavior of welan gum solutions remained virtually unchanged. The transcriptional levels of the key genes in the electron transfer chain, TCA cycle and welan gum synthesis pathway, as well as ATP level revealed that the VHb expression in Sphingomonas sp. HT-1 enhanced welan gum biosynthesis by improving respiration and ATP supply. This study would pave the genetic manipulation way for enhancing welan gum yield, and it's also of great importance for the industrial applications of welan gum under harsh conditions. Copyright © 2016 Elsevier Ltd. All rights reserved.

The effect of red light and far-red light conditions on secondary metabolism in agarwood.

PubMed

Kuo, Tony Chien-Yen; Chen, Chuan-Hung; Chen, Shu-Hwa; Lu, I-Hsuan; Chu, Mei-Ju; Huang, Li-Chun; Lin, Chung-Yen; Chen, Chien-Yu; Lo, Hsiao-Feng; Jeng, Shih-Tong; Chen, Long-Fang O

2015-06-12

Agarwood, a heartwood derived from Aquilaria trees, is a valuable commodity that has seen prevalent use among many cultures. In particular, it is widely used in herbal medicine and many compounds in agarwood are known to exhibit medicinal properties. Although there exists much research into medicinal herbs and extraction of high value compounds, few have focused on increasing the quantity of target compounds through stimulation of its related pathways in this species. In this study, we observed that cucurbitacin yield can be increased through the use of different light conditions to stimulate related pathways and conducted three types of high-throughput sequencing experiments in order to study the effect of light conditions on secondary metabolism in agarwood. We constructed genome-wide profiles of RNA expression, small RNA, and DNA methylation under red light and far-red light conditions. With these profiles, we identified a set of small RNA which potentially regulates gene expression via the RNA-directed DNA methylation pathway. We demonstrate that light conditions can be used to stimulate pathways related to secondary metabolism, increasing the yield of cucurbitacins. The genome-wide expression and methylation profiles from our study provide insight into the effect of light on gene expression for secondary metabolism in agarwood and provide compelling new candidates towards the study of functional secondary metabolic components.

Expression and functional analysis of the lysine decarboxylase and copper amine oxidase genes from the endophytic fungus Colletotrichum gloeosporioides ES026.

PubMed

Zhang, Xiangmei; Wang, Zhangqian; Jan, Saad; Yang, Qian; Wang, Mo

2017-06-05

Huperzine A (HupA) isolated from Huperzia serrata is an important compound used to treat Alzheimer's disease (AD). Recently, HupA was reported in various endophytic fungi, with Colletotrichum gloeosporioides ES026 previously isolated from H. serrata shown to produce HupA. In this study, we performed next-generation sequencing and de novo RNA sequencing of C. gloeosporioides ES026 to elucidate the molecular functions, biological processes, and biochemical pathways of these unique sequences. Gene ontology and Kyoto Encyclopedia of Genes and Genomes assignments allowed annotation of lysine decarboxylase (LDC) and copper amine oxidase (CAO) for their conversion of L-lysine to 5-aminopentanal during HupA biosynthesis. Additionally, we constructed a stable, high-yielding HupA-expression system resulting from the overexpression of CgLDC and CgCAO from the HupA-producing endophytic fungus C. gloeosporioides ES026 in Escherichia coli. Quantitative reverse transcription polymerase chain reaction analysis confirmed CgLDC and CgCAO expression, and quantitative determination of HupA levels was assessed by liquid chromatography high-resolution mass spectrometry, which revealed that elevated expression of CgLDC and CgCAO produced higher yields of HupA than those derived from C. gloeosporioides ES026. These results revealed CgLDC and CgCAO involvement in HupA biosynthesis and their key role in regulating HupA content in C. gloeosporioides ES026.

Purification and immunogenicity of hemagglutinin from highly pathogenic avian influenza virus H5N1 expressed in Nicotiana benthamiana

PubMed Central

Pua, Teen-Lee; Chan, Xiao Ying; Loh, Hwei-San; Omar, Abdul Rahman; Yusibov, Vidadi; Musiychuk, Konstantin; Hall, Alexandra C.; Coffin, Megan V.; Shoji, Yoko; Chichester, Jessica A.; Bi, Hong; Streatfield, Stephen J.

2017-01-01

ABSTRACT Highly pathogenic avian influenza (HPAI) H5N1 is an ongoing global health concern due to its severe sporadic outbreaks in Asia, Africa and Europe, which poses a potential pandemic threat. The development of safe and cost-effective vaccine candidates for HPAI is considered the best strategy for managing the disease and addressing the pandemic preparedness. The most potential vaccine candidate is the antigenic determinant of influenza A virus, hemagglutinin (HA). The present research was aimed at developing optimized expression in Nicotiana benthamiana and protein purification process for HA from the Malaysian isolate of H5N1 as a vaccine antigen for HPAI H5N1. Expression of HA from the Malaysian isolate of HPAI in N. benthamiana was confirmed, and more soluble protein was expressed as truncated HA, the HA1 domain over the entire ectodomain of HA. Two different purification processes were evaluated for efficiency in terms of purity and yield. Due to the reduced yield, protein degradation and length of the 3-column purification process, the 2-column method was chosen for target purification. Purified HA1 was found immunogenic in mice inducing H5 HA-specific IgG and a hemagglutination inhibition antibody. This paper offers an alternative production system of a vaccine candidate against a locally circulating HPAI, which has a regional significance. PMID:27929750

Data on enhanced expression and purification of camelid single domain antibodies from Escherichia coli classical inclusion bodies.

PubMed

Maggi, Maristella; Scotti, Claudia

2017-06-01

Heterologous expression of high amounts of recombinant proteins is a milestone for research and industrial purposes. Single domain antibodies (sdAbs) are heavy-chain only antibody fragments with applications in the biotechnological, medical and industrial fields. The simple nature and small size of sdAbs allows for efficient expression of the soluble molecule in different hosts. However, in some cases, it results in low functional protein yield. To overcome this limitation, expression of a 6xHistag sdAb was attempted in different conditions in Escherichia coli BL21(DE3) cells. Data showed that high amount of sdAb can be expressed in E. coli classical inclusion bodies, efficiently extracted by urea in a short-time, and properly purified by metal ion affinity chromatography. These data originate from the research article "Enhanced expression and purification of camelid single domain VHH antibodies from classical inclusion bodies" Maggi and Scotti (2017) [1] (DOI: http://dx.doi.org/10.1016/j.pep.2017.02.007).

Decreased histone deacetylase 2 impairs Nrf2 activation by oxidative stress

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mercado, Nicolas; Thimmulappa, Rajesh; Thomas, Catherine M.R.

2011-03-11

Research highlights: {yields} Nrf2 anti-oxidant function is impaired when HDAC activity is inhibited. {yields} HDAC inhibition decreases Nrf2 protein stability. {yields} HDAC2 is involved in reduced Nrf2 stability and both correlate in COPD samples. {yields} HDAC inhibition increases Nrf2 acetylation. -- Abstract: Nuclear factor erythroid 2-related factor 2 (Nrf2) plays a crucial role in cellular defence against oxidative stress by inducing the expression of multiple anti-oxidant genes. However, where high levels of oxidative stress are observed, such as chronic obstructive pulmonary disease (COPD), Nrf2 activity is reduced, although the molecular mechanism for this defect is uncertain. Here, we show thatmore » down-regulation of histone deacetylase (HDAC) 2 causes Nrf2 instability, resulting in reduced anti-oxidant gene expression and increase sensitivity to oxidative stress. Although Nrf2 protein was clearly stabilized after hydrogen peroxide (H{sub 2}O{sub 2}) stimulation in a bronchial epithelial cell line (BEAS2B), Nrf2 stability was decreased and Nrf2 acetylation increased in the presence of an HDAC inhibitor, trichostatin A (TSA). TSA also reduced Nrf2-regulated heme-oxygenase-1 (HO-1) expression in these cells, and this was confirmed in acute cigarette-smoke exposed mice in vivo. HDAC2 knock-down by RNA interference resulted in reduced H{sub 2}O{sub 2}-induced Nrf2 protein stability and activity in BEAS2B cells, whereas HDAC1 knockdown had no effect. Furthermore, monocyte-derived macrophages obtained from healthy volunteers (non-smokers and smokers) and COPD patients showed a significant correlation between HDAC2 expression and Nrf2 expression (r = 0.92, p < 0.0001). Thus, reduced HDAC2 activity in COPD may account for increased Nrf2 acetylation, reduced Nrf2 stability and impaired anti oxidant defences.« less

Metabolic engineering of biomass for high energy density: oilseed-like triacylglycerol yields from plant leaves

PubMed Central

Vanhercke, Thomas; El Tahchy, Anna; Liu, Qing; Zhou, Xue-Rong; Shrestha, Pushkar; Divi, Uday K; Ral, Jean-Philippe; Mansour, Maged P; Nichols, Peter D; James, Christopher N; Horn, Patrick J; Chapman, Kent D; Beaudoin, Frederic; Ruiz-López, Noemi; Larkin, Philip J; de Feyter, Robert C; Singh, Surinder P; Petrie, James R

2014-01-01

High biomass crops have recently attracted significant attention as an alternative platform for the renewable production of high energy storage lipids such as triacylglycerol (TAG). While TAG typically accumulates in seeds as storage compounds fuelling subsequent germination, levels in vegetative tissues are generally low. Here, we report the accumulation of more than 15% TAG (17.7% total lipids) by dry weight in Nicotiana tabacum (tobacco) leaves by the co-expression of three genes involved in different aspects of TAG production without severely impacting plant development. These yields far exceed the levels found in wild-type leaf tissue as well as previously reported engineered TAG yields in vegetative tissues of Arabidopsis thaliana and N. tabacum. When translated to a high biomass crop, the current levels would translate to an oil yield per hectare that exceeds those of most cultivated oilseed crops. Confocal fluorescence microscopy and mass spectrometry imaging confirmed the accumulation of TAG within leaf mesophyll cells. In addition, we explored the applicability of several existing oil-processing methods using fresh leaf tissue. Our results demonstrate the technical feasibility of a vegetative plant oil production platform and provide for a step change in the bioenergy landscape, opening new prospects for sustainable food, high energy forage, biofuel and biomaterial applications. PMID:24151938

Engineering crop nutrient efficiency for sustainable agriculture.

PubMed

Chen, Liyu; Liao, Hong

2017-10-01

Increasing crop yields can provide food, animal feed, bioenergy feedstocks and biomaterials to meet increasing global demand; however, the methods used to increase yield can negatively affect sustainability. For example, application of excess fertilizer can generate and maintain high yields but also increases input costs and contributes to environmental damage through eutrophication, soil acidification and air pollution. Improving crop nutrient efficiency can improve agricultural sustainability by increasing yield while decreasing input costs and harmful environmental effects. Here, we review the mechanisms of nutrient efficiency (primarily for nitrogen, phosphorus, potassium and iron) and breeding strategies for improving this trait, along with the role of regulation of gene expression in enhancing crop nutrient efficiency to increase yields. We focus on the importance of root system architecture to improve nutrient acquisition efficiency, as well as the contributions of mineral translocation, remobilization and metabolic efficiency to nutrient utilization efficiency. © 2017 Institute of Botany, Chinese Academy of Sciences.

High-yield production of herbicidal thaxtomins and analogs in a nonpathogenic Streptomyces strain.

PubMed

Jiang, Guangde; Zhang, Yucheng; Powell, Magan M; Zhang, Peilan; Zuo, Ran; Zhang, Yi; Kallifidas, Dimitrios; Tieu, Albert M; Luesch, Hendrik; Loria, Rosemary; Ding, Yousong

2018-03-30

Thaxtomins are virulence factors of most plant pathogenic Streptomyces strains. Due to their potent herbicidal activity, attractive environmental compatibility and inherent biodegradability, thaxtomins are key active ingredients of bioherbicides approved by the United States Environmental Protection Agency. However, the low yield of thaxtomins in native Streptomyces producers limits their wide agricultural applications. Here, we describe the high-yield production of thaxtomins in a heterologous host. The thaxtomin gene cluster from S. scabiei 87.22 was cloned and expressed in S. albus J1074 after chromosomal integration. The production of thaxtomins and nitro-tryptophan analogs were observed using LC-MS analysis. When culturing the engineered S. albus J1074 in the minimal medium TMDc, the yield of the most abundant and herbicidal analog, thaxtomin A, was 10 times higher than S. scabiei 87.22, and optimization of the medium resulted in the highest yield of thaxtomin analogs at about 222 mg/L. Further engineering of the thaxtomin biosynthetic gene cluster through gene deletion led to the production of multiple biosynthetic intermediates important to the chemical synthesis of new analogs. Additionally, the versatility of the thaxtomin biosynthetic system in S. albus J1074 was capitalized to produce one unnatural fluorinated analog 5-F-thaxtomin A, whose structure was elucidated by a combination of MS and 1D and 2D NMR analyses. Natural and unnatural thaxtomins demonstrated potent herbicidal activity in radish seedling assays. These results indicated that S. albus J1074 has the potential to produce thaxtomins and thereof with high yield, fostering their agricultural applications. IMPORTANCE Thaxtomins are agriculturally valuable herbicidal natural products but the productivity of native producers is limiting. Heterologous expression of thaxtomin gene cluster in S. albus J1074 resulted in the highest yield of thaxtomins ever reported, representing a significant leap forward in its wide agricultural use. Furthermore, current synthetic routes to thaxtomins and analogs are lengthy, and two thaxtomin biosynthetic intermediates produced at high yields in this work can provide precursors and building blocks to advanced synthetic routes. Importantly, the production of 5-F-thaxtomin A in engineered S. albus J1074 demonstrated a viable alternative to chemical methods in the synthesis of new thaxtomin analogs. Moreover, our work presents an attractive synthetic biology strategy to improve the supply of herbicidal thaxtomins, likely finding general applications in the discovery and production of many other bioactive natural products. Copyright © 2018 American Society for Microbiology.

Differential gene expression of wheat progeny with contrasting levels of transpiration efficiency.

PubMed

Xue, Gang-Ping; McIntyre, C Lynne; Chapman, Scott; Bower, Neil I; Way, Heather; Reverter, Antonio; Clarke, Bryan; Shorter, Ray

2006-08-01

High water use efficiency or transpiration efficiency (TE) in wheat is a desirable physiological trait for increasing grain yield under water-limited environments. The identification of genes associated with this trait would facilitate the selection for genotypes with higher TE using molecular markers. We performed an expression profiling (microarray) analysis of approximately 16,000 unique wheat ESTs to identify genes that were differentially expressed between wheat progeny lines with contrasting TE levels from a cross between Quarrion (high TE) and Genaro 81 (low TE). We also conducted a second microarray analysis to identify genes responsive to drought stress in wheat leaves. Ninety-three genes that were differentially expressed between high and low TE progeny lines were identified. One fifth of these genes were markedly responsive to drought stress. Several potential growth-related regulatory genes, which were down-regulated by drought, were expressed at a higher level in the high TE lines than the low TE lines and are potentially associated with a biomass production component of the Quarrion-derived high TE trait. Eighteen of the TE differentially expressed genes were further analysed using quantitative RT-PCR on a separate set of plant samples from those used for microarray analysis. The expression levels of 11 of the 18 genes were positively correlated with the high TE trait, measured as carbon isotope discrimination (Delta(13)C). These data indicate that some of these TE differentially expressed genes are candidates for investigating processes that underlie the high TE trait or for use as expression quantitative trait loci (eQTLs) for TE.

The TCP4 transcription factor of Arabidopsis blocks cell division in yeast at G1 {yields} S transition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aggarwal, Pooja; Padmanabhan, Bhavna; Bhat, Abhay

2011-07-01

Highlights: {yields} TCP4 is a class II TCP transcription factor, that represses cell division in Arabidopsis. {yields} TCP4 expression in yeast retards cell division by blocking G1 {yields} S transition. {yields} Genome-wide expression studies and Western analysis reveals stabilization of cell cycle inhibitor Sic1, as possible mechanism. -- Abstract: The TCP transcription factors control important aspects of plant development. Members of class I TCP proteins promote cell cycle by regulating genes directly involved in cell proliferation. In contrast, members of class II TCP proteins repress cell division. While it has been postulated that class II proteins induce differentiation signal, theirmore » exact role on cell cycle has not been studied. Here, we report that TCP4, a class II TCP protein from Arabidopsis that repress cell proliferation in developing leaves, inhibits cell division by blocking G1 {yields} S transition in budding yeast. Cells expressing TCP4 protein with increased transcriptional activity fail to progress beyond G1 phase. By analyzing global transcriptional status of these cells, we show that expression of a number of cell cycle genes is altered. The possible mechanism of G1 {yields} S arrest is discussed.« less

Playing Digital: Music Instruction for the Next Generation.

ERIC Educational Resources Information Center

Hardy, Lawrence

2001-01-01

Active involvement in music can yield significant intellectual and emotional benefits. A Washington-area high school features a digitally literate music teacher and a piano lab with 25 workstations allowing music-loving students to express their creativity. MIDI sequencers and synthesizers aid young composers' efforts. (MLH)

Effects of H2 and Formate on Growth Yield and Regulation of Methanogenesis in Methanococcus maripaludis

PubMed Central

Costa, Kyle C.; Yoon, Sung Ho; Pan, Min; Burn, June A.; Baliga, Nitin S.

2013-01-01

Hydrogenotrophic methanogenic Archaea are defined by an H2 requirement for growth. Despite this requirement, many hydrogenotrophs are also capable of growth with formate as an electron donor for methanogenesis. While certain responses of these organisms to hydrogen availability have been characterized, responses to formate starvation have not been reported. Here we report that during continuous culture of Methanococcus maripaludis under defined nutrient conditions, growth yields relative to methane production decreased markedly with either H2 excess or formate excess. Analysis of the growth yields of several mutants suggests that this phenomenon occurs independently of the storage of intracellular carbon or a transcriptional response to methanogenesis. Using microarray analysis, we found that the expression of genes encoding coenzyme F420-dependent steps of methanogenesis, including one of two formate dehydrogenases, increased with H2 starvation but with formate occurred at high levels regardless of limitation or excess. One gene, encoding H2-dependent methylene-tetrahydromethanopterin dehydrogenase, decreased in expression with either H2 limitation or formate limitation. Expression of genes for the second formate dehydrogenase, molybdenum-dependent formylmethanofuran dehydrogenase, and molybdenum transport increased specifically with formate limitation. Of the two formate dehydrogenases, only the first could support growth on formate in batch culture where formate was in excess. PMID:23335420

Epigenetic regulation of the transcription factor Foxa2 directs differential elafin expression in melanocytes and melanoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu, Kyung Sook; Jo, Ji Yoon; Kim, Su Jin

2011-04-29

Highlights: {yields} Elafin expression is epigenetically silenced in human melanoma cells. {yields} Foxa2 expression in melanoma cells is silenced by promoter hypermethylation. {yields} Foxa2 directs activation of the elafin promoter in vivo. {yields} Foxa2 expression induces apoptosis of melanoma cells via elafin re-expression. -- Abstract: Elafin, a serine protease inhibitor, induces the intrinsic apoptotic pathway in human melanoma cells, where its expression is transcriptionally silenced. However, it remains unknown how the elafin gene is repressed in melanoma cells. We here demonstrate that elafin expression is modulated via epigenetically regulated expression of the transcription factor Foxa2. Treatment of melanoma cells withmore » a DNA methyltransferase inhibitor induced elafin expression, which was specifically responsible for reduced proliferation and increased apoptosis. Suppression of Foxa2 transcription, mediated by DNA hypermethylation in its promoter region, was released in melanoma cells upon treatment with the demethylating agent. Luciferase reporter assays indicated that the Foxa2 binding site in the elafin promoter was critical for the activation of the promoter. Chromatin immunoprecipitation assays further showed that Foxa2 bound to the elafin promoter in vivo. Analyses of melanoma cells with varied levels of Foxa2 revealed a correlated expression between Foxa2 and elafin and the ability of Foxa2 to induce apoptosis. Our results collectively suggest that, in melanoma cells, Foxa2 expression is silenced and therefore elafin is maintained unexpressed to facilitate cell proliferation in the disease melanoma.« less

[Construction of high-yield strain by optimizing lycopene cyclase for β-carotene production].

PubMed

Jin, Yingfu; Han, Li; Zhang, Shasha; Li, Shizhong; Liu, Weifeng; Tao, Yong

2017-11-25

To optimize key enzymes, such as to explore the gene resources and to modify the expression level, can maximize metabolic pathways of target products. β-carotene is a terpenoid compound with important application value. Lycopene cyclase (CrtY) is the key enzyme in β-carotene biosynthesis pathway, catalyzing flavin adenine dinucleotide (FAD)-dependent cyclization reaction and β-carotene synthesis from lycopene precursor. We optimized lycopene cyclase (CrtY) to improve the synthesis of β-carotene and determined the effect of CrtY expression on metabolic pathways. Frist, we developed a β-carotene synthesis module by coexpressing the lycopene β-cyclase gene crtY with crtEBI module in Escherichia coli. Then we simultaneously optimized the ribosome-binding site (RBS) intensity and the species of crtY using oligo-linker mediated DNA assembly method (OLMA). Five strains with high β-carotene production capacity were screened out from the OLMA library. The β-carotene yields of these strains were up to 15.79-18.90 mg/g DCW (Dry cell weight), 65% higher than that of the original strain at shake flask level. The optimal strain CP12 was further identified and evaluated for β-carotene production at 5 L fermentation level. After process optimization, the final β-carotene yield could reach to 1.9 g/L. The results of RBS strength and metabolic intermediate analysis indicated that an appropriate expression level of CrtY could be beneficial for the function of the β-carotene synthesis module. The results of this study provide important insight into the optimization of β-carotene synthesis pathway in metabolic engineering.

Heterologous expression of the immunomodulatory protein gene from Ganoderma sinense in the basidiomycete Coprinopsis cinerea.

PubMed

Han, F; Liu, Y; Guo, L Q; Zeng, X L; Liu, Z M; Lin, J F

2010-11-01

FIP-gsi, a fungal immunomodulatory protein found in Ganoderma sinense, has antitumour, anti-allergy and immunomodulatory activities and is regulated by the fip-gsi gene. In this study, we aimed to express the fip-gsi gene from G. sinense in Coprinopsis cinerea to increase yield of FIPs-gsi. A fungal expression vector pBfip-gsi containing the gpd promoter from Agaricus bisporus and the fip-gsi gene from the G. sinense was constructed and transformed into C. cinerea. PCR and Southern blotting analysis verified the successful integration of the exogenous gene fip-gsi into the genome of C. cinerea. RT-PCR and Northern blotting analysis confirmed that the fip-gsi gene was transcribed in C. cinerea. The yield of the FIP-gsi protein reached 314mg kg(-1) fresh mycelia. The molecular weight of the FIP-gsi was 13kDa, and the FIP-gsi was capable of hemagglutinating mouse red blood cells, but no such activity was observed towards human red blood cells in vitro. The fip-gsi from G. sinense has been successfully translated in C. cinerea, and the yield of bioactive FIP-gsi protein was high. This is the first report using the C. cinerea for the heterologous expression of FIP-gsi protein and it might supply a basis for large-scale production of the protein. © 2010 The Authors. Journal of Applied Microbiology © 2010 The Society for Applied Microbiology.

Optimization of human granulocyte macrophage-colony stimulating factor (hGM-CSF) expression using asparaginase and xylanase gene's signal sequences in Escherichia coli.

PubMed

Khasa, Yogender Pal; Khushoo, Amardeep; Tapryal, Suman; Mukherjee, K J

2011-09-01

The toxicity of the recombinant protein towards the expression host remains a significant deterrent for bioprocess development. In this study, the expression of human granulocyte macrophage-colony stimulating factor (hGM-CSF), which is known to be toxic to its host, was enhanced many folds using a combination of genetic and bioprocess strategies in Escherichia coli. The N terminus attachment of endoxylanase and asparaginase signal sequences from Bacillus subtilis and E. coli, respectively, in combination with and without His-tag, considerably improved expression levels. Induction and media optimization studies in shake flask cultures resulted in a maximal hGM-CSF concentration of 365 mg/L in the form of inclusion bodies (IBs) with a specific product yield (Y (P/X)) of 120 mg/g dry cell weight in case of the asparaginase signal. Culturing the cells in nutrient rich Terrific broth maintained the specific product yields (Y (P/X)) while a 6.6-fold higher volumetric concentration of both product and biomass was obtained. The purification and refolding steps were optimized resulting in a 95% pure protein with a fairly high refolding yield of 45%. The biological activity of the refolded protein was confirmed by a cell proliferation assay on hGM-CSF dependent human erythroleukemia TF-1 cells. This study demonstrated that this indeed is a viable route for the efficient production of hGM-CSF.

Human Ocular Epithelial Cells Endogenously Expressing SOX2 and OCT4 Yield High Efficiency of Pluripotency Reprogramming.

PubMed

Poon, Ming-Wai; He, Jia; Fang, Xiaowei; Zhang, Zhao; Wang, Weixin; Wang, Junwen; Qiu, Fangfang; Tse, Hung-Fat; Li, Wei; Liu, Zuguo; Lian, Qizhou

2015-01-01

A variety of pluripotency reprogramming frequencies from different somatic cells has been observed, indicating cell origin is a critical contributor for efficiency of pluripotency reprogramming. Identifying the cell sources for efficient induced pluripotent stem cells (iPSCs) generation, and defining its advantages or disadvantages on reprogramming, is therefore important. Human ocular tissue-derived conjunctival epithelial cells (OECs) exhibited endogenous expression of reprogramming factors OCT4A (the specific OCT 4 isoform on pluripotency reprogramming) and SOX2. We therefore determined whether OECs could be used for high efficiency of iPSCs generation. We compared the endogenous expression levels of four pluripotency factors and the pluripotency reprograming efficiency of human OECs with that of ocular stromal cells (OSCs). Real-time PCR, microarray analysis, Western blotting and immunostaining assays were employed to compare OECiPSCs with OSCiPSCs on molecular bases of reprogramming efficiency and preferred lineage-differentiation potential. Using the traditional KMOS (KLF4, C-MYC, OCT4 and SOX2) reprogramming protocol, we confirmed that OECs, endogenously expressing reprogramming factors OCT4A and SOX2, yield very high efficiency of iPSCs generation (~1.5%). Furthermore, higher efficiency of retinal pigmented epithelial differentiation (RPE cells) was observed in OECiPSCs compared to OSCiPSCs or skin fibroblast iMR90iPSCs. The findings in this study suggest that conjunctival-derived epithelial (OECs) cells can be easier converted to iPSCs than conjunctival-derived stromal cells (OSCs). This cell type may also have advantages in retinal pigmented epithelial differentiation.

Loss of p21{sup Sdi1} expression in senescent cells after DNA damage accompanied with increase of miR-93 expression and reduced p53 interaction with p21{sup Sdi1} gene promoter

DOE Office of Scientific and Technical Information (OSTI.GOV)

Choi, Ok Ran; Lim, In Kyoung, E-mail: iklim@ajou.ac.kr

2011-04-08

Highlights: {yields} Reduced p21 expression in senescent cells treated with DNA damaging agents. {yields} Increase of [{sup 3}H]thymidine and BrdU incorporations in DNA damaged-senescent cells. {yields} Upregulation of miR-93 expression in senescent cells in response to DSB. {yields} Failure of p53 binding to p21 promoter in senescent cells in response to DSB. {yields} Molecular mechanism of increased cancer development in aged than young individuals. -- Abstract: To answer what is a critical event for higher incidence of tumor development in old than young individuals, primary culture of human diploid fibroblasts were employed and DNA damage was induced by doxorubicin ormore » X-ray irradiation. Response to the damage was different between young and old cells; loss of p21{sup sdi1} expression in spite of p53{sup S15} activation in old cells along with [{sup 3}H]thymidine and BrdU incorporation, but not in young cells. The phenomenon was confirmed by other tissue fibroblasts obtained from different donor ages. Induction of miR-93 expression and reduced p53 binding to p21 gene promoter account for loss of p21{sup sdi1} expression in senescent cells after DNA damage, suggesting a mechanism of in vivo carcinogenesis in aged tissue without repair arrest.« less

ADAM15 expression is downregulated in melanoma metastasis compared to primary melanoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ungerer, Christopher; Doberstein, Kai; Buerger, Claudia

2010-10-22

Research highlights: {yields} Strong ADAM15 expression is found in normal melanocytes. {yields} ADAM15 expression is significantly downregulated in patients with melanoma metastasis. {yields} TGF-{beta} can downregulate ADAM15 expression in melanoma cells. {yields} Overexpression of ADAM15 in melanoma cells inhibits migration, proliferation and invasion of melanoma cells. {yields} Conclusion: ADAM15 represents an tumor suppressor protein in melanoma. -- Abstract: In a mouse melanoma metastasis model it has been recently shown that ADAM15 overexpression in melanoma cells significantly reduced the number of metastatic nodules on the lung. Unfortunately, the expression of ADAM15 in human melanoma tissue has not been determined so far.more » In our study, we characterized the expression of ADAM15 in tissue micro-arrays of patients with primary melanoma with melanoma metastasis. ADAM15 was expressed in melanocytes and endothelial cells of benign nevi and melanoma tissue. Importantly, ADAM15 was significantly downregulated in melanoma metastasis compared to primary melanoma. We further demonstrate that IFN-{gamma} and TGF-{beta} downregulate ADAM15 protein levels in melanoma cells. To investigate the role of ADAM15 in melanoma progression, we overexpressed ADAM15 in melanoma cells. Importantly, overexpression of ADAM15 in melanoma cells reduced the migration, invasion and the anchorage dependent and independent cell growth of melanoma cells. In summary, the downregulation of ADAM15 plays an important role in melanoma progression and ADAM15 act as a tumorsuppressor in melanoma.« less

Viscoplasticity of simulated high-level radioactive waste glass containing platinum group metal particles

NASA Astrophysics Data System (ADS)

Uruga, Kazuyoshi; Usami, Tsuyoshi; Tsukada, Takeshi; Komamine, Satoshi; Ochi, Eiji

2014-09-01

The shear rate dependency of the viscosity of three simulated high-level radioactive waste glasses containing 0, 1.2 and 4.5 wt% platinum group metals (PGMs) was examined at a temperature range of 1173-1473 K by a rotating viscometer. Shear stress when the shear rate equals zero, i.e. yield stress, was also measured by capillary method. The viscosity of the glass containing no PGM was shear rate-independent Newtonian fluid. On the other hand, the apparent viscosity of the glasses containing PGMs increased with decreasing shear rate, and nonzero amount of yield stresses were detected from both glasses. The viscosity and yield stress of the glass containing 4.5 wt% PGMs was roughly one to two orders of magnitude greater than the glass containing 1.2 wt% PGMs. These viscoplastic properties were numerically expressed by Casson equation.

Skn-1a/Oct-11 and {Delta}Np63{alpha} exert antagonizing effects on human keratin expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lena, Anna Maria; Cipollone, Rita; Amelio, Ivano

2010-10-29

Research highlights: {yields} Skn-1a markedly downregulates {Delta}Np63-driven K14 expression. {yields} {Delta}Np63 inhibits Skn-1a-mediated K10 expression. {yields} {Delta}Np63, mutated in SAM domain, is less effecting in K10 downregulation. {yields} Immunolocalization in human skin of the two transcription factors is partially overlapping. {yields} The antagonistic effects of Skn-1a and p63 is through competition for overlapping responsive elements or through an indirect interaction. -- Abstract: The formation of a stratified epidermis requires a carefully controlled balance between keratinocyte proliferation and differentiation. Here, we report the reciprocal effect on keratin expression of {Delta}Np63, pivotal in normal epidermal morphogenesis and maintenance, and Skn-1a/Oct-11, a POUmore » transcription factor that triggers and regulates the differentiation of keratinocytes. The expression of Skn-1a markedly downregulated {Delta}Np63-driven K14 expression in luciferase reporter assays. The extent of downregulation was comparable to the inhibition of Skn-1a-mediated K10 expression upon expression of {Delta}Np63. {Delta}Np63, mutated in the protein-protein interaction domain (SAM domain; mutated in human ectodermal dysplasia syndrome), was significantly less effecting in downregulating K10, raising the possibility of a direct interaction among Skn-1a and {Delta}Np63. Immunolocalization in human skin biopsies revealed that the expression of the two transcription factors is partially overlapping. Co-immunoprecipitation experiments did not, however, demonstrate a direct interaction between {Delta}Np63 and Skn-1a, suggesting that the antagonistic effects of Skn-1a and p63 on keratin promoter transactivation is probably through competition for overlapping binding sites on target gene promoter or through an indirect interaction.« less

Rehosting of Bacterial Chaperones for High-Quality Protein Production▿

PubMed Central

Martínez-Alonso, Mónica; Toledo-Rubio, Verónica; Noad, Rob; Unzueta, Ugutz; Ferrer-Miralles, Neus; Roy, Polly; Villaverde, Antonio

2009-01-01

Coproduction of DnaK/DnaJ in Escherichia coli enhances solubility but promotes proteolytic degradation of their substrates, minimizing the yield of unstable polypeptides. Higher eukaryotes have orthologs of DnaK/DnaJ but lack the linked bacterial proteolytic system. By coexpression of DnaK and DnaJ in insect cells with inherently misfolding-prone recombinant proteins, we demonstrate simultaneous improvement of soluble protein yield and quality and proteolytic stability. Thus, undesired side effects of bacterial folding modulators can be avoided by appropriate rehosting in heterologous cell expression systems. PMID:19820142

Negative regulation of parathyroid hormone-related protein expression by steroid hormones

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kajitani, Takashi; Tamamori-Adachi, Mimi; Okinaga, Hiroko

Highlights: {yields} Steroid hormones repress expression of PTHrP in the cell lines where the corresponding nuclear receptors are expressed. {yields} Nuclear receptors are required for suppression of PTHrP expression by steroid hormones, except for androgen receptor. {yields} Androgen-induced suppression of PTHrP expression appears to be mediated by estrogen receptor. -- Abstract: Elevated parathyroid hormone-related protein (PTHrP) is responsible for humoral hypercalcemia of malignancy (HHM), which is of clinical significance in treatment of terminal patients with malignancies. Steroid hormones were known to cause suppression of PTHrP expression. However, detailed studies linking multiple steroid hormones to PTHrP expression are lacking. Here wemore » studied PTHrP expression in response to steroid hormones in four cell lines with excessive PTHrP production. Our study established that steroid hormones negatively regulate PTHrP expression. Vitamin D receptor, estrogen receptor {alpha}, glucocorticoid receptor, and progesterone receptor, were required for repression of PTHrP expression by the cognate ligands. A notable exception was the androgen receptor, which was dispensable for suppression of PTHrP expression in androgen-treated cells. We propose a pathway(s) involving nuclear receptors to suppress PTHrP expression.« less

High activity and stability of codon-optimized phosphoenolpyruvate carboxylase from Photobacterium profundum SS9 at low temperatures and its application for in vitro production of oxaloacetate.

PubMed

Park, Soohyun; Hong, Soohye; Pack, Seung Pil; Lee, Jinwon

2014-02-01

Phosphoenolpyruvate carboxylase (PEPC) of Photobacterium profundum SS9 can be expressed and purified using the Escherichia coli expression system. In this study, a codon-optimized PEPC gene (OPPP) was used to increase expression levels. We confirmed OPPP expression and purified it from extracts of recombinant E. coli SGJS117 harboring the OPPP gene. The purified OPPP showed a specific activity value of 80.3 U/mg protein. The OPPP was stable under low temperature (5-30 °C) and weakly basic conditions (pH 8.5-10). The enzymatic ability of OPPP was investigated for in vitro production of oxaloacetate using phosphoenolpyruvate (PEP) and bicarbonate. Only samples containing the OPPP, PEP, and bicarbonate resulted in oxaloacetate production. OPPP production system using E. coli could be a platform technology to produce high yields of heterogeneous gene and provide the PEPC enzyme, which has high enzyme activity.

A Simple And Rapid Minicircle DNA Vector Manufacturing System

PubMed Central

Kay, Mark A; He, Cheng-Yi; Chen, Zhi-Ying

2010-01-01

Minicircle DNA vectors consisting of a circular expression cassette devoid of the bacterial plasmid DNA backbone provides several advantages including sustained transgene expression in quiescent cells/tissues. Their use has been limited by labor-intensive production. We report on a strategy for making multiple genetic modifications in E.coli to construct a producer strain that stably expresses a set of inducible minicircle-assembly enzymes, the øC31-integrase and I-SceI homing-endonuclease. This bacterial strain is capable of producing highly purified minicircle yields in the same time frame as routine plasmid DNA. It is now feasible for minicircle DNA vectors to replace routine plasmids in mammalian transgene expression studies. PMID:21102455

Information Along Contours and Object Boundaries

ERIC Educational Resources Information Center

Feldman, Jacob; Singh, Manish

2005-01-01

F. Attneave (1954) famously suggested that information along visual contours is concentrated in regions of high magnitude of curvature, rather than being distributed uniformly along the contour. Here the authors give a formal derivation of this claim, yielding an exact expression for information, in C. Shannon's (1948) sense, as a function of…

Modifying lignin composition and content of sorghum biomass for improved bioenergy conversion

USDA-ARS?s Scientific Manuscript database

Sorghum (Sorghum bicolor) is an opportune crop for bioenergy due to its high yield potential, and lower nitrogen and water requirements. Transgenic constructs expressing monolignol biosynthetic genes under control of 35S promoter have been developed and used for sorghum transformation to examine the...

Representation and presentation of requirements knowledge

NASA Technical Reports Server (NTRS)

Johnson, W. L.; Feather, Martin S.; Harris, David R.

1992-01-01

An approach to representation and presentation of knowledge used in the ARIES, an experimental requirements/specification environment, is described. The approach applies the notion of a representation architecture to the domain of software engineering and incorporates a strong coupling to a transformation system. It is characterized by a single highly expressive underlying representation, interfaced simultaneously to multiple presentations, each with notations of differing degrees of expressivity. This enables analysts to use multiple languages for describing systems and have these descriptions yield a single consistent model of the system.

Purification and Characterization of Functional Human Paraoxonase-1 Expressed in Trichoplusia ni Larvae

DTIC Science & Technology

2010-01-01

purified from Trichoplusia ni (T. ni) larvae infected with an orally active form of bac- ulovirus. SDS-PAGE and anti-HuPON1 Western blot analyses yielded...Organophosphorus (OP) nerve agents readily bind covalently o acetylcholinesterase (AChE) at the active site serine and inhibit he ability of AChE to terminate...The results demon- trate that T. ni larvae are capable of producing high quantities of unctionally active recombinant HuPON1, and larvae expressing

High-level conversion of L-lysine into 5-aminovalerate that can be used for nylon 6,5 synthesis.

PubMed

Park, Si Jae; Oh, Young Hoon; Noh, Won; Kim, Hye Young; Shin, Jae Ho; Lee, Eun Gyo; Lee, Seungwoon; David, Yokimiko; Baylon, Mary Grace; Song, Bong Keun; Jegal, Jonggeon; Lee, Sang Yup; Lee, Seung Hwan

2014-10-01

L-Lysine is a potential feedstock for the production of bio-based precursors for engineering plastics. In this study, we developed a microbial process for high-level conversion of L-lysine into 5-aminovalerate (5AVA) that can be used as a monomer in nylon 6,5 synthesis. Recombinant Escherichia coli WL3110 strain expressing Pseudomonas putida delta-aminovaleramidase (DavA) and lysine 2-monooxygenase (DavB) was grown to high density in fed-batch culture and used as a whole cell catalyst. High-density E. coli WL3110 expressing DavAB, grown to an optical density at 600 nm (OD600 ) of 30, yielded 36.51 g/L 5AVA from 60 g/L L-lysine in 24 h. Doubling the cell density of E. coli WL3110 improved the conversion yield to 47.96 g/L 5AVA from 60 g/L of L-lysine in 24 h. 5AVA production was further improved by doubling the L-lysine concentration from 60 to 120 g/L. The highest 5AVA titer (90.59 g/L; molar yield 0.942) was obtained from 120 g/L L-lysine by E. coli WL3110 cells grown to OD600 of 60. Finally, nylon 6,5 was synthesized by bulk polymerization of ϵ-caprolactam and δ-valerolactam prepared from microbially synthesized 5AVA. The hybrid system demonstrated here has promising possibilities for application in the development of industrial bio-nylon production processes. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Lumber Grade Yields for Graded Aspen Logs and Trees

Treesearch

Leland F. Hanks; Robert L. Brisbin

1978-01-01

Green lumber grade yields for aspen were determined for use with the U.S. Forest Service hardwood log and tree grades. The yields for logs are expressed in percent of total lumber tally volume, and those for trees are expressed in board feet. Overruns for the International 1/4-inch and Scribner log rules along with lumber recovery factors are shown by log grade.

Expression of proteins in Escherichia coli as fusions with maltose-binding protein to rescue non-expressed targets in a high-throughput protein-expression and purification pipeline

PubMed Central

Hewitt, Stephen N.; Choi, Ryan; Kelley, Angela; Crowther, Gregory J.; Napuli, Alberto J.; Van Voorhis, Wesley C.

2011-01-01

Despite recent advances, the expression of heterologous proteins in Escherichia coli for crystallization remains a nontrivial challenge. The present study investigates the efficacy of maltose-binding protein (MBP) fusion as a general strategy for rescuing the expression of target proteins. From a group of sequence-verified clones with undetectable levels of protein expression in an E. coli T7 expression system, 95 clones representing 16 phylogenetically diverse organisms were selected for recloning into a chimeric expression vector with an N-terminal histidine-tagged MBP. PCR-amplified inserts were annealed into an identical ligation-independent cloning region in an MBP-fusion vector and were analyzed for expression and solubility by high-throughput nickel-affinity binding. This approach yielded detectable expression of 72% of the clones; soluble expression was visible in 62%. However, the solubility of most proteins was marginal to poor upon cleavage of the MBP tag. This study offers large-scale evidence that MBP can improve the soluble expression of previously non-expressing proteins from a variety of eukaryotic and prokaryotic organisms. While the behavior of the cleaved proteins was disappointing, further refinements in MBP tagging may permit the more widespread use of MBP-fusion proteins in crystallographic studies. PMID:21904041

Construction of minitransposons for constitutive and inducible expression of pertussis toxin in bvg-negative Bordetella bronchiseptica.

PubMed Central

Walker, M J; Rohde, M; Wehland, J; Timmis, K N

1991-01-01

Appropriately detoxified pertussis toxin (PT) of Bordetella pertussis is considered to be an essential component of new-generation whooping cough vaccines, but the development of a procedure to obtain high levels of purified toxin has been and continues to be a major difficulty. To produce a system enabling the biological separation of PT from other virulence determinants of B. pertussis and the attainment of high yields of the toxin, minitransposons containing the PT operon were constructed and stably integrated into the chromosome of Bordetella virulence regulatory gene (bvg)-negative Bordetella bronchiseptica ATCC 10580. Since the minitransposons introduced into Bordetella spp. lack the cognate transposase function, they are unable to undergo further transposition events or mediate gene deletions and rearrangements that lead to strain instability. The TnPtacPT minitransposon contains the PT operon under the control of the tac promoter and directs IPTG (isopropyl-beta-D-thiogalactopyranoside)-inducible expression of PT in B. bronchiseptica ATCC 10580. The level of IPTG-induced PT expression was, however, lower than that found for the wild-type B. pertussis Tohama I strain. The TnfusPT minitransposon contains a promoterless PT operon which is only expressed after insertion of the transposon downstream of an appropriately oriented indigenous promoter. After "promoter probing" of B. bronchiseptica with the transposon, clones were screened for PT production by immunoblotting with specific monoclonal antibodies. One clone, designated B. bronchiseptica 10580:: TnfusPT1, expresses significantly higher levels of PT than does B. pertussis Tohama I. The recombinant toxin produced was biologically active in the Chinese hamster ovary cell-clustering assay. High-level expression of PT from a B. bronchiseptica host promoter should provide better yields of the toxin from bacteria not producing other bvg-regulated pathogenesis factors that may play a role in the undesired side effects of current pertussis vaccine preparations. Images PMID:1682257

Biochar derived from corn straw affected availability and distribution of soil nutrients and cotton yield.

PubMed

Tian, Xiaofei; Li, Chengliang; Zhang, Min; Wan, Yongshan; Xie, Zhihua; Chen, Baocheng; Li, Wenqing

2018-01-01

Biochar application as a soil amendment has been proposed as a strategy to improve soil fertility and increase crop yields. However, the effects of successive biochar applications on cotton yields and nutrient distribution in soil are not well documented. A three-year field study was conducted to investigate the effects of successive biochar applications at different rates on cotton yield and on the soil nutrient distribution in the 0-100 cm soil profile. Biochar was applied at 0, 5, 10, and 20 t ha-1 (expressed as Control, BC5, BC10, and BC20, respectively) for each cotton season, with identical doses of chemical fertilizers. Biochar enhanced the cotton lint yield by 8.0-15.8%, 9.3-13.9%, and 9.2-21.9% in 2013, 2014, and 2015, respectively, and high levels of biochar application achieved high cotton yields each year. Leaching of soil nitrate was reduced, while the pH values, soil organic carbon, total nitrogen (N), and available K content of the 0-20 cm soil layer were increased in 2014 and 2015. However, the changes in the soil available P content were less substantial. This study suggests that successive biochar amendments have the potential to enhance cotton productivity and soil fertility while reducing nitrate leaching.

Transcriptome Sequencing Identified Genes and Gene Ontologies Associated with Early Freezing Tolerance in Maize

PubMed Central

Li, Zhao; Hu, Guanghui; Liu, Xiangfeng; Zhou, Yao; Li, Yu; Zhang, Xu; Yuan, Xiaohui; Zhang, Qian; Yang, Deguang; Wang, Tianyu; Zhang, Zhiwu

2016-01-01

Originating in a tropical climate, maize has faced great challenges as cultivation has expanded to the majority of the world's temperate zones. In these zones, frost and cold temperatures are major factors that prevent maize from reaching its full yield potential. Among 30 elite maize inbred lines adapted to northern China, we identified two lines of extreme, but opposite, freezing tolerance levels—highly tolerant and highly sensitive. During the seedling stage of these two lines, we used RNA-seq to measure changes in maize whole genome transcriptome before and after freezing treatment. In total, 19,794 genes were expressed, of which 4550 exhibited differential expression due to either treatment (before or after freezing) or line type (tolerant or sensitive). Of the 4550 differently expressed genes, 948 exhibited differential expression due to treatment within line or lines under freezing condition. Analysis of gene ontology found that these 948 genes were significantly enriched for binding functions (DNA binding, ATP binding, and metal ion binding), protein kinase activity, and peptidase activity. Based on their enrichment, literature support, and significant levels of differential expression, 30 of these 948 genes were selected for quantitative real-time PCR (qRT-PCR) validation. The validation confirmed our RNA-Seq-based findings, with squared correlation coefficients of 80% and 50% in the tolerance and sensitive lines, respectively. This study provided valuable resources for further studies to enhance understanding of the molecular mechanisms underlying maize early freezing response and enable targeted breeding strategies for developing varieties with superior frost resistance to achieve yield potential. PMID:27774095

Detection of differentially expressed genes in broiler pectoralis major muscle affected by White Striping - Wooden Breast myopathies.

PubMed

Zambonelli, Paolo; Zappaterra, Martina; Soglia, Francesca; Petracci, Massimiliano; Sirri, Federico; Cavani, Claudio; Davoli, Roberta

2016-12-01

White Striping and Wooden Breast (WS/WB) are abnormalities increasingly occurring in the fillets of high breast yield and growth rate chicken hybrids. These defects lead to consistent economic losses for poultry meat industry, as affected broiler fillets present an impaired visual appearance that negatively affects consumers' acceptability. Previous studies have highlighted in affected fillets a severely damaged muscle, showing profound inflammation, fibrosis, and lipidosis. The present study investigated the differentially expressed genes and pathways linked to the compositional changes observed in WS/WB breast muscles, in order to outline a more complete framework of the gene networks related to the occurrence of this complex pathological picture. The biochemical composition was performed on 20 pectoralis major samples obtained from high breast yield and growth rate broilers (10 affected vs. 10 normal) and 12 out of the 20 samples were used for the microarray gene expression profiling (6 affected vs. 6 normal). The obtained results indicate strong changes in muscle mineral composition, coupled to an increased deposition of fat. In addition, 204 differentially expressed genes (DEG) were found: 102 up-regulated and 102 down-regulated in affected breasts. The gene expression pathways found more altered in WS/WB muscles are those related to muscle development, polysaccharide metabolic processes, proteoglycans synthesis, inflammation, and calcium signaling pathway. On the whole, the findings suggest that a multifactorial and complex etiology is associated with the occurrence of WS/WB muscle abnormalities, contributing to further defining the transcription patterns associated with these myopathies. © 2016 Poultry Science Association Inc.

High-yield expression and purification of isotopically labeled cytochrome P450 monooxygenases for solid-state NMR spectroscopy

PubMed Central

Rupasinghe, Sanjeewa G.; Duan, Hui; Frericks Schmidt, Heather L.; Berthold, Deborah A.; Rienstra, Chad M.; Schuler, Mary A.

2008-01-01

Cytochrome P450 monooxygenases (P450s), which represent the major group of drug metabolizing enzymes in humans, also catalyze important synthetic and detoxicative reactions in insects, plants and many microbes. Flexibilities in their catalytic sites and membrane associations are thought to play central roles in substrate binding and catalytic specificity. To date, E. coli expression strategies for structural analysis of eukaryotic membrane-bound P450s by X-ray crystallography have necessitated full or partial removal of their N-terminal signal anchor domain (SAD) and, often, replacement of residues more peripherally associated with the membrane (such as the F-G loop region). Even with these modifications, investigations of P450 structural flexibility remain challenging with multiple single crystal conditions needed to identify spatial variations between substrate-free and different substrate-bound forms. To overcome these limitations, we have developed methods for the efficient expression of 13C- and 15N-labeled P450s and analysis of their structures by magic-angle spinning solid-state NMR (SSNMR) spectroscopy. In the presence of co-expressed GroEL and GroES chaperones, full-length (53 kDa) Arabidopsis 13C,15N-labeled CYP98A3 is expressed at yields of 2–4 mg per liter of minimal media without the necessity of generating side chain modifications or N-terminal deletions. Precipitated CYP98A3 generates high quality SSNMR spectra consistent with a homogeneous, folded protein. These data highlight the potential of these methodologies to contribute to the structural analysis of membrane-bound proteins. PMID:18005930

Contribution of morphoagronomic traits to grain yield and earliness in grain sorghum.

PubMed

da Silva, K J; Teodoro, P E; de Menezes, C B; Júlio, M P M; de Souza, V F; da Silva, M J; Pimentel, L D; Borém, A

2017-05-04

Given the importance of selecting lines to obtain hybrids, we aimed to verify the relationship between morphological traits that can be used as the criteria for the selection of sorghum lines with high grain yield and earliness. A total of 18 traits were evaluated in 160 sorghum elite lines, in an incomplete block design with two replicates. A correlation network was used to graphically express the estimates of phenotypic and genotypic correlations between the traits. Two path analyses were processed, the first considering grain yield and the second considering flowering as the principle dependent variable. In general, most of the variation in the grain yield and flowering of sorghum lines was explained by the traits evaluated. Selecting sorghum lines with greater width of the third leaf blade from flag leaf, panicle weight, and panicle harvest index might lead to increased grain yield, and selecting sorghum genotypes with higher plant height might lead to reduced earliness and increased grain yield. Thus, the results suggest the establishment of selection indices aiming at simultaneously increasing the grain yield and earliness in sorghum genotypes.

Senescence marker protein 30 (SMP30)/regucalcin (RGN) expression decreases with aging, acute liver injuries and tumors in zebrafish

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fujisawa, Koichi; Terai, Shuji, E-mail: terais@yamaguchi-u.ac.jp; Hirose, Yoshikazu

2011-10-22

Highlights: {yields} Zebrafish SMP30/RGN mRNA expression decreases with aging. {yields} Decreased expression was observed in liver tumors as compared to the surrounding area. {yields} SMP30/RGN is important for liver proliferation and tumorigenesis. -- Abstract: Senescence marker protein 30 (SMP30)/regucalcin (RGN) is known to be related to aging, hepatocyte proliferation and tumorigenesis. However, expression and function of non-mammalian SMP30/RGN is poorly understood. We found that zebrafish SMP30/RGN mRNA expression decreases with aging, partial hepatectomy and thioacetamide-induced acute liver injury. SMP30/RGN expression was also greatly decreased in a zebrafish liver cell line. In addition, we induced liver tumors in adult zebrafish bymore » administering diethylnitrosamine. Decreased expression was observed in foci, hepatocellular carcinomas, cholangiocellular carcinomas and mixed tumors as compared to the surrounding area. We thus showed the importance of SMP30/RGN in liver proliferation and tumorigenesis.« less

TRACING CO-REGULATORY NETWORK DYNAMICS IN NOISY, SINGLE-CELL TRANSCRIPTOME TRAJECTORIES.

PubMed

Cordero, Pablo; Stuart, Joshua M

2017-01-01

The availability of gene expression data at the single cell level makes it possible to probe the molecular underpinnings of complex biological processes such as differentiation and oncogenesis. Promising new methods have emerged for reconstructing a progression 'trajectory' from static single-cell transcriptome measurements. However, it remains unclear how to adequately model the appreciable level of noise in these data to elucidate gene regulatory network rewiring. Here, we present a framework called Single Cell Inference of MorphIng Trajectories and their Associated Regulation (SCIMITAR) that infers progressions from static single-cell transcriptomes by employing a continuous parametrization of Gaussian mixtures in high-dimensional curves. SCIMITAR yields rich models from the data that highlight genes with expression and co-expression patterns that are associated with the inferred progression. Further, SCIMITAR extracts regulatory states from the implicated trajectory-evolvingco-expression networks. We benchmark the method on simulated data to show that it yields accurate cell ordering and gene network inferences. Applied to the interpretation of a single-cell human fetal neuron dataset, SCIMITAR finds progression-associated genes in cornerstone neural differentiation pathways missed by standard differential expression tests. Finally, by leveraging the rewiring of gene-gene co-expression relations across the progression, the method reveals the rise and fall of co-regulatory states and trajectory-dependent gene modules. These analyses implicate new transcription factors in neural differentiation including putative co-factors for the multi-functional NFAT pathway.

Phenotypic and functional characterization of a CD4+ CD25high FOXP3high regulatory T-cell population in the dog

PubMed Central

Pinheiro, Dammy; Singh, Yogesh; Grant, Charlotte R; Appleton, Richard C; Sacchini, Flavio; Walker, Kate R L; Chadbourne, Alden H; Palmer, Charlotte A; Armitage-Chan, Elizabeth; Thompson, Ian; Williamson, Lina; Cunningham, Fiona; Garden, Oliver A

2011-01-01

Relatively little is known about regulatory T (Treg) cells and their functional responses in dogs. We have used the cross-reactive anti-mouse/rat Foxp3 antibody clone FJK-16s to identify a population of canine CD4+ FOXP3high T cells in both the peripheral blood (PB) and popliteal lymph node (LN). FOXP3+ cells in both PB and LN yielded positive staining with the newly developed anti-murine/human Helios antibody clone 22F6, consistent with the notion that they were naturally occurring Treg cells. Stimulation of mononuclear cells of LN origin with concanavalin A (Con A) in vitro yielded increased proportions and median fluorescence intensity of FOXP3 expression by both CD4+ and CD8+ T cells. Removal of the Con A and continued culture disclosed a CD4+ FOXP3high population, distinct from the CD4+ FOXP3intermediate T cells; very few CD8+ FOXP3high T cells were observed, though CD8+ FOXP3intermediate cells were present in equal abundance to CD4+ FOXP3intermediate cells. The CD4+ FOXP3high T cells were thought to represent activated Treg cells, in contrast to the FOXP3intermediate cells, which were thought to be a more heterogeneous population comprising predominantly activated conventional T cells. Co-staining with interferon-γ (IFN-γ) supported this notion, because the FOXP3high T cells were almost exclusively IFN-γ−, whereas the FOXP3intermediate cells expressed a more heterogeneous IFN-γ phenotype. Following activation of mononuclear cells with Con A and interleukin-2, the 5% of CD4+ T cells showing the highest CD25 expression (CD4+ CD25high) were enriched in cells expressing FOXP3. These cells were anergic in vitro, in contrast to the 20% of CD4+ T cells with the lowest CD25 expression (CD4+ CD25−), which proliferated readily. The CD4+ CD25high FOXP3high T cells were able to suppress the proliferation of responder CD4+ T cells in vitro, in contrast to the CD4+ CD25− cells, which showed no regulatory properties. PMID:20880379

High level transient expression of a chloramphenicol acetyl transferase gene by DEAE-dextran mediated DNA transfection coupled with a dimethyl sulfoxide or glycerol shock treatment.

PubMed Central

Lopata, M A; Cleveland, D W; Sollner-Webb, B

1984-01-01

Using a plasmid containing the bacterial chloramphenicol acetyl transferase gene, we have assayed for transient expression of DNA introduced into mouse L cells by a variety of transfection conditions. High efficiency uptake and expression of this foreign DNA have been achieved by modifying the DEAE dextran mediated transfection procedure of McCutchan and Pagano (1) to include a shock with either dimethyl sulfoxide or glycerol. Inclusion of the shock step can increase expression of the transfected gene a surprising approximately 50 fold. With plasmid constructs that do not replicate after transfection, we can readily detect CAT activity in an overnight autoradiographic exposure from less than 0.1% of an extract from a 60 mm dish of transfected cells. We have determined the amounts of DNA, the amount and time course of DEAE-dextran and dimethyl sulfoxide treatments, the effects of additional DNA, and the time after transfection which yield maximal expression. Overall, this transfection protocol using DEAE-dextran coupled to a shock treatment is simple, straightforward, and gives consistently high levels of expression of the input DNA. Images PMID:6589587

Tombusvirus-based vector systems to permit over-expression of genes or that serve as sensors of antiviral RNA silencing in plants.

PubMed

Shamekova, Malika; Mendoza, Maria R; Hsieh, Yi-Cheng; Lindbo, John; Omarov, Rustem T; Scholthof, Herman B

2014-03-01

A next generation Tomato bushy stunt virus (TBSV) coat protein gene replacement vector system is described that can be applied by either RNA inoculation or through agroinfiltration. A vector expressing GFP rapidly yields high levels of transient gene expression in inoculated leaves of various plant species, as illustrated for Nicotiana benthamiana, cowpea, tomato, pepper, and lettuce. A start-codon mutation to down-regulate the dose of the P19 silencing suppressor reduces GFP accumulation, whereas mutations that result in undetectable levels of P19 trigger rapid silencing of GFP. Compared to existing virus vectors the TBSV system has a unique combination of a very broad host range, rapid and high levels of replication and gene expression, and the ability to regulate its suppressor. These features are attractive for quick transient assays in numerous plant species for over-expression of genes of interest, or as a sensor to monitor the efficacy of antiviral RNA silencing. Copyright © 2014. Published by Elsevier Inc.

Efficient production of Trastuzumab Fab antibody fragments in Brevibacillus choshinensis expression system.

PubMed

Mizukami, Makoto; Onishi, Hiromasa; Hanagata, Hiroshi; Miyauchi, Akira; Ito, Yuji; Tokunaga, Hiroko; Ishibashi, Matsujiro; Arakawa, Tsutomu; Tokunaga, Masao

2018-10-01

The Brevibacillus expression system has been successfully employed for the efficient productions of a variety of recombinant proteins, including enzymes, cytokines, antigens and antibody fragments. Here, we succeeded in secretory expression of Trastuzumab Fab antibody fragments using B. choshinensis/BIC (Brevibacillus in vivocloning) expression system. In the fed-batch high-density cell culture, recombinant Trastuzumab Fab with amino-terminal His-tag (His-BcFab) was secreted at high level, 1.25 g/liter, and Fab without His-tag (BcFab) at ∼145 mg/L of culture supernatant. His-BcFab and BcFab were purified to homogeneity using combination of conventional column chromatographies with a yield of 10-13%. This BcFab preparation exhibited native structure and functions evaluated by enzyme-linked immunosorbent assay, surface plasmon resonance, circular dichroism measurements and size exclusion chromatography. To our knowledge, this is the highest production of Fab antibody fragments in gram-positive bacterial expression/secretion systems. Copyright © 2018 Elsevier Inc. All rights reserved.

Elevated HLA-A expression impairs HIV control through inhibition of NKG2A-expressing cells.

PubMed

Ramsuran, Veron; Naranbhai, Vivek; Horowitz, Amir; Qi, Ying; Martin, Maureen P; Yuki, Yuko; Gao, Xiaojiang; Walker-Sperling, Victoria; Del Prete, Gregory Q; Schneider, Douglas K; Lifson, Jeffrey D; Fellay, Jacques; Deeks, Steven G; Martin, Jeffrey N; Goedert, James J; Wolinsky, Steven M; Michael, Nelson L; Kirk, Gregory D; Buchbinder, Susan; Haas, David; Ndung'u, Thumbi; Goulder, Philip; Parham, Peter; Walker, Bruce D; Carlson, Jonathan M; Carrington, Mary

2018-01-05

The highly polymorphic human leukocyte antigen ( HLA ) locus encodes cell surface proteins that are critical for immunity. HLA-A expression levels vary in an allele-dependent manner, diversifying allele-specific effects beyond peptide-binding preference. Analysis of 9763 HIV-infected individuals from 21 cohorts shows that higher HLA-A levels confer poorer control of HIV. Elevated HLA-A expression provides enhanced levels of an HLA-A-derived signal peptide that specifically binds and determines expression levels of HLA-E, the ligand for the inhibitory NKG2A natural killer (NK) cell receptor. HLA-B haplotypes that favor NKG2A-mediated NK cell licensing (i.e., education) exacerbate the deleterious effect of high HLA-A on HIV control, consistent with NKG2A-mediated inhibition impairing NK cell clearance of HIV-infected targets. Therapeutic blockade of HLA-E:NKG2A interaction may yield benefit in HIV disease. Copyright © 2017, American Association for the Advancement of Science.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Saito, Yuri, E-mail: saito-yu@bldon.med.osaka-u.ac.jp; Shibayama, Hirohiko; Tanaka, Hirokazu

Research highlights: {yields} Anamorsin (AM) (also called CIAPIN-1) is a cell-death-defying factor. {yields} Biological mechanisms of AM functions have not been elucidated yet. {yields} PKC{theta} , PKC{delta} and p38MAPK were more phosphorylated in AM deficient MEF cells. {yields} AM may negatively regulates PKCs and p38MAPK in MEF cells. -- Abstract: Anamorsin (AM) plays crucial roles in hematopoiesis and embryogenesis. AM deficient (AM KO) mice die during late gestation; AM KO embryos are anemic and very small compared to wild type (WT) embryos. To determine which signaling pathways AM utilizes for these functions, we used murine embryonic fibroblast (MEF) cells generatedmore » from E-14.5 AM KO or WT embryos. Proliferation of AM KO MEF cells was markedly retarded, and PKC{theta}, PKC{delta}, and p38MAPK were more highly phosphorylated in AM KO MEF cells. Expression of cyclinD1, the target molecule of p38MAPK, was down-regulated in AM KO MEF cells. p38MAPK inhibitor as well as PKC inhibitor restored expression of cyclinD1 and cell growth in AM KO MEF cells. These data suggest that PKC{theta}, PKC{delta}, and p38MAPK activation lead to cell cycle retardation in AM KO MEF cells, and that AM may negatively regulate novel PKCs and p38MAPK in MEF cells.« less

Tigutcystatin, a cysteine protease inhibitor from Triatoma infestans midgut expressed in response to Trypanosoma cruzi

DOE Office of Scientific and Technical Information (OSTI.GOV)

Buarque, Diego S.; Spindola, Leticia M.N.; Martins, Rafael M.

2011-09-23

Highlights: {yields} Tigutcystatin inhibits Trypanosoma cruzi cysteine proteases with high specificity. {yields} Tigutcystatin expression is up-regulated in response to T. cruzi infection. {yields} It is the first cysteine proteases inhibitor characterized from a triatomine insect. -- Abstract: The insect Triatoma infestans is a vector of Trypanosoma cruzi, the etiological agent of Chagas disease. A cDNA library was constructed from T. infestans anterior midgut, and 244 clones were sequenced. Among the EST sequences, an open reading frame (ORF) with homology to a cystatin type 2 precursor was identified. Then, a 288-bp cDNA fragment encoding mature cystatin (lacking signal peptide) named Tigutcystatinmore » was cloned fused to a N-terminal His tag in pET-14b vector, and the protein expressed in Escherichia coli strain Rosetta gami. Tigutcystatin purified and cleaved by thrombin to remove His tag presented molecular mass of 11 kDa and 10,137 Da by SDS-PAGE and MALDI-TOF mass spectrometry, respectively. Purified Tigutcystatin was shown to be a tight inhibitor towards cruzain, a T. cruzi cathepsin L-like enzyme (K{sub i} = 3.29 nM) and human cathepsin L (K{sub i} = 3.78 nM). Tissue specific expression analysis showed that Tigutcystatin was mostly expressed in anterior midgut, although amplification in small intestine was also detected by semi quantitative RT-PCR. qReal time PCR confirmed that Tigutcystatin mRNA is significantly up-regulated in anterior midgut when T. infestans is infected with T. cruzi. Together, these results indicate that Tigutcystatin may be involved in modulation of T. cruzi in intestinal tract by inhibiting parasite cysteine proteases, which represent the virulence factors of this protozoan.« less

Animal component-free Agrobacterium tumefaciens cultivation media for better GMP-compliance increases biomass yield and pharmaceutical protein expression in Nicotiana benthamiana.

PubMed

Houdelet, Marcel; Galinski, Anna; Holland, Tanja; Wenzel, Kathrin; Schillberg, Stefan; Buyel, Johannes Felix

2017-04-01

Transient expression systems allow the rapid production of recombinant proteins in plants. Such systems can be scaled up to several hundred kilograms of biomass, making them suitable for the production of pharmaceutical proteins required at short notice, such as emergency vaccines. However, large-scale transient expression requires the production of recombinant Agrobacterium tumefaciens strains with the capacity for efficient gene transfer to plant cells. The complex media often used for the cultivation of this species typically include animal-derived ingredients that can contain human pathogens, thus conflicting with the requirements of good manufacturing practice (GMP). We replaced all the animal-derived components in yeast extract broth (YEB) cultivation medium with soybean peptone, and then used a design-of-experiments approach to optimize the medium composition, increasing the biomass yield while maintaining high levels of transient expression in subsequent infiltration experiments. The resulting plant peptone Agrobacterium medium (PAM) achieved a two-fold increase in OD 600 compared to YEB medium during a 4-L batch fermentation lasting 18 h. Furthermore, the yields of the monoclonal antibody 2G12 and the fluorescent protein DsRed were maintained when the cells were cultivated in PAM rather than YEB. We have thus demonstrated a simple, efficient and scalable method for medium optimization that reduces process time and costs. The final optimized medium for the cultivation of A. tumefaciens completely lacks animal-derived components, thus facilitating the GMP-compliant large-scale transient expression of recombinant proteins in plants. © 2017 The Authors. Biotechnology Journal published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Expression of adhA from different organisms in Clostridium thermocellum.

PubMed

Zheng, Tianyong; Cui, Jingxuan; Bae, Hye Ri; Lynd, Lee R; Olson, Daniel G

2017-01-01

Clostridium thermocellum is a cellulolytic anaerobic thermophile that is a promising candidate for consolidated bioprocessing of lignocellulosic biomass into biofuels such as ethanol. It was previously shown that expressing Thermoanaerobacterium saccharolyticum adhA in C. thermocellum increases ethanol yield.In this study, we investigated expression of adhA genes from different organisms in Clostridium thermocellum . Based on sequence identity to T. saccharolyticum adhA , we chose adhA genes from 10 other organisms: Clostridium botulinum , Methanocaldococcus bathoardescens , Thermoanaerobacterium ethanolicus , Thermoanaerobacter mathranii , Thermococcus strain AN1, Thermoanaerobacterium thermosaccharolyticum , Caldicellulosiruptor saccharolyticus , Fervidobacterium nodosum , Marinitoga piezophila , and Thermotoga petrophila . All 11 adhA genes (including T. saccharolyticum adhA ) were expressed in C. thermocellum and fermentation end products were analyzed. All 11 adhA genes increased C. thermocellum ethanol yield compared to the empty-vector control. C. botulinum and T. ethanolicus adhA genes generated significantly higher ethanol yield than T. saccharolyticum adhA . Our results indicated that expressing adhA is an effective method of increasing ethanol yield in wild-type C. thermocellum , and that this appears to be a general property of adhA genes.

Nordihydroguaiaretic acid protects against high-fat diet-induced fatty liver by activating AMP-activated protein kinase in obese mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Myoung-Su; Kim, Daeyoung; Jo, Keunae

Research highlights: {yields} NDGA decreases high-fat diet-induced body weight gain and adiposity. {yields} NDGA reduces high-fat diet-induced triglyceride accumulation in liver. {yields} NDGA improves lipid storage in vitro through altering lipid regulatory proteins. {yields} Inhibition of lipid storage in vivo and in vitro is mediated by AMPK activation. -- Abstract: Nonalcoholic fatty liver disease, one of the most common causes of chronic liver disease, is strongly associated with metabolic syndrome. Nordihydroguaiaretic acid (NDGA) has been reported to inhibit lipoprotein lipase; however, the effect of NDGA on hepatic lipid metabolism remains unclear. We evaluated body weight, adiposity, liver histology, and hepaticmore » triglyceride content in high-fat diet (HFD)-fed C57BL/6J mice treated with NDGA. In addition, we characterized the underlying mechanism of NDGA's effects in HepG2 hepatocytes by Western blot and RT-PCR analysis. NDGA (100 or 200 mg/kg/day) reduced weight gain, fat pad mass, and hepatic triglyceride accumulation, and improved serum lipid parameters in mice fed a HFD for 8 weeks. NDGA significantly increased AMP-activated protein kinase (AMPK) phosphorylation in the liver and in HepG2 hepatocytes. NDGA downregulated the level of mature SREBP-1 and its target genes (acetyl-CoA carboxylase and fatty acid synthase), but, it upregulated expression of genes involved in fatty acid oxidation, such as peroxisome proliferator-activated receptor (PPAR){alpha}, PPAR{gamma} coactivator-1, carnitine palmitoyl transferase-1, and uncoupling protein-2. The specific AMPK inhibitor compound C attenuated the effects of NDGA on expression of lipid metabolism-related proteins in HepG2 hepatocytes. The beneficial effects of NDGA on HFD-induced hepatic triglyceride accumulation are mediated through AMPK signaling pathways, suggesting a potential target for preventing NAFLD.« less

Improvement of FK506 Production in the High-Yielding Strain Streptomyces sp. RM7011 by Engineering the Supply of Allylmalonyl-CoA Through a Combination of Genetic and Chemical Approach.

PubMed

Mo, SangJoon; Lee, Sung-Kwon; Jin, Ying-Yu; Suh, Joo-Won

2016-02-01

FK506, a widely used immunosuppressant, is a 23-membered polyketide macrolide that is produced by several Streptomyces species. FK506 high-yielding strain Streptomyces sp. RM7011 was developed from the discovered Streptomyces sp. KCCM 11116P by random mutagenesis in our previous study. The results of transcript expression analysis showed that the transcription levels of tcsA, B, C, and D were increased in Streptomyces sp. RM7011 by 2.1-, 3.1-, 3.3-, and 4.1- fold, respectively, compared with Streptomyces sp. KCCM 11116P. The overexpression of tcsABCD genes in Streptomyces sp. RM7011 gave rise to approximately 2.5-fold (238.1 μg/ml) increase in the level of FK506 production compared with that of Streptomyces sp. RM7011. When vinyl pentanoate was added into the culture broth of Streptomyces sp. RM7011, the level of FK506 production was approximately 2.2-fold (207.7 μg/ml) higher than that of the unsupplemented fermentation. Furthermore, supplementing the culture broth of Streptomyces sp. RM7011 expressing tcsABCD genes with vinyl pentanoate resulted in an additional 1.7-fold improvement in the FK506 titer (498.1 μg/ml) compared with that observed under nonsupplemented condition. Overall, the level of FK506 production was increased approximately 5.2-fold by engineering the supply of allylmalonyl-CoA in the high-yielding strain Streptomyces sp. RM7011, using a combination of overexpressing tcsABCD genes and adding vinyl pentanoate, as compared with Streptomyces sp. RM7011 (95.3 μg/ml). Moreover, among the three precursors analyzed, pentanoate was the most effective precursor, supporting the highest titer of FK506 in the FK506 high-yielding strain Streptomyces sp. RM7011.

Metabolic engineering for the high-yield production of isoprenoid-based C 5 alcohols in E. coli

DOE Office of Scientific and Technical Information (OSTI.GOV)

George, Kevin W.; Thompson, Mitchell G.; Kang, Aram

2015-06-08

Branched five carbon (C 5) alcohols are attractive targets for microbial production due to their desirable fuel properties and importance as platform chemicals. In this study, we engineered a heterologous isoprenoid pathway in E. coli for the high-yield production of 3-methyl-3-buten-1-ol, 3-methyl-2-buten-1-ol, and 3-methyl-1-butanol, three C 5 alcohols that serve as potential biofuels. We first constructed a pathway for 3-methyl-3-buten-1-ol, where metabolite profiling identified NudB, a promiscuous phosphatase, as a likely pathway bottleneck. We achieved a 60% increase in the yield of 3-methyl-3-buten-1-ol by engineering the Shine-Dalgarno sequence of nudB, which increased protein levels by 9-fold and reduced isopentenyl diphosphatemore » (IPP) accumulation by 4-fold. To further optimize the pathway, we adjusted mevalonate kinase (MK) expression and investigated MK enzymes from alternative microbes such as Methanosarcina mazei. Next, we expressed a fusion protein of IPP isomerase and the phosphatase (Idi1~NudB) along with a reductase (NemA) to diversify production to 3-methyl-2-buten-1-ol and 3-methyl-1-butanol. Lastly, we used an oleyl alcohol overlay to improve alcohol recovery, achieving final titers of 2.23 g/L of 3-methyl-3-buten-1-ol (~70% of pathway-dependent theoretical yield), 150 mg/L of 3-methyl-2-buten-1-ol, and 300 mg/L of 3-methyl-1-butanol.« less

Metabolic engineering for the high-yield production of isoprenoid-based C5 alcohols in E. coli

PubMed Central

George, Kevin W.; Thompson, Mitchell G.; Kang, Aram; Baidoo, Edward; Wang, George; Chan, Leanne Jade G.; Adams, Paul D.; Petzold, Christopher J.; Keasling, Jay D.; Soon Lee, Taek

2015-01-01

Branched five carbon (C5) alcohols are attractive targets for microbial production due to their desirable fuel properties and importance as platform chemicals. In this study, we engineered a heterologous isoprenoid pathway in E. coli for the high-yield production of 3-methyl-3-buten-1-ol, 3-methyl-2-buten-1-ol, and 3-methyl-1-butanol, three C5 alcohols that serve as potential biofuels. We first constructed a pathway for 3-methyl-3-buten-1-ol, where metabolite profiling identified NudB, a promiscuous phosphatase, as a likely pathway bottleneck. We achieved a 60% increase in the yield of 3-methyl-3-buten-1-ol by engineering the Shine-Dalgarno sequence of nudB, which increased protein levels by 9-fold and reduced isopentenyl diphosphate (IPP) accumulation by 4-fold. To further optimize the pathway, we adjusted mevalonate kinase (MK) expression and investigated MK enzymes from alternative microbes such as Methanosarcina mazei. Next, we expressed a fusion protein of IPP isomerase and the phosphatase (Idi1~NudB) along with a reductase (NemA) to diversify production to 3-methyl-2-buten-1-ol and 3-methyl-1-butanol. Finally, we used an oleyl alcohol overlay to improve alcohol recovery, achieving final titers of 2.23 g/L of 3-methyl-3-buten-1-ol (~70% of pathway-dependent theoretical yield), 150 mg/L of 3-methyl-2-buten-1-ol, and 300 mg/L of 3-methyl-1-butanol. PMID:26052683

Angiotensin II modulates interleukin-1{beta}-induced inflammatory gene expression in vascular smooth muscle cells via interfering with ERK-NF-{kappa}B crosstalk

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Shanqin; Zhi, Hui; Hou, Xiuyun

2011-07-08

Highlights: {yields} We examine how angiotensin II modulates ERK-NF-{kappa}B crosstalk and gene expression. {yields} Angiotensin II suppresses IL-1{beta}-induced prolonged ERK and NF-{kappa}B activation. {yields} ERK-RSK1 signaling is required for IL-1{beta}-induced prolonged NF-{kappa}B activation. {yields} Angiotensin II modulates NF-{kappa}B responsive genes via regulating ERK-NF-{kappa}B crosstalk. {yields} ERK-NF-{kappa}B crosstalk is a novel mechanism regulating inflammatory gene expression. -- Abstract: Angiotensin II is implicated in cardiovascular diseases, which is associated with a role in increasing vascular inflammation. The present study investigated how angiotensin II modulates vascular inflammatory signaling and expression of inducible nitric oxide synthase (iNOS) and vascular cell adhesion molecule (VCAM)-1. Inmore » cultured rat aortic vascular smooth muscle cells (VSMCs), angiotensin II suppressed interleukin-1{beta}-induced prolonged phosphorylation of extracellular signal-regulated kinase (ERK) and ribosomal S6 kinase (RSK)-1, and nuclear translocation of nuclear factor (NF)-{kappa}B, leading to decreased iNOS but enhanced VCAM-1 expression, associated with an up-regulation of mitogen-activated protein kinase phosphatase-1 expression. Knock-down of RSK1 selectively down regulated interleukin-1{beta}-induced iNOS expression without influencing VCAM-1 expression. In vivo experiments showed that interleukin-1{beta}, iNOS, and VCAM-1 expression were detectable in the aortic arches of both wild-type and apolipoprotein E-deficient (ApoE{sup -/-}) mice. VCAM-1 and iNOS expression were higher in ApoE{sup -/-} than in wild type mouse aortic arches. Angiotensin II infusion (3.2 mg/kg/day, for 6 days, via subcutaneous osmotic pump) in ApoE{sup -/-} mice enhanced endothelial and adventitial VCAM-1 and iNOS expression, but reduced medial smooth muscle iNOS expression associated with reduced phosphorylation of ERK and RSK-1. These results indicate that angiotensin II can differentially modulate inflammatory gene expression in aortic smooth muscle cells through influencing ERK-NF-{kappa}B crosstalk, which may contribute to angiotensin II-induced inflammatory disorders related to cardiovascular diseases.« less

The Expression of TaRca2-α Gene Associated with Net Photosynthesis Rate, Biomass and Grain Yield in Bread Wheat (Triticum aestivum L.) under Field Conditions

PubMed Central

Saeed, Iqbal; Bachir, Daoura Goudia; Chen, Liang; Hu, Yin-Gang

2016-01-01

Improvement in activation of Rubisco by Rubisco activase can potentially enhance CO2 assimilation and photosynthetic efficiency in plants. The three homoeologous copies of TaRca2-α were identified on chromosomes 4AL, 4BS and 4DS (TaRca2-α-4AL, TaRca2-α-4BS, and TaRca2-α-4DS) in bread wheat. Expression patterns of the three copies at heading (Z55), anthesis (Z67) and grain-filling (Z73) stages were investigated through qRT-PCR analyses in a panel of 59 bread wheat genotypes and their effects on net photosynthesis rate (Pn), biomass plant-1 (BMPP) and grain yield plant-1 (GYPP) were further explored. Different but similar expression patterns were observed for the three copies of TaRca2-α at the three growth stages with highest expression at grain-filling stage. TaRca2-α-4BS expressed higher at the three stages than TaRca2-α-4AL and TaRca2-α-4DS. The 59 genotypes could be clustered into three groups as high (7 genotypes), intermediate (41 genotypes) and low (11 genotypes) expression based on the expression of the three copies of TaRca2-α at three growth stages. Significant variations (P<0.01) were observed among the three groups of bread wheat genotypes for Pn, BMPP and GYPP. Generally, the genotypes with higher TaRca2-α expression also showed higher values for Pn, BMPP and GYPP. The expressions of the three copies of TaRca2-α at heading, anthesis and grain-filling stages were positively correlated with Pn, BMPP and GYPP (P<0.01) with stronger association for TaRca2-α-4BS at grain-filling stage. These results revealed that the expression of TaRca2-α contribute substantially to Pn, BMPP and GYPP, and suggested that manipulating TaRca-α expression may efficiently improve Pn, BMPP and GYPP in bread wheat and detecting TaRca-α expression levels with emphasis on TaRca2-α-4BS may be a positive strategy for selection in improving photosynthetic efficiency and grain yield of bread wheat. PMID:27548477

The orphan nuclear receptor Nur77 regulates decidual prolactin expression in human endometrial stromal cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jiang, Yue; Hu, Yali; Zhao, Jing

2011-01-14

Research highlights: {yields} Decidually produced PRL plays a key role during pregnancy. {yields} Overexpression of Nur77 increased PRL mRNA expression and enhanced decidual PRL promoter activity. {yields} Knockdown of Nur77 decreased decidual PRL secretion induced by 8-Br-cAMP and MPA. {yields} Nur77 is a novel transcription factor that plays an active role in decidual prolactin expression. -- Abstract: Prolactin (PRL) is synthesized and released by several extrapituitary tissues, including decidualized stromal cells. Despite the important role of decidual PRL during pregnancy, little is understood about the factors involved in the proper regulation of decidual PRL expression. Here we present evidence thatmore » the transcription factor Nur77 plays an active role in decidual prolactin expression in human endometrial stromal cells (hESCs). Nur77 mRNA expression in hESCs was significantly increased after decidualization stimulated by 8-Br-cAMP and medroxyprogesterone acetate (MPA). Adenovirus-mediated overexpression of Nur77 in hESCs markedly increased PRL mRNA expression and enhanced decidual PRL promoter (dPRL/-332Luc) activity in a concentration-dependent manner. Furthermore, knockdown of Nur77 in hESCs significantly decreased decidual PRL promoter activation and substantially attenuated PRL mRNA expression and PRL secretion (P < 0.01) induced by 8-Br-cAMP and MPA. These results demonstrate that Nur77 is a novel transcription factor that contributes significantly to the regulation of prolactin gene expression in human endometrial stromal cells.« less

Efficient osmolyte-based procedure to increase expression level and solubility of infectious hematopoietic necrosis virus (IHNV) nucleoprotein in E. coli.

PubMed

Mohammadinezhad, Rezvan; Farahmand, Hamid; Jalali, Seyed Amir Hossein; Mirvaghefi, Alireza

2018-05-01

The nucleoprotein of infectious hematopoietic necrosis virus (IHNV) is considered as the main target antigen for detection of IHNV infection in salmonid fish. This study aimed at improving the expression and solubility of IHNV nucleoprotein (IHNV-NP) in E. coli expression system. The effects of several expression strategies including host strain type, protein expression temperature, heat-shock treatment prior to protein induction, and additives in the growth medium and in the cell lysis buffer were examined. Results showed that bacterial strain type had a great impact on protein expression level, whereas it was not effective in preventing protein aggregation. Production of soluble IHNV-NP was proportionally increased with decreased incubation temperature. Heat-shock treatment prior to protein induction did not change the percent of solubility. For cells grown at low temperature, the presence of additives in the lysis buffer enhanced the solubility of IHNV-NP up to 24%. The highest yield of soluble protein was obtained via incorporation of osmolytes in the growth medium of cells exposed to a mild salt stress, in the following order: sucrose > sorbitol > glycerol > glycine. Soluble protein obtained by the optimized condition was efficiently purified in high yield and successfully detected by two monoclonal antibodies in a sandwich ELISA. Taken together, a combination of proper host strain, low-temperature expression, and timely application of osmolytes in the growth medium provided sufficient quantities of soluble recombinant IHNV-NP that has the potential to be used for diagnostic purposes.

Competitive Performance of Transgenic Wheat Resistant to Powdery Mildew

PubMed Central

Kalinina, Olena; Zeller, Simon L.; Schmid, Bernhard

2011-01-01

Genetically modified (GM) plants offer an ideal model system to study the influence of single genes that confer constitutive resistance to pathogens on the ecological behaviour of plants. We used phytometers to study competitive interactions between GM lines of spring wheat Triticum aestivum carrying such genes and control lines. We hypothesized that competitive performance of GM lines would be reduced due to enhanced transgene expression under pathogen levels typically encountered in the field. The transgenes pm3b from wheat (resistance against powdery mildew Blumeria graminis) or chitinase and glucanase genes from barley (resistance against fungi in general) were introduced with the ubiquitin promoter from maize (pm3b and chitinase genes) or the actin promoter from rice (glucanase gene). Phytometers of 15 transgenic and non-transgenic wheat lines were transplanted as seedlings into plots sown with the same 15 lines as competitive environments and subject to two soil nutrient levels. Pm3b lines had reduced mildew incidence compared with control lines. Chitinase and chitinase/glucanase lines showed the same high resistance to mildew as their control in low-nutrient treatment and slightly lower mildew rates than the control in high-nutrient environment. Pm3b lines were weaker competitors than control lines. This resulted in reduced yield and seed number. The Pm3b line with the highest transgene expression had 53.2% lower yield than the control whereas the Pm3b line which segregated in resistance and had higher mildew rates showed only minor costs under competition. The line expressing both chitinase and glucanase genes also showed reduced yield and seed number under competition compared with its control. Our results suggest that single transgenes conferring constitutive resistance to pathogens can have ecological costs and can weaken plant competitiveness even in the presence of the pathogen. The magnitude of these costs appears related to the degree of expression of the transgenes. PMID:22132219

Metabolic engineering of biomass for high energy density: oilseed-like triacylglycerol yields from plant leaves.

PubMed

Vanhercke, Thomas; El Tahchy, Anna; Liu, Qing; Zhou, Xue-Rong; Shrestha, Pushkar; Divi, Uday K; Ral, Jean-Philippe; Mansour, Maged P; Nichols, Peter D; James, Christopher N; Horn, Patrick J; Chapman, Kent D; Beaudoin, Frederic; Ruiz-López, Noemi; Larkin, Philip J; de Feyter, Robert C; Singh, Surinder P; Petrie, James R

2014-02-01

High biomass crops have recently attracted significant attention as an alternative platform for the renewable production of high energy storage lipids such as triacylglycerol (TAG). While TAG typically accumulates in seeds as storage compounds fuelling subsequent germination, levels in vegetative tissues are generally low. Here, we report the accumulation of more than 15% TAG (17.7% total lipids) by dry weight in Nicotiana tabacum (tobacco) leaves by the co-expression of three genes involved in different aspects of TAG production without severely impacting plant development. These yields far exceed the levels found in wild-type leaf tissue as well as previously reported engineered TAG yields in vegetative tissues of Arabidopsis thaliana and N. tabacum. When translated to a high biomass crop, the current levels would translate to an oil yield per hectare that exceeds those of most cultivated oilseed crops. Confocal fluorescence microscopy and mass spectrometry imaging confirmed the accumulation of TAG within leaf mesophyll cells. In addition, we explored the applicability of several existing oil-processing methods using fresh leaf tissue. Our results demonstrate the technical feasibility of a vegetative plant oil production platform and provide for a step change in the bioenergy landscape, opening new prospects for sustainable food, high energy forage, biofuel and biomaterial applications. © 2013 CSIRO. Plant Biotechnology Journal published by Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Prospective Surface Marker-Based Isolation and Expansion of Fetal Endothelial Colony-Forming Cells From Human Term Placenta

PubMed Central

Patel, Jatin; Seppanen, Elke; Chong, Mark S.K.; Yeo, Julie S.L.; Teo, Erin Y.L.; Chan, Jerry K.Y.; Fisk, Nicholas M.

2013-01-01

The term placenta is a highly vascularized tissue and is usually discarded upon birth. Our objective was to isolate clinically relevant quantities of fetal endothelial colony-forming cells (ECFCs) from human term placenta and to compare them to the well-established donor-matched umbilical cord blood (UCB)-derived ECFCs. A sorting strategy was devised to enrich for CD45−CD34+CD31Lo cells prior to primary plating to obtain pure placental ECFCs (PL-ECFCs) upon culture. UCB-ECFCs were derived using a well-described assay. PL-ECFCs were fetal in origin and expressed the same cell surface markers as UCB-ECFCs. Most importantly, a single term placenta could yield as many ECFCs as 27 UCB donors. PL-ECFCs and UCB-ECFCs had similar in vitro and in vivo vessel forming capacities and restored mouse hind limb ischemia in similar proportions. Gene expression profiles were only minimally divergent between PL-ECFCs and UCB-ECFCs, probably reflecting a vascular source versus a circulating source. Finally, PL-ECFCs and UCB-ECFCs displayed similar hierarchies between high and low proliferative colonies. We report a robust strategy to isolate ECFCs from human term placentas based on their cell surface expression. This yielded much larger quantities of ECFCs than UCB, but the cells were comparable in immunophenotype, gene expression, and in vivo functional ability. We conclude that PL-ECFCs have significant bio-banking and clinical translatability potential. PMID:24106336

Framework for the rapid optimization of soluble protein expression in Escherichia coli combining microscale experiments and statistical experimental design.

PubMed

Islam, R S; Tisi, D; Levy, M S; Lye, G J

2007-01-01

A major bottleneck in drug discovery is the production of soluble human recombinant protein in sufficient quantities for analysis. This problem is compounded by the complex relationship between protein yield and the large number of variables which affect it. Here, we describe a generic framework for the rapid identification and optimization of factors affecting soluble protein yield in microwell plate fermentations as a prelude to the predictive and reliable scaleup of optimized culture conditions. Recombinant expression of firefly luciferase in Escherichia coli was used as a model system. Two rounds of statistical design of experiments (DoE) were employed to first screen (D-optimal design) and then optimize (central composite face design) the yield of soluble protein. Biological variables from the initial screening experiments included medium type and growth and induction conditions. To provide insight into the impact of the engineering environment on cell growth and expression, plate geometry, shaking speed, and liquid fill volume were included as factors since these strongly influence oxygen transfer into the wells. Compared to standard reference conditions, both the screening and optimization designs gave up to 3-fold increases in the soluble protein yield, i.e., a 9-fold increase overall. In general the highest protein yields were obtained when cells were induced at a relatively low biomass concentration and then allowed to grow slowly up to a high final biomass concentration, >8 g.L-1. Consideration and analysis of the model results showed 6 of the original 10 variables to be important at the screening stage and 3 after optimization. The latter included the microwell plate shaking speeds pre- and postinduction, indicating the importance of oxygen transfer into the microwells and identifying this as a critical parameter for subsequent scale translation studies. The optimization process, also known as response surface methodology (RSM), predicted there to be a distinct optimum set of conditions for protein expression which could be verified experimentally. This work provides a generic approach to protein expression optimization in which both biological and engineering variables are investigated from the initial screening stage. The application of DoE reduces the total number of experiments needed to be performed, while experimentation at the microwell scale increases experimental throughput and reduces cost.

Comparative interrogation of the developing xylem transcriptomes of two wood-forming species: Populus trichocarpa and Eucalyptus grandis.

PubMed

Hefer, Charles A; Mizrachi, Eshchar; Myburg, Alexander A; Douglas, Carl J; Mansfield, Shawn D

2015-06-01

Wood formation is a complex developmental process governed by genetic and environmental stimuli. Populus and Eucalyptus are fast-growing, high-yielding tree genera that represent ecologically and economically important species suitable for generating significant lignocellulosic biomass. Comparative analysis of the developing xylem and leaf transcriptomes of Populus trichocarpa and Eucalyptus grandis together with phylogenetic analyses identified clusters of homologous genes preferentially expressed during xylem formation in both species. A conserved set of 336 single gene pairs showed highly similar xylem preferential expression patterns, as well as evidence of high functional constraint. Individual members of multi-gene orthologous clusters known to be involved in secondary cell wall biosynthesis also showed conserved xylem expression profiles. However, species-specific expression as well as opposite (xylem versus leaf) expression patterns observed for a subset of genes suggest subtle differences in the transcriptional regulation important for xylem development in each species. Using sequence similarity and gene expression status, we identified functional homologs likely to be involved in xylem developmental and biosynthetic processes in Populus and Eucalyptus. Our study suggests that, while genes involved in secondary cell wall biosynthesis show high levels of gene expression conservation, differential regulation of some xylem development genes may give rise to unique xylem properties. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

Geminivirus vectors for high-level expression of foreign proteins in plant cells.

PubMed

Mor, Tsafrir S; Moon, Yong-Sun; Palmer, Kenneth E; Mason, Hugh S

2003-02-20

Bean yellow dwarf virus (BeYDV) is a monopartite geminivirus that can infect dicotyledonous plants. We have developed a high-level expression system that utilizes elements of the replication machinery of this single-stranded DNA virus. The replication initiator protein (Rep) mediates release and replication of a replicon from a DNA construct ("LSL vector") that contains an expression cassette for a gene of interest flanked by cis-acting elements of the virus. We used tobacco NT1 cells and biolistic delivery of plasmid DNA for evaluation of replication and expression of reporter genes contained within an LSL vector. By codelivery of a GUS reporter-LSL vector and a Rep-supplying vector, we obtained up to 40-fold increase in expression levels compared to delivery of the reporter-LSL vectors alone. High-copy replication of the LSL vector was correlated with enhanced expression of GUS. Rep expression using a whole BeYDV clone, a cauliflower mosaic virus 35S promoter driving either genomic rep or an intron-deleted rep gene, or 35S-rep contained in the LSL vector all achieved efficient replication and enhancement of GUS expression. We anticipate that this system can be adapted for use in transgenic plants or plant cell cultures with appropriately regulated expression of Rep, with the potential to greatly increase yield of recombinant proteins. Copyright 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 430-437, 2003.

Genetic architecture, inter-relationship and selection criteria for yield improvement in rice (Oryza sativa L.).

PubMed

Yadav, S K; Pandey, P; Kumar, B; Suresh, B G

2011-05-01

This study has been conducted to determine the extent of genetic association between yield of Rice (Oryza sativa L.) and its components. The present experiment was carried out with 40 Rice (Oryza sativa L.) genotypes which were evaluated in a randomized block design with 3 replications during wet season of 2007 and 2008. Results showed that sufficient amount of variability was found in the entire gene pool for all traits studied. Higher magnitude of genotypic and phenotypic coefficients of variation was recorded for seed yield, harvest index, biological yield, number of spikelets per panicle, flag leaf length, plant height and number of tillers indicates that these characters are least influence by environment. High heritability coupled with high genetic advance as percent of mean was registered for seed yield, harvest index, number of spikelets per panicle, biological yield and flag leaf length, suggesting preponderance of additive gene action in the expression of these characters. Grain yield was significantly and positively associated with harvest index, number of tillers per hill, number of panicle per plant, panicle length, number of spikelet's per panicle and test weight at both genotypic and phenotypic levels. Path coefficient analysis revealed that harvest index, biological yield, number of tillers per hill, panicle length, number of spikelets per panicle, plant height and test weight had direct positive effect on seed yield, indicating these are the main contributors to yield. From this study it may be concluded that harvest index, number of tillers per hill, panicle length and number of spikelet per panicle and test weight are the most important characters that contributed directly to yield. Thus, these characters may serve selection criteria for improving genetic potential of rice.

Assessing water-related plant traits to explain slow-wilting in soybean PI 471938

USDA-ARS?s Scientific Manuscript database

Exotic soybean accession PI 471938 from Nepal expresses a slow-wilting phenotype in the field, and the progeny of this genotype have been shown to have high yield under water-deficit conditions. However, the physiological basis for the slow-wilting trait in PI 471938 remains unclear and failure to ...

Identification and functional expression of ZIP1 transporter protein in Triticum dicoccoides

USDA-ARS?s Scientific Manuscript database

Zinc (Zn) deficiency is a common problem, especially in cereal-growing areas, leading to severe decreases in grain yield and nutritional quality. Among the cereal species, durum wheat is the most sensitive crop to Zn deficiency. One major reason for this high sensitivity of durum wheat is its poor ...

PAY1 improves plant architecture and enhances grain yield in rice.

PubMed

Zhao, Lei; Tan, Lubin; Zhu, Zuofeng; Xiao, Langtao; Xie, Daoxin; Sun, Chuanqing

2015-08-01

Plant architecture, a complex of the important agronomic traits that determine grain yield, is a primary target of artificial selection of rice domestication and improvement. Some important genes affecting plant architecture and grain yield have been isolated and characterized in recent decades; however, their underlying mechanism remains to be elucidated. Here, we report genetic identification and functional analysis of the PLANT ARCHITECTURE AND YIELD 1 (PAY1) gene in rice, which affects plant architecture and grain yield in rice. Transgenic plants over-expressing PAY1 had twice the number of grains per panicle and consequently produced nearly 38% more grain yield per plant than control plants. Mechanistically, PAY1 could improve plant architecture via affecting polar auxin transport activity and altering endogenous indole-3-acetic acid distribution. Furthermore, introgression of PAY1 into elite rice cultivars, using marker-assisted background selection, dramatically increased grain yield compared with the recipient parents. Overall, these results demonstrated that PAY1 could be a new beneficial genetic resource for shaping ideal plant architecture and breeding high-yielding rice varieties. © 2015 The Authors The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.

Development of an efficient E. coli expression and purification system for a catalytically active, human Cullin3-RINGBox1 protein complex and elucidation of its quaternary structure with Keap1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Small, Evan; Eggler, Aimee; Mesecar, Andrew D., E-mail: amesecar@purdue.edu

2010-10-01

Research highlights: {yields} A novel expression strategy was used to purify Cul3-Rbx1 from E. coli. {yields} The Cul3-Rbx1 complex is fully active and catalyzes ubiquitination of Nrf2 in vitro. {yields} Cul3, Rbx1, and Keap1 form a complex with unique stoichiometry of 1:1:2. -- Abstract: The Cullin3-based E3 ubiquitin ligase complex is thought to play an important role in the cellular response to oxidative stress and xenobiotic assault. While limited biochemical studies of the ligase's role in these complex signaling pathways are beginning to emerge, structural studies are lagging far behind due to the inability to acquire sufficient quantities of full-length,more » highly pure and active Cullin3. Here we describe the design and construction of an optimized expression and purification system for the full-length, human Cullin3-RINGBox 1 (Rbx1) protein complex from Escherichia coli. The dual-expression system is comprised of codon-optimized Cullin3 and Rbx1 genes co-expressed from a single pET-Duet-1 plasmid. Rapid purification of the Cullin3-Rbx1 complex is achieved in two steps via an affinity column followed by size-exclusion chromatography. Approximately 15 mg of highly pure and active Cullin3-Rbx1 protein from 1 L of E. coli culture can be achieved. Analysis of the quaternary structure of the Cullin3-Rbx1 and Cullin3-Rbx1-Keap1 complexes by size-exclusion chromatography and analytical ultracentrifugation indicates a 1:1 stoichiometry for the Cullin3-Rbx1 complex (MW = 111 kDa), and a 1:1:2 stoichiometry for the Cullin3-Rbx1-Keap1 complex (MW = 280 kDa). This latter complex has a novel quaternary structural organization for cullin E3 ligases, and it is fully active based on an in vitro Cullin3-Rbx1-Keap1-Nrf2 ubiquitination activity assay that was developed and optimized in this study.« less

Can parent training for parents with high levels of expressed emotion have a positive effect on their child's social anxiety improvement?

PubMed

Garcia-Lopez, Luis Joaquín; Díaz-Castela, Maria del Mar; Muela-Martinez, Jose Antonio; Espinosa-Fernandez, Lourdes

2014-12-01

The role that parents' involvement may play in improving their child's social anxiety is still under debate. This paper aimed to investigate whether training parents with high expressed emotion (EE) could improve outcomes for adolescent social anxiety intervention. Fifty-two socially anxious adolescents (aged 13-18 years), whose parents exhibited high levels of expressed emotion, were assigned to either (a) a school-based intervention with an added parent training component, or (b) a school-based program focused solely on intervening with the adolescent (no parental involvement). Post-treatment and 12-month follow-up findings showed that school-based intervention with parent training was superior to the adolescent-specific program, yielding significant reductions in diagnosis remission, social and depressive symptomatology, particularly when the EE status of parents changed. Overall, the findings suggest that high-EE parents of children with social anxiety need to be involved in their child's therapy. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kimura, Rino; Takahashi, Nobuyuki, E-mail: nobu@kais.kyoto-u.ac.jp; Murota, Kaeko

Highlights: {yields} PPAR{alpha} activation increased mRNA expression levels of fatty acid oxidation-related genes in human intestinal epithelial Caco-2 cells. {yields} PPAR{alpha} activation also increased oxygen consumption rate and CO{sub 2} production and decreased secretion of triglyceride and ApoB from Caco-2 cells. {yields} Orally administration of bezafibrate increased mRNA expression levels of fatty acid oxidation-related genes and CO{sub 2} production in small intestinal epithelial cells. {yields} Treatment with bezafibrate decreased postprandial serum concentration of triglyceride after oral injection of olive oil in mice. {yields} It suggested that intestinal lipid metabolism regulated by PPAR{alpha} activation suppresses postprandial lipidemia. -- Abstract: Activation ofmore » peroxisome proliferator-activated receptor (PPAR)-{alpha} which regulates lipid metabolism in peripheral tissues such as the liver and skeletal muscle, decreases circulating lipid levels, thus improving hyperlipidemia under fasting conditions. Recently, postprandial serum lipid levels have been found to correlate more closely to cardiovascular diseases than fasting levels, although fasting hyperlipidemia is considered an important risk of cardiovascular diseases. However, the effect of PPAR{alpha} activation on postprandial lipidemia has not been clarified. In this study, we examined the effects of PPAR{alpha} activation in enterocytes on lipid secretion and postprandial lipidemia. In Caco-2 enterocytes, bezafibrate, a potent PPAR{alpha} agonist, increased mRNA expression levels of fatty acid oxidation-related genes, such as acyl-CoA oxidase, carnitine palmitoyl transferase, and acyl-CoA synthase, and oxygen consumption rate (OCR) and suppressed secretion levels of both triglycerides and apolipoprotein B into the basolateral side. In vivo experiments revealed that feeding high-fat-diet containing bezafibrate increased mRNA expression levels of fatty acid oxidation-related genes and production of CO{sub 2} and acid soluble metabolites in enterocytes. Moreover, bezafibrate treatment suppressed postprandial lipidemia after oral administration of olive oil to the mice. These findings indicate that PPAR{alpha} activation suppresses postprandial lipidemia through enhancement of fatty acid oxidation in enterocytes, suggesting that intestinal lipid metabolism regulated by PPAR{alpha} activity is a novel target of PPAR{alpha} agonist for decreasing circulating levels of lipids under postprandial conditions.« less

Characterization of the in vitro expressed autoimmune rippling muscle disease immunogenic domain of human titin encoded by TTN exons 248-249

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zelinka, L.; McCann, S.; Budde, J.

2011-08-05

Highlights: {yields} Affinity purification of the autoimmune rippling muscle disease immunogenic domain of titin. {yields} Partial sequence analysis confirms that the peptides is in the I band region of titin. {yields} This region of the human titin shows high degree of homology to mouse titin N2-A. -- Abstract: Autoimmune rippling muscle disease (ARMD) is an autoimmune neuromuscular disease associated with myasthenia gravis (MG). Past studies in our laboratory recognized a very high molecular weight skeletal muscle protein antigen identified by ARMD patient antisera as the titin isoform. These past studies used antisera from ARMD and MG patients as probes tomore » screen a human skeletal muscle cDNA library and several pBluescript clones revealed supporting expression of immunoreactive peptides. This study characterizes the products of subcloning the titin immunoreactive domain into pGEX-3X and the subsequent fusion protein. Sequence analysis of the fusion gene indicates the cloned titin domain (GenBank ID: (EU428784)) is in frame and is derived from a sequence of N2-A spanning the exons 248-250 an area that encodes the fibronectin III domain. PCR and EcoR1 restriction mapping studies have demonstrated that the inserted cDNA is of a size that is predicted by bioinformatics analysis of the subclone. Expression of the fusion protein result in the isolation of a polypeptide of 52 kDa consistent with the predicted inferred amino acid sequence. Immunoblot experiments of the fusion protein, using rippling muscle/myasthenia gravis antisera, demonstrate that only the titin domain is immunoreactive.« less

Improved Method for Isolation of Microbial RNA from Biofuel Feedstock for Metatranscriptomics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Piao, Hailan; Markillie, Lye Meng; Culley, David E.

2013-03-28

Metatranscriptomics—gene express profiling via DNA sequencing—is a powerful tool to identify genes that are ac- tively expressed and might contribute to the phenotype of individual organisms or the phenome (the sum of several phenotypes) of a microbial community. Furthermore, metatranscriptome studies can result in extensive catalogues of genes that encode for enzymes of industrial relevance. In both cases, a major challenge for generating a high quality metatranscriptome is the extreme lability of RNA and its susceptibility to ubiquitous RNAses. The microbial commu- nity (the microbiome) of the cow rumen efficiently degrades lignocelullosic biomass, generates significant amounts of methane, a greenhousemore » gas twenty times more potent than carbon dioxide, and is of general importance for the physio- logical wellbeing of the host animal. Metatranscriptomes of the rumen microbiome from animals kept under different conditions and from various types of rumen-incubated biomass can be expected to provide new insights into these highly interesting phenotypes and subsequently provide the framework for an enhanced understanding of this socio- economically important ecosystem. The ability to isolate large amounts of intact RNA will significantly facilitate accu- rate transcript annotation and expression profiling. Here we report a method that combines mechanical disruption with chemical homogenization of the sample material and consistently yields 1 mg of intact RNA from 1 g of rumen-in- cubated biofuel feedstock. The yield of total RNA obtained with our method exceeds the RNA yield achieved with pre- viously reported isolation techniques, which renders RNA isolated with the method presented here as an ideal starting material for metatranscriptomic analyses and other molecular biology applications that require significant amounts of starting material.« less

Directed evolution and expression tuning of geraniol synthase for efficient geraniol production in Escherichia coli.

PubMed

Tashiro, Miki; Fujii, Akira; Kawai-Noma, Shigeko; Saito, Kyoichi; Umeno, Daisuke

2017-11-17

To achieve an efficient production of geraniol and its derivatives in Escherichia coli, we aimed to improve the activity of geraniol synthase (GES) through a single round of mutagenesis and screening for higher substrate consumption. We isolated GES variants that outperform their parent in geraniol production. The analysis of GES variants indicated that the expression level of GES was the bottleneck for geraniol synthesis. Over-expression of the mutant GES M53 with a 5'-untranslated sequence designed for high translational efficiency, along with the additional expression of mevalonate pathway enzymes, isopentenyl pyrophosphate isomerase, and geranyl pyrophosphate synthase, yielded 300 mg/L/12 h geraniol and its derivatives (>1000 mg/L/42 h in total) in a shaking flask.

Reconstruction of cytosolic fumaric acid biosynthetic pathways in Saccharomyces cerevisiae

PubMed Central

2012-01-01

Background Fumaric acid is a commercially important component of foodstuffs, pharmaceuticals and industrial materials, yet the current methods of production are unsustainable and ecologically destructive. Results In this study, the fumarate biosynthetic pathway involving reductive reactions of the tricarboxylic acid cycle was exogenously introduced in S. cerevisiae by a series of simple genetic modifications. First, the Rhizopus oryzae genes for malate dehydrogenase (RoMDH) and fumarase (RoFUM1) were heterologously expressed. Then, expression of the endogenous pyruvate carboxylase (PYC2) was up-regulated. The resultant yeast strain, FMME-001 ↑PYC2 + ↑RoMDH, was capable of producing significantly higher yields of fumarate in the glucose medium (3.18 ± 0.15 g liter-1) than the control strain FMME-001 empty vector. Conclusions The results presented here provide a novel strategy for fumarate biosynthesis, which represents an important advancement in producing high yields of fumarate in a sustainable and ecologically-friendly manner. PMID:22335940

Heterologous laccase production and its role in industrial applications

PubMed Central

Pezzella, Cinzia; Giardina, Paola; Faraco, Vincenza; Sannia, Giovanni

2010-01-01

Laccases are blue multicopper oxidases, catalyzing the oxidation of an array of aromatic substrates concomitantly with the reduction of molecular oxygen to water. These enzymes are implicated in a variety of biological activities. Most of the laccases studied thus far are of fungal origin. The large range of substrates oxidized by laccases has raised interest in using them within different industrial fields, such as pulp delignification, textile dye bleaching and bioremediation. Laccases secreted from native sources are usually not suitable for large-scale purposes, mainly due to low production yields and high cost of preparation/purification procedures. Heterologous expression may provide higher enzyme yields and may permit to produce laccases with desired properties (such as different substrate specificities, or improved stabilities) for industrial applications. This review surveys researches on heterologous laccase expression focusing on the pivotal role played by recombinant systems towards the development of robust tools for greening modern industry. PMID:21327057

Automated Structure- and Sequence-Based Design of Proteins for High Bacterial Expression and Stability.

PubMed

Goldenzweig, Adi; Goldsmith, Moshe; Hill, Shannon E; Gertman, Or; Laurino, Paola; Ashani, Yacov; Dym, Orly; Unger, Tamar; Albeck, Shira; Prilusky, Jaime; Lieberman, Raquel L; Aharoni, Amir; Silman, Israel; Sussman, Joel L; Tawfik, Dan S; Fleishman, Sarel J

2016-07-21

Upon heterologous overexpression, many proteins misfold or aggregate, thus resulting in low functional yields. Human acetylcholinesterase (hAChE), an enzyme mediating synaptic transmission, is a typical case of a human protein that necessitates mammalian systems to obtain functional expression. We developed a computational strategy and designed an AChE variant bearing 51 mutations that improved core packing, surface polarity, and backbone rigidity. This variant expressed at ∼2,000-fold higher levels in E. coli compared to wild-type hAChE and exhibited 20°C higher thermostability with no change in enzymatic properties or in the active-site configuration as determined by crystallography. To demonstrate broad utility, we similarly designed four other human and bacterial proteins. Testing at most three designs per protein, we obtained enhanced stability and/or higher yields of soluble and active protein in E. coli. Our algorithm requires only a 3D structure and several dozen sequences of naturally occurring homologs, and is available at http://pross.weizmann.ac.il. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Purification of Single-Stranded cDNA Based on RNA Degradation Treatment and Adsorption Chromatography.

PubMed

Trujillo-Esquivel, Elías; Franco, Bernardo; Flores-Martínez, Alberto; Ponce-Noyola, Patricia; Mora-Montes, Héctor M

2016-08-02

Analysis of gene expression is a common research tool to study networks controlling gene expression, the role of genes with unknown function, and environmentally induced responses of organisms. Most of the analytical tools used to analyze gene expression rely on accurate cDNA synthesis and quantification to obtain reproducible and quantifiable results. Thus far, most commercial kits for isolation and purification of cDNA target double-stranded molecules, which do not accurately represent the abundance of transcripts. In the present report, we provide a simple and fast method to purify single-stranded cDNA, exhibiting high purity and yield. This method is based on the treatment with RNase H and RNase A after cDNA synthesis, followed by separation in silica spin-columns and ethanol precipitation. In addition, our method avoids the use of DNase I to eliminate genomic DNA from RNA preparations, which improves cDNA yield. As a case report, our method proved to be useful in the purification of single-stranded cDNA from the pathogenic fungus Sporothrix schenckii.

Estimates of genetics and phenotypics parameters for the yield and quality of soybean seeds.

PubMed

Zambiazzi, E V; Bruzi, A T; Guilherme, S R; Pereira, D R; Lima, J G; Zuffo, A M; Ribeiro, F O; Mendes, A E S; Godinho, S H M; Carvalho, M L M

2017-09-27

Estimating genotype x environment (GxE) parameters for quality and yield in soybean seed grown in different environments in Minas Gerais State was the goal of this study, as well as to evaluate interaction effects of GxE for soybean seeds yield and quality. Seeds were produced in three locations in Minas Gerais State (Lavras, Inconfidentes, and Patos de Minas) in 2013/14 and 2014/15 seasons. Field experiments were conducted in randomized blocks in a factorial 17 x 6 (GxE), and three replications. Seed yield and quality were evaluated for germination in substrates paper and sand, seedling emergence, speed emergency index, mechanical damage by sodium hypochlorite, electrical conductivity, speed aging, vigor and viability of seeds by tetrazolium test in laboratory using completely randomized design. Quadratic component genotypic, GXE variance component, genotype determination coefficient, genetic variation coefficient and environmental variation coefficient were estimated using the Genes software. Percentage analysis of genotypes contribution, environments and genotype x environment interaction were conducted by sites combination two by two and three sites combination, using the R software. Considering genotypes selection of broad adaptation, TMG 1179 RR, CD 2737 RR, and CD 237 RR associated better yield performance at high physical and physiological potential of seed. Environmental effect was more expressive for most of the characters related to soybean seed quality. GxE interaction effects were expressive though genotypes did not present coincidental behavior in different environments.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sugi, Yutaka; Takahashi, Kyoko, E-mail: ktaka@brs.nihon-u.ac.jp; Nakano, Kou

Highlights: {yields} Transcriptional activation of the Tollitip gene is higher in IECs than in monocytes. {yields} Nt -194/-186 region acts as a cis-element and is recognized by Elf-1. {yields} Elf-1 suppresses Tollip gene transcription in monocytes but not in IECs. {yields} O-GlcNAc modification is necessary for nuclear translocation of Elf-1. {yields} O-GlcNAcylation-dependent nuclear translocation of Elf-1 is impaired in IECs. -- Abstract: Intestinal epithelial cells (IECs) must be tolerant of the large number of commensal bacteria inhabiting the intestinal tract to avoid excessive inflammatory reactions. Toll-interacting protein (Tollip), a negative regulator of Toll-like receptor signaling, is known to be expressedmore » at high levels in IECs, and to thereby contribute to the hyporesponsiveness of IECs to commensals. In this study, we analyzed the underlying mechanisms for elevated transcription of the Tollip gene in IECs using a human IEC line, Caco-2, and a human monocyte line, THP-1, as a control. Elf-1 was identified as a transcription factor that negatively regulates Tollip gene expression. The transcription factor Elf-1 was localized in the nucleus by O-linked N-acetylglucosamine (O-GlcNAc) modification, whereas the unmodified form was detected only in the cytoplasm. Comparison of Caco-2 and THP-1 cells revealed that O-GlcNAc modification of Elf-1 was significantly lower in IECs than in monocytes. Collectively, the results indicate that insufficient O-GlcNAc modification prevents Elf-1-mediated transcriptional repression and thereby upregulates Tollip gene expression in IECs.« less

Expression of POEM, a positive regulator of osteoblast differentiation, is suppressed by TNF-{alpha}

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tsukasaki, Masayuki; Yamada, Atsushi, E-mail: yamadaa@dent.showa-u.ac.jp; Suzuki, Dai

2011-07-15

Highlights: {yields} TNF-{alpha} inhibits POEM gene expression. {yields} Inhibition of POEM gene expression is caused by NF-{kappa}B activation by TNF-{alpha}. {yields} Over-expression of POEM recovers inhibition of osteoblast differentiation by TNF-{alpha}. -- Abstract: POEM, also known as nephronectin, is an extracellular matrix protein considered to be a positive regulator of osteoblast differentiation. In the present study, we found that tumor necrosis factor-{alpha} (TNF-{alpha}), a key regulator of bone matrix properties and composition that also inhibits terminal osteoblast differentiation, strongly inhibited POEM expression in the mouse osteoblastic cell line MC3T3-E1. TNF-{alpha}-induced down-regulation of POEM gene expression occurred in both time- andmore » dose-dependent manners through the nuclear factor kappa B (NF-{kappa}B) pathway. In addition, expressions of marker genes in differentiated osteoblasts were down-regulated by TNF-{alpha} in a manner consistent with our findings for POEM, while over-expression of POEM recovered TNF-{alpha}-induced inhibition of osteoblast differentiation. These results suggest that TNF-{alpha} inhibits POEM expression through the NF-{kappa}B signaling pathway and down-regulation of POEM influences the inhibition of osteoblast differentiation by TNF-{alpha}.« less

Optimization of the Production Process and Characterization of the Yeast-Expressed SARS-CoV Recombinant Receptor-Binding Domain (RBD219-N1), a SARS Vaccine Candidate.

PubMed

Chen, Wen-Hsiang; Chag, Shivali M; Poongavanam, Mohan V; Biter, Amadeo B; Ewere, Ebe A; Rezende, Wanderson; Seid, Christopher A; Hudspeth, Elissa M; Pollet, Jeroen; McAtee, C Patrick; Strych, Ulrich; Bottazzi, Maria Elena; Hotez, Peter J

2017-08-01

From 2002 to 2003, a global pandemic of severe acute respiratory syndrome (SARS) spread to 5 continents and caused 8000 respiratory infections and 800 deaths. To ameliorate the effects of future outbreaks as well as to prepare for biodefense, a process for the production of a recombinant protein vaccine candidate is under development. Previously, we reported the 5 L scale expression and purification of a promising recombinant SARS vaccine candidate, RBD219-N1, the 218-amino acid residue receptor-binding domain (RBD) of SARS coronavirus expressed in yeast-Pichia pastoris X-33. When adjuvanted with aluminum hydroxide, this protein elicited high neutralizing antibody titers and high RBD-specific antibody titers. However, the yield of RBD219-N1 (60 mg RBD219-N1 per liter of fermentation supernatant; 60 mg/L FS) still required improvement to reach our target of >100 mg/L FS. In this study, we optimized the 10 L scale production process and increased the fermentation yield 6- to 7-fold to 400 mg/L FS with purification recovery >50%. A panel of characterization tests indicated that the process is reproducible and that the purified, tag-free RBD219-N1 protein has high purity and a well-defined structure and is therefore a suitable candidate for production under current Good Manufacturing Practice and future phase-1 clinical trials. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Identification of Useful Nanobodies by Phage Display of Immune Single Domain Libraries Derived from Camelid Heavy Chain Antibodies.

PubMed

Romao, Ema; Morales-Yanez, Francisco; Hu, Yaozhong; Crauwels, Maxine; De Pauw, Pieter; Hassanzadeh, Gholamreza Ghassanzadeh; Devoogdt, Nick; Ackaert, Chloe; Vincke, Cecile; Muyldermans, Serge

2016-01-01

The discovery of functional heavy chain-only antibodies devoid of light chains in sera of camelids and sharks in the early nineties provided access to the generation of minimal-sized, single-domain, in vivo affinity-matured, recombinant antigenbinding fragments, also known as Nanobodies. Recombinant DNA technology and adaptation of phage display vectors form the basis to construct large naïve, synthetic or medium sized immune libraries from where multiple Nanobodies have been retrieved. Alternative selection methods (i.e. bacterial display, bacterial two-hybrid, Cis-display and ribosome display) have also been developed to identify Nanobodies. The antigen affinity, stability, expression yields and structural details of the Nanobodies have been determined by standard technology. Nanobodies were subsequently engineered for higher stability and affinity, to have a sequence closer to that of human immunoglobulin domains, or to add designed effector functions. Antigen specific Nanobodies recognizing with high affinity their cognate antigen were retrieved from various libraries. High expression yields are obtained from microorganisms, even when expressed in the cytoplasm. The purified Nanobodies are shown to possess beneficial biochemical and biophysical properties. The crystal structure of Nanobody::antigen complexes reveal the preference of Nanobodies for cavities on the antigen surface. Thanks to the properties described above, Nanobodies became a highly valued and versatile tool for biomolecular research. Moreover, numerous diagnostic and therapeutic Nanobody-based applications have been developed in the past decade. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Optimal Scaling of Digital Transcriptomes

PubMed Central

Glusman, Gustavo; Caballero, Juan; Robinson, Max; Kutlu, Burak; Hood, Leroy

2013-01-01

Deep sequencing of transcriptomes has become an indispensable tool for biology, enabling expression levels for thousands of genes to be compared across multiple samples. Since transcript counts scale with sequencing depth, counts from different samples must be normalized to a common scale prior to comparison. We analyzed fifteen existing and novel algorithms for normalizing transcript counts, and evaluated the effectiveness of the resulting normalizations. For this purpose we defined two novel and mutually independent metrics: (1) the number of “uniform” genes (genes whose normalized expression levels have a sufficiently low coefficient of variation), and (2) low Spearman correlation between normalized expression profiles of gene pairs. We also define four novel algorithms, one of which explicitly maximizes the number of uniform genes, and compared the performance of all fifteen algorithms. The two most commonly used methods (scaling to a fixed total value, or equalizing the expression of certain ‘housekeeping’ genes) yielded particularly poor results, surpassed even by normalization based on randomly selected gene sets. Conversely, seven of the algorithms approached what appears to be optimal normalization. Three of these algorithms rely on the identification of “ubiquitous” genes: genes expressed in all the samples studied, but never at very high or very low levels. We demonstrate that these include a “core” of genes expressed in many tissues in a mutually consistent pattern, which is suitable for use as an internal normalization guide. The new methods yield robustly normalized expression values, which is a prerequisite for the identification of differentially expressed and tissue-specific genes as potential biomarkers. PMID:24223126

Expression of codon-optmized phosphoenolpyruvate carboxylase gene from Glaciecola sp. HTCC2999 in Escherichia coli and its application for C4 chemical production.

PubMed

Park, Soohyun; Pack, Seung Pil; Lee, Jinwon

2012-08-01

We examined the expression of the phosphoenolpyruvate carboxylase (PEPC) gene from marine bacteria in Escherichia coli using codon optimization. The codon-optimized PEPC gene was expressed in the E. coli K-12 strain W3110. SDS-PAGE analysis revealed that the codon-optimized PEPC gene was only expressed in E. coli, and measurement of enzyme activity indicated the highest PEPC activity in the E. coli SGJS112 strain that contained the codon-optimized PEPC gene. In fermentation assays, the E. coli SGJS112 produced the highest yield of oxaloacetate using glucose as the source and produced a 20-times increase in the yield of malate compared to the control. We concluded that the codon optimization enabled E. coli to express the PEPC gene derived from the Glaciecola sp. HTCC2999. Also, the expressed protein exhibited an enzymatic activity similar to that of E. coli PEPC and increased the yield of oxaloacetate and malate in an E. coli system.

Engineering Camelina sativa (L.) Crantz for enhanced oil and seed yields by combining diacylglycerol acyltransferase1 and glycerol-3-phosphate dehydrogenase expression.

PubMed

Chhikara, Sudesh; Abdullah, Hesham M; Akbari, Parisa; Schnell, Danny; Dhankher, Om Parkash

2018-05-01

Plant seed oil-based liquid transportation fuels (i.e., biodiesel and green diesel) have tremendous potential as environmentally, economically and technologically feasible alternatives to petroleum-derived fuels. Due to their nutritional and industrial importance, one of the major objectives is to increase the seed yield and oil production of oilseed crops via biotechnological approaches. Camelina sativa, an emerging oilseed crop, has been proposed as an ideal crop for biodiesel and bioproduct applications. Further increase in seed oil yield by increasing the flux of carbon from increased photosynthesis into triacylglycerol (TAG) synthesis will make this crop more profitable. To increase the oil yield, we engineered Camelina by co-expressing the Arabidopsis thaliana (L.) Heynh. diacylglycerol acyltransferase1 (DGAT1) and a yeast cytosolic glycerol-3-phosphate dehydrogenase (GPD1) genes under the control of seed-specific promoters. Plants co-expressing DGAT1 and GPD1 exhibited up to 13% higher seed oil content and up to 52% increase in seed mass compared to wild-type plants. Further, DGAT1- and GDP1-co-expressing lines showed significantly higher seed and oil yields on a dry weight basis than the wild-type controls or plants expressing DGAT1 and GPD1 alone. The oil harvest index (g oil per g total dry matter) for DGTA1- and GPD1-co-expressing lines was almost twofold higher as compared to wild type and the lines expressing DGAT1 and GPD1 alone. Therefore, combining the overexpression of TAG biosynthetic genes, DGAT1 and GPD1, appears to be a positive strategy to achieve a synergistic effect on the flux through the TAG synthesis pathway, and thereby further increase the oil yield. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Strategies for achieving high-level expression of genes in Escherichia coli.

PubMed Central

Makrides, S C

1996-01-01

Progress in our understanding of several biological processes promises to broaden the usefulness of Escherichia coli as a tool for gene expression. There is an expanding choice of tightly regulated prokaryotic promoters suitable for achieving high-level gene expression. New host strains facilitate the formation of disulfide bonds in the reducing environment of the cytoplasm and offer higher protein yields by minimizing proteolytic degradation. Insights into the process of protein translocation across the bacterial membranes may eventually make it possible to achieve robust secretion of specific proteins into the culture medium. Studies involving molecular chaperones have shown that in specific cases, chaperones can be very effective for improved protein folding, solubility, and membrane transport. Negative results derived from such studies are also instructive in formulating different strategies. The remarkable increase in the availability of fusion partners offers a wide range of tools for improved protein folding, solubility, protection from proteases, yield, and secretion into the culture medium, as well as for detection and purification of recombinant proteins. Codon usage is known to present a potential impediment to high-level gene expression in E. coli. Although we still do not understand all the rules governing this phenomenon, it is apparent that "rare" codons, depending on their frequency and context, can have an adverse effect on protein levels. Usually, this problem can be alleviated by modification of the relevant codons or by coexpression of the cognate tRNA genes. Finally, the elucidation of specific determinants of protein degradation, a plethora of protease-deficient host strains, and methods to stabilize proteins afford new strategies to minimize proteolytic susceptibility of recombinant proteins in E. coli. PMID:8840785

Expression Variants of the Lipogenic AGPAT6 Gene Affect Diverse Milk Composition Phenotypes in Bos taurus

PubMed Central

Littlejohn, Mathew D.; Tiplady, Kathryn; Lopdell, Thomas; Law, Tania A.; Scott, Andrew; Harland, Chad; Sherlock, Ric; Henty, Kristen; Obolonkin, Vlad; Lehnert, Klaus; MacGibbon, Alistair; Spelman, Richard J.; Davis, Stephen R.; Snell, Russell G.

2014-01-01

Milk is composed of a complex mixture of lipids, proteins, carbohydrates and various vitamins and minerals as a source of nutrition for young mammals. The composition of milk varies between individuals, with lipid composition in particular being highly heritable. Recent reports have highlighted a region of bovine chromosome 27 harbouring variants affecting milk fat percentage and fatty acid content. We aimed to further investigate this locus in two independent cattle populations, consisting of a Holstein-Friesian x Jersey crossbreed pedigree of 711 F2 cows, and a collection of 32,530 mixed ancestry Bos taurus cows. Bayesian genome-wide association mapping using markers imputed from the Illumina BovineHD chip revealed a large quantitative trait locus (QTL) for milk fat percentage on chromosome 27, present in both populations. We also investigated a range of other milk composition phenotypes, and report additional associations at this locus for fat yield, protein percentage and yield, lactose percentage and yield, milk volume, and the proportions of numerous milk fatty acids. We then used mammary RNA sequence data from 212 lactating cows to assess the transcript abundance of genes located in the milk fat percentage QTL interval. This analysis revealed a strong eQTL for AGPAT6, demonstrating that high milk fat percentage genotype is also additively associated with increased expression of the AGPAT6 gene. Finally, we used whole genome sequence data from six F1 sires to target a panel of novel AGPAT6 locus variants for genotyping in the F2 crossbreed population. Association analysis of 58 of these variants revealed highly significant association for polymorphisms mapping to the 5′UTR exons and intron 1 of AGPAT6. Taken together, these data suggest that variants affecting the expression of AGPAT6 are causally involved in differential milk fat synthesis, with pleiotropic consequences for a diverse range of other milk components. PMID:24465687

Ghrelin inhibits proliferation and increases T-type Ca{sup 2+} channel expression in PC-3 human prostate carcinoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Diaz-Lezama, Nundehui; Hernandez-Elvira, Mariana; Sandoval, Alejandro

Research highlights: {yields} Ghrelin decreases prostate carcinoma PC-3 cells proliferation. {yields} Ghrelin favors apoptosis in PC-3 cells. {yields} Ghrelin increase in intracellular free Ca{sup 2+} levels in PC-3 cells. {yields} Grelin up-regulates expression of T-type Ca{sup 2+} channels in PC-3 cells. {yields} PC-3 cells express T-channels of the Ca{sub V}3.1 and Ca{sub V}3.2 subtype. -- Abstract: Ghrelin is a multifunctional peptide hormone with roles in growth hormone release, food intake and cell proliferation. With ghrelin now recognized as important in neoplastic processes, the aim of this report is to present findings from a series of in vitro studies evaluating themore » cellular mechanisms involved in ghrelin regulation of proliferation in the PC-3 human prostate carcinoma cells. The results showed that ghrelin significantly decreased proliferation and induced apoptosis. Consistent with a role in apoptosis, an increase in intracellular free Ca{sup 2+} levels was observed in the ghrelin-treated cells, which was accompanied by up-regulated expression of T-type voltage-gated Ca{sup 2+} channels. Interestingly, T-channel antagonists were able to prevent the effects of ghrelin on cell proliferation. These results suggest that ghrelin inhibits proliferation and may promote apoptosis by regulating T-type Ca{sup 2+} channel expression.« less

Seasonal Changes in Bacterial and Archaeal Gene Expression Patterns across Salinity Gradients in the Columbia River Coastal Margin

PubMed Central

Smith, Maria W.; Herfort, Lydie; Tyrol, Kaitlin; Suciu, Dominic; Campbell, Victoria; Crump, Byron C.; Peterson, Tawnya D.; Zuber, Peter; Baptista, Antonio M.; Simon, Holly M.

2010-01-01

Through their metabolic activities, microbial populations mediate the impact of high gradient regions on ecological function and productivity of the highly dynamic Columbia River coastal margin (CRCM). A 2226-probe oligonucleotide DNA microarray was developed to investigate expression patterns for microbial genes involved in nitrogen and carbon metabolism in the CRCM. Initial experiments with the environmental microarrays were directed toward validation of the platform and yielded high reproducibility in multiple tests. Bioinformatic and experimental validation also indicated that >85% of the microarray probes were specific for their corresponding target genes and for a few homologs within the same microbial family. The validated probe set was used to query gene expression responses by microbial assemblages to environmental variability. Sixty-four samples from the river, estuary, plume, and adjacent ocean were collected in different seasons and analyzed to correlate the measured variability in chemical, physical and biological water parameters to differences in global gene expression profiles. The method produced robust seasonal profiles corresponding to pre-freshet spring (April) and late summer (August). Overall relative gene expression was high in both seasons and was consistent with high microbial abundance measured by total RNA, heterotrophic bacterial production, and chlorophyll a. Both seasonal patterns involved large numbers of genes that were highly expressed relative to background, yet each produced very different gene expression profiles. April patterns revealed high differential gene expression in the coastal margin samples (estuary, plume and adjacent ocean) relative to freshwater, while little differential gene expression was observed along the river-to-ocean transition in August. Microbial gene expression profiles appeared to relate, in part, to seasonal differences in nutrient availability and potential resource competition. Furthermore, our results suggest that highly-active particle-attached microbiota in the Columbia River water column may perform dissimilatory nitrate reduction (both dentrification and DNRA) within anoxic particle microniches. PMID:20967204

Expression of the human blood coagulation protein factor XIIIa in Saccharomyces cerevisiae: dependence of the expression levels from host-vector systems and medium conditions.

PubMed

Bröker, M; Bäuml, O; Göttig, A; Ochs, J; Bodenbenner, M; Amann, E

1991-03-01

The human blood coagulation protein Factor XIIIa (FXIIIa) was expressed in Saccharomyces cerevisiae employing Escherichia coli-yeast shuttle vectors based on a 2-mu plasmid. Several factors affecting high production yield of recombinant FXIIIa were analysed. The use of the regulatable GAL-CYC1 hybrid promoter resulted in higher FXIIIa expression when compared with the constitutive ADCI promoter. Screening for suitable yeast strains for expression of FXIIIa under the transcriptional control of the GAL-CYC1 hybrid promoter revealed a broad spectrum of productivity. No obvious correlation between the expression rate and the genetic markers of the strains could be identified. The medium composition markedly influenced the FXIIIa expression rates. The expression of FXIIIa was strictly regulated by the carbon source. Glucose as the only sugar and energy source repressed the synthesis of FXIIIa, whereas addition of galactose induced FXIIIa expression. Special feeding schemes resulted in a productivity of up to 100 mg FXIIIa/l in shake flasks.

Highly efficient mesophyll protoplast isolation and PEG-mediated transient gene expression for rapid and large-scale gene characterization in cassava (Manihot esculenta Crantz).

PubMed

Wu, Jun-Zheng; Liu, Qin; Geng, Xiao-Shan; Li, Kai-Mian; Luo, Li-Juan; Liu, Jin-Ping

2017-03-14

Cassava (Manihot esculenta Crantz) is a major crop extensively cultivated in the tropics as both an important source of calories and a promising source for biofuel production. Although stable gene expression have been used for transgenic breeding and gene function study, a quick, easy and large-scale transformation platform has been in urgent need for gene functional characterization, especially after the cassava full genome was sequenced. Fully expanded leaves from in vitro plantlets of Manihot esculenta were used to optimize the concentrations of cellulase R-10 and macerozyme R-10 for obtaining protoplasts with the highest yield and viability. Then, the optimum conditions (PEG4000 concentration and transfection time) were determined for cassava protoplast transient gene expression. In addition, the reliability of the established protocol was confirmed for subcellular protein localization. In this work we optimized the main influencing factors and developed an efficient mesophyll protoplast isolation and PEG-mediated transient gene expression in cassava. The suitable enzyme digestion system was established with the combination of 1.6% cellulase R-10 and 0.8% macerozyme R-10 for 16 h of digestion in the dark at 25 °C, resulting in the high yield (4.4 × 10 7 protoplasts/g FW) and vitality (92.6%) of mesophyll protoplasts. The maximum transfection efficiency (70.8%) was obtained with the incubation of the protoplasts/vector DNA mixture with 25% PEG4000 for 10 min. We validated the applicability of the system for studying the subcellular localization of MeSTP7 (an H + /monosaccharide cotransporter) with our transient expression protocol and a heterologous Arabidopsis transient gene expression system. We optimized the main influencing factors and developed an efficient mesophyll protoplast isolation and transient gene expression in cassava, which will facilitate large-scale characterization of genes and pathways in cassava.

Retroviral vectors encoding ADA regulatory locus control region provide enhanced T-cell-specific transgene expression.

PubMed

Trinh, Alice T; Ball, Bret G; Weber, Erin; Gallaher, Timothy K; Gluzman-Poltorak, Zoya; Anderson, French; Basile, Lena A

2009-12-30

Murine retroviral vectors have been used in several hundred gene therapy clinical trials, but have fallen out of favor for a number of reasons. One issue is that gene expression from viral or internal promoters is highly variable and essentially unregulated. Moreover, with retroviral vectors, gene expression is usually silenced over time. Mammalian genes, in contrast, are characterized by highly regulated, precise levels of expression in both a temporal and a cell-specific manner. To ascertain if recapitulation of endogenous adenosine deaminase (ADA) expression can be achieved in a vector construct we created a new series of Moloney murine leukemia virus (MuLV) based retroviral vector that carry human regulatory elements including combinations of the ADA promoter, the ADA locus control region (LCR), ADA introns and human polyadenylation sequences in a self-inactivating vector backbone. A MuLV-based retroviral vector with a self-inactivating (SIN) backbone, the phosphoglycerate kinase promoter (PGK) and the enhanced green fluorescent protein (eGFP), as a reporter gene, was generated. Subsequent vectors were constructed from this basic vector by deletion or addition of certain elements. The added elements that were assessed are the human ADA promoter, human ADA locus control region (LCR), introns 7, 8, and 11 from the human ADA gene, and human growth hormone polyadenylation signal. Retroviral vector particles were produced by transient three-plasmid transfection of 293T cells. Retroviral vectors encoding eGFP were titered by transducing 293A cells, and then the proportion of GFP-positive cells was determined using fluorescence-activated cell sorting (FACS). Non T-cell and T-cell lines were transduced at a multiplicity of infection (MOI) of 0.1 and the yield of eGFP transgene expression was evaluated by FACS analysis using mean fluorescent intensity (MFI) detection. Vectors that contained the ADA LCR were preferentially expressed in T-cell lines. Further improvements in T-cell specific gene expression were observed with the incorporation of additional cis-regulatory elements, such as a human polyadenylation signal and intron 7 from the human ADA gene. These studies suggest that the combination of an authentically regulated ADA gene in a murine retroviral vector, together with additional locus-specific regulatory refinements, will yield a vector with a safer profile and greater efficacy in terms of high-level, therapeutic, regulated gene expression for the treatment of ADA-deficient severe combined immunodeficiency.

Phosphatidylinositol 5-phosphate 4-kinase type II beta is required for vitamin D receptor-dependent E-cadherin expression in SW480 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kouchi, Zen, E-mail: zkouchi@toyaku.ac.jp; Fujiwara, Yuki; Yamaguchi, Hideki

2011-05-20

Highlights: {yields} We analyzed Phosphatidylinositol 5-phosphate kinase II{beta} (PIPKII{beta}) function in cancer. {yields} PIPKII{beta} is required for vitamin D receptor-mediated E-cadherin upregulation in SW480. {yields} PIPKII{beta} suppresses cellular motility through E-cadherin induction in SW480 cells. {yields} Nuclear PIP{sub 2} but not plasma membrane-localized PIP{sub 2} mediates E-cadherin upregulation. -- Abstract: Numerous epidemiological data indicate that vitamin D receptor (VDR) signaling induced by its ligand or active metabolite 1{alpha},25-dihydroxyvitamin D{sub 3} (1{alpha},25(OH){sub 2}D{sub 3}) has anti-cancer activity in several colon cancers. 1{alpha},25(OH){sub 2}D{sub 3} induces the epithelial differentiation of SW480 colon cancer cells expressing VDR (SW480-ADH) by upregulating E-cadherin expression; however,more » its precise mechanism remains unknown. We found that phosphatidylinositol-5-phosphate 4-kinase type II beta (PIPKII{beta}) but not PIPKII{alpha} is required for VDR-mediated E-cadherin induction in SW480-ADH cells. The syntenin-2 postsynaptic density protein/disc large/zona occludens (PDZ) domain and pleckstrin homology domain of phospholipase C-delta1 (PLC{delta}1 PHD) possess high affinity for phosphatidylinositol-4,5-bisphosphate (PI(4,5)P{sub 2}) mainly localized to the nucleus and plasma membrane, respectively. The expression of syntenin-2 PDZ but not PLC{delta}1 PHD inhibited 1{alpha},25(OH){sub 2}D{sub 3}-induced E-cadherin upregulation, suggesting that nuclear PI(4,5)P{sub 2} production mediates E-cadherin expression through PIPKII{beta} in a VDR-dependent manner. PIPKII{beta} is also involved in the suppression of the cell motility induced by 1{alpha},25(OH){sub 2}D{sub 3}. These results indicate that PIPKII{beta}-mediated PI(4,5)P{sub 2} signaling is important for E-cadherin upregulation and inhibition of cellular motility induced by VDR activation.« less

Loss of DLK expression in WI-38 human diploid fibroblasts induces a senescent-like proliferation arrest

DOE Office of Scientific and Technical Information (OSTI.GOV)

Daviau, Alex; Couture, Jean-Philippe; Blouin, Richard, E-mail: Richard.Blouin@USherbrooke.ca

Highlights: {yields} Role of DLK in cell proliferation. {yields} Modulation of DLK expression during cell cycle progression. {yields} DLK knockdown induces proliferation arrest and senescence. {yields} DLK-depleted cells display loss of cyclin D1 and up-regulation of p21. {yields} DLK participates in cell proliferation by modulating cell cycle regulator expression. -- Abstract: DLK, a serine/threonine kinase that functions as an upstream activator of the mitogen-activated protein kinase (MAPK) pathways, has been shown to play a role in development, cell differentiation, apoptosis and neuronal response to injury. Interestingly, recent studies have shown that DLK may also be required for cell proliferation, althoughmore » little is known about its specific functions. To start addressing this issue, we studied how DLK expression is modulated during cell cycle progression and what effect DLK depletion has on cell proliferation in WI-38 fibroblasts. Our results indicate that DLK protein levels are low in serum-starved cells, but that serum addition markedly stimulated it. Moreover, RNA interference experiments demonstrate that DLK is required for ERK activity, expression of the cell cycle regulator cyclin D1 and proliferation of WI-38 cells. DLK-depleted cells also show a senescent phenotype as revealed by senescence-associated galactosidase activity and up-regulation of the senescence pathway proteins p53 and p21. Consistent with a role for p53 in this response, inhibition of p53 expression by RNA interference significantly alleviated senescence induced by DLK knockdown. Together, these findings indicate that DLK participates in cell proliferation and/or survival, at least in part, by modulating the expression of cell cycle regulatory proteins.« less

Over-expression of 3-hydroxy-3- methylglutaryl-coenzyme A reductase 1 (hmgr1) gene under super-promoter for enhanced latex biosynthesis in rubber tree (Hevea brasiliensis Muell. Arg.).

PubMed

Jayashree, R; Nazeem, P A; Rekha, K; Sreelatha, S; Thulaseedharan, A; Krishnakumar, R; Kala, R G; Vineetha, M; Leda, P; Jinu, U; Venkatachalam, P

2018-06-01

Natural rubber (cis-1, 4-polyisoprene) is being produced from bark laticifer cells of Hevea brasiliensis and the popular high latex yielding Indian rubber clones are easily prone to onset of tapping panel dryness syndrome (TPD) which is considered as a physiological syndrome affecting latex production either partially or completely. This report describes an efficient protocol for development of transgenic rubber plants by over-expression of 3-hydroxy 3-methylglutaryl Co-enzyme A reductase 1 (hmgr1) gene which is considered as rate limiting factor for latex biosynthesis via Agrobacterium-mediated transformation. The pBIB plasmid vector containing hmgr1 gene cloned under the control of a super-promoter was used for genetic transformation using embryogenic callus. Putatively transgenic cell lines were obtained on selection medium and produced plantlets with 44% regeneration efficiency. Transgene integration was confirmed by PCR amplification of 1.8 kb hmgr1 and 0.6 kb hpt genes from all putatively transformed callus lines as well as transgenic plants. Southern blot analysis showed the stable integration and presence of transgene in the transgenic plants. Over expression of hmgr1 transgene was determined by Northern blot hybridization, semi-quantitative PCR and real-time PCR (qRT-PCR) analysis. Accumulation of hmgr1 mRNA transcripts was more abundant in transgenic plants than control. Increased level of photosynthetic pigments, protein contents and HMGR enzyme activity was also noticed in transgenic plants over control. Interestingly, the latex yield was significantly enhanced in all transgenic plants compared to the control. The qRT-PCR results exhibit that the hmgr1 mRNA transcript levels was 160-fold more abundance in transgenic plants over untransformed control. These results altogether suggest that there is a positive correlation between latex yield and accumulation of mRNA transcripts level as well as HMGR enzyme activity in transgenic rubber plants. It is presumed that there is a possibility for enhanced level of latex biosynthesis in transgenic plants as the level of mRNA transcripts and HMGR enzyme activity is directly correlated with latex yield in rubber tree. Further, the present results clearly suggest that the quantification of HMGR enzyme activity in young seedlings will be highly beneficial for early selection of high latex yielding plants in rubber breeding programs. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Molecular Design of Performance Proteins With Repetitive Sequences

NASA Astrophysics Data System (ADS)

Vendrely, Charlotte; Ackerschott, Christian; Römer, Lin; Scheibel, Thomas

Most performance proteins responsible for the mechanical stability of cells and organisms reveal highly repetitive sequences. Mimicking such performance proteins is of high interest for the design of nanostructured biomaterials. In this article, flagelliform silk is exemplary introduced to describe a general principle for designing genes of repetitive performance proteins for recombinant expression in Escherichia coli . In the first step, repeating amino acid sequence motifs are reversely transcripted into DNA cassettes, which can in a second step be seamlessly ligated, yielding a designed gene. Recombinant expression thereof leads to proteins mimicking the natural ones. The recombinant proteins can be assembled into nanostructured materials in a controlled manner, allowing their use in several applications.

Overcoming codon bias: a method for high-level overexpression of Plasmodium and other AT-rich parasite genes in Escherichia coli.

PubMed

Baca, A M; Hol, W G

2000-02-01

Parasite genes often use codons which are rarely used in the highly expressed genes of Escherichia coli, possibly resulting in translational stalling and lower yields of recombinant protein. We have constructed the "RIG" plasmid to overcome the potential codon-bias problem seen in Plasmodium genes. RIG contains the genes that encode three tRNAs (Arg, Ile, Gly), which recognise rare codons found in parasite genes. When co-transformed into E. coli along with expression plasmids containing parasite genes, RIG can greatly increase levels of overexpressed protein. Codon frequency analysis suggests that RIG may be applied to a variety of protozoan and helminth genes.

High-throughput DNA extraction of forensic adhesive tapes.

PubMed

Forsberg, Christina; Jansson, Linda; Ansell, Ricky; Hedman, Johannes

2016-09-01

Tape-lifting has since its introduction in the early 2000's become a well-established sampling method in forensic DNA analysis. Sampling is quick and straightforward while the following DNA extraction is more challenging due to the "stickiness", rigidity and size of the tape. We have developed, validated and implemented a simple and efficient direct lysis DNA extraction protocol for adhesive tapes that requires limited manual labour. The method uses Chelex beads and is applied with SceneSafe FAST tape. This direct lysis protocol provided higher mean DNA yields than PrepFiler Express BTA on Automate Express, although the differences were not significant when using clothes worn in a controlled fashion as reference material (p=0.13 and p=0.34 for T-shirts and button-down shirts, respectively). Through in-house validation we show that the method is fit-for-purpose for application in casework, as it provides high DNA yields and amplifiability, as well as good reproducibility and DNA extract stability. After implementation in casework, the proportion of extracts with DNA concentrations above 0.01ng/μL increased from 71% to 76%. Apart from providing higher DNA yields compared with the previous method, the introduction of the developed direct lysis protocol also reduced the amount of manual labour by half and doubled the potential throughput for tapes at the laboratory. Generally, simplified manual protocols can serve as a cost-effective alternative to sophisticated automation solutions when the aim is to enable high-throughput DNA extraction of complex crime scene samples. Copyright © 2016 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

Conversion of the high-yield salinomycin producer Streptomyces albus BK3-25 into a surrogate host for polyketide production.

PubMed

Zhang, Xiaojie; Lu, Chenyang; Bai, Linquan

2017-09-01

An ideal surrogate host for heterologous production of various natural products is expected to have efficient nutrient utilization, fast growth, abundant precursors and energy supply, and a pronounced gene expression. Streptomyces albus BK3-25 is a high-yield industrial strain producing type-I polyketide salinomycin, with a unique ability of bean oil utilization. Its potential of being a surrogate host for heterologous production of PKS was engineered and evaluated herein. Firstly, introduction of a three-gene cassette for the biosynthesis of ethylmalonyl-CoA resulted in accumulation of ethylmalonyl-CoA precursor and salinomycin, and subsequent deletion of the salinomycin biosynthetic gene cluster resulted in a host with rich supplies of common polyketide precursors, including malonyl-CoA, methylmalonyl-CoA, and ethylmalonyl-CoA. Secondly, the energy and reducing force were measured, and the improved accumulation of ATP and NADPH was observed in the mutant. Furthermore, the strength of a series of selected endogenous promoters based on microarray data was assessed at different growth phases, and a strong constitutive promoter was identified, providing a useful tool for further engineered gene expression. Finally, the potential of the BK3-25 derived host ZXJ-6 was evaluated with the introduction of the actinorhodin biosynthetic gene cluster from Streptomyces coelicolor, and the heterologous production of actinorhodin was obtained. This work clearly indicated the potential of the high-yield salinomycin producer as a surrogate host for heterologous production of polyketides, although more genetic manipulation should be conducted to streamline its performance.

High-Level Expression of Recombinant Bovine Lactoferrin in Pichia pastoris with Antimicrobial Activity

PubMed Central

Iglesias-Figueroa, Blanca; Valdiviezo-Godina, Norberto; Siqueiros-Cendón, Tania; Sinagawa-García, Sugey; Arévalo-Gallegos, Sigifredo; Rascón-Cruz, Quintín

2016-01-01

In this study, bovine lactoferrin (bLf), an iron-binding glycoprotein considered an important nutraceutical protein because of its several properties, was expressed in Pichia pastoris KM71-H under AOX1 promoter control, using pJ902 as the recombinant plasmid. Dot blotting analysis revealed the expression of recombinant bovine lactoferrin (rbLf) in Pichia pastoris. After Bach fermentation and purification by molecular exclusion, we obtained an expression yield of 3.5 g/L of rbLf. rbLf and predominantly pepsin-digested rbLf (rbLfcin) demonstrated antibacterial activity against Escherichia coli (E. coli) BL21DE3, Staphylococcus aureus (S. aureus) FRI137, and, in a smaller percentage, Pseudomonas aeruginosa (Ps. Aeruginosa) ATCC 27833. The successful expression and characterization of functional rbLf expressed in Pichia pastoris opens a prospect for the development of natural antimicrobial agents produced recombinantly. PMID:27294912

Biochar derived from corn straw affected availability and distribution of soil nutrients and cotton yield

PubMed Central

Tian, Xiaofei; Zhang, Min; Wan, Yongshan; Xie, Zhihua; Chen, Baocheng; Li, Wenqing

2018-01-01

Biochar application as a soil amendment has been proposed as a strategy to improve soil fertility and increase crop yields. However, the effects of successive biochar applications on cotton yields and nutrient distribution in soil are not well documented. A three-year field study was conducted to investigate the effects of successive biochar applications at different rates on cotton yield and on the soil nutrient distribution in the 0–100 cm soil profile. Biochar was applied at 0, 5, 10, and 20 t ha-1 (expressed as Control, BC5, BC10, and BC20, respectively) for each cotton season, with identical doses of chemical fertilizers. Biochar enhanced the cotton lint yield by 8.0–15.8%, 9.3–13.9%, and 9.2–21.9% in 2013, 2014, and 2015, respectively, and high levels of biochar application achieved high cotton yields each year. Leaching of soil nitrate was reduced, while the pH values, soil organic carbon, total nitrogen (N), and available K content of the 0–20 cm soil layer were increased in 2014 and 2015. However, the changes in the soil available P content were less substantial. This study suggests that successive biochar amendments have the potential to enhance cotton productivity and soil fertility while reducing nitrate leaching. PMID:29324750

Maize histone H2B-mCherry: a new fluorescent chromatin marker for somatic and meiotic chromosome research.

PubMed

Howe, Elizabeth S; Clemente, Thomas E; Bass, Hank W

2012-06-01

Cytological studies of fluorescent proteins are rapidly yielding insights into chromatin structure and dynamics. Here we describe the production and cytological characterization of new transgenic maize lines expressing a fluorescent histone fusion protein, H2B-mCherry. The transgene is expressed under the control of the maize ubiquitin1 promoter, including its first exon and intron. Polymerase chain reaction-based genotyping and root-tip microscopy showed that most of the lines carrying the transgene also expressed it, producing bright uniform staining of nuclei. Further, plants showing expression in root tips at the seedling stage also showed expression during meiosis, late in the life cycle. Detailed high-resolution three-dimensional imaging of cells and nuclei from various somatic and meiotic cell types showed that H2B-mCherry produced remarkably clear images of chromatin and chromosome fiber morphology, as seen in somatic, male meiotic prophase, and early microgametophyte cells. H2B-mCherry also yielded distinct nucleolus staining and was shown to be compatible with fluorescence in situ hybridization. We found several instances where H2B-mCherry was superior to DAPI as a generalized chromatin stain. Our study establishes these histone H2B-mCherry lines as new biological reagents for visualizing chromatin structure, chromosome morphology, and nuclear dynamics in fixed and living cells in a model plant genetic system.

Production of phytotoxic cationic α-helical antimicrobial peptides in plant cells using inducible promoters.

PubMed

Company, Nuri; Nadal, Anna; Ruiz, Cristina; Pla, Maria

2014-01-01

Synthetic linear antimicrobial peptides with cationic α-helical structures, such as BP100, have potent and specific activities against economically important plant pathogenic bacteria. They are also recognized as valuable therapeutics and preservatives. However, highly active BP100 derivatives are often phytotoxic when expressed at high levels as recombinant peptides in plants. Here we demonstrate that production of recombinant phytotoxic peptides in transgenic plants is possible by strictly limiting transgene expression to certain tissues and conditions, and specifically that minimization of this expression during transformation and regeneration of transgenic plants is essential to obtain viable plant biofactories. On the basis of whole-genome transcriptomic data available online, we identified the Os.hsp82 promoter that fulfilled this requirement and was highly induced in response to heat shock. Using this strategy, we generated transgenic rice lines producing moderate yields of severely phytotoxic BP100 derivatives on exposure to high temperature. In addition, a threshold for gene expression in selected tissues and stages was experimentally established, below which the corresponding promoters should be suitable for driving the expression of recombinant phytotoxic proteins in genetically modified plants. In view of the growing transcriptomics data available, this approach is of interest to assist promoter selection for specific purposes.

Production of Phytotoxic Cationic α-Helical Antimicrobial Peptides in Plant Cells Using Inducible Promoters

PubMed Central

Company, Nuri; Nadal, Anna; Ruiz, Cristina; Pla, Maria

2014-01-01

Synthetic linear antimicrobial peptides with cationic α-helical structures, such as BP100, have potent and specific activities against economically important plant pathogenic bacteria. They are also recognized as valuable therapeutics and preservatives. However, highly active BP100 derivatives are often phytotoxic when expressed at high levels as recombinant peptides in plants. Here we demonstrate that production of recombinant phytotoxic peptides in transgenic plants is possible by strictly limiting transgene expression to certain tissues and conditions, and specifically that minimization of this expression during transformation and regeneration of transgenic plants is essential to obtain viable plant biofactories. On the basis of whole-genome transcriptomic data available online, we identified the Os.hsp82 promoter that fulfilled this requirement and was highly induced in response to heat shock. Using this strategy, we generated transgenic rice lines producing moderate yields of severely phytotoxic BP100 derivatives on exposure to high temperature. In addition, a threshold for gene expression in selected tissues and stages was experimentally established, below which the corresponding promoters should be suitable for driving the expression of recombinant phytotoxic proteins in genetically modified plants. In view of the growing transcriptomics data available, this approach is of interest to assist promoter selection for specific purposes. PMID:25387106

Effect of the tyrosine kinase inhibitor lapatinib on CUB-domain containing protein (CDCP1)-mediated breast cancer cell survival and migration

DOE Office of Scientific and Technical Information (OSTI.GOV)

Seidel, Jeanette; Kunc, Klaudia; Possinger, Kurt

2011-10-14

Highlights: {yields} CDCP1 downregulation reduces anchorage free survival of breast cancer cells. {yields} Anoikis of CDCP1-positive breast cancer cells is increased after CDCP1 downregulation. {yields} CDCP1 knockdown decreases migration and extensively reduces invasiveness in vitro. {yields} Proliferation rate does not correlate with CDCP1 expression. {yields} Lapatinib does not influence tyrosine kinases of CDCP1 signal transduction. -- Abstract: The surface receptor CUB domain-containing protein 1 (CDCP1) is highly expressed in several adenocarcinomas and speculated to participate in anchorage-independent cell survival and cell motility. Tyrosine kinase phosphorylation seems to be crucial for intracellular signaling of CDCP1. Lapatinib, a tyrosine kinase inhibitor (TKI),more » is approved for treatment of HER-2/neu overexpressing metastatic breast cancer and functions by preventing autophosphorylation following HER-2/neu receptor activation. This study aimed to investigate the effect of CDCP1 expression on anchorage-independent growth and cell motility of breast cancer cells. Moreover, studies were performed to examine if lapatinib provided any beneficial effect on HER-2/neu{sup (+)/-}/CDCP1{sup +} breast cancer cell lines. In our studies, we affirmed that CDCP1 prevents cells from undergoing apoptosis when cultured in the absence of cell-substratum anchorage and that migratory and invasive properties of these cells were decreased when CDCP1 was down-regulated. However, only HER-2/neu{sup +}, but not HER-2/neu{sup (+)/-} cells showed decreased proliferation and invasion and an enhanced level of apoptosis towards loss of anchorage when treated with lapatinib. Therefore, we conclude that CDCP1 might be involved in regulating adhesion and motility of breast cancer cells but that lapatinib has no effect on tyrosine kinases regulating CDCP1. Nonetheless, other TKIs might offer therapeutic approaches for CDCP1-targeted breast cancer therapy and should be studied considering this aspect.« less

Heterologous Expression of ATG8c from Soybean Confers Tolerance to Nitrogen Deficiency and Increases Yield in Arabidopsis

PubMed Central

Liu, Dong; Chai, Wenting; Gong, Qingqiu; Wang, Ning Ning

2012-01-01

Nitrogen is an essential element for plant growth and yield. Improving Nitrogen Use Efficiency (NUE) of crops could potentially reduce the application of chemical fertilizer and alleviate environmental damage. To identify new NUE genes is therefore an important task in molecular breeding. Macroautophagy (autophagy) is an intracellular process in which damaged or obsolete cytoplasmic components are encapsulated in double membraned vesicles termed autophagosomes, then delivered to the vacuole for degradation and nutrient recycling. One of the core components of autophagosome formation, ATG8, has been shown to directly mediate autophagosome expansion, and the transcript of which is highly inducible upon starvation. Therefore, we postulated that certain homologs of Saccharomyces cerevisiae ATG8 (ScATG8) from crop species could have potential for NUE crop breeding. A soybean (Glycine max, cv. Zhonghuang-13) ATG8, GmATG8c, was selected from the 11 family members based on transcript analysis upon nitrogen deprivation. GmATG8c could partially complement the yeast atg8 mutant. Constitutive expression of GmATG8c in soybean callus cells not only enhanced nitrogen starvation tolerance of the cells but accelerated the growth of the calli. Transgenic Arabidopsis over-expressing GmATG8c performed better under extended nitrogen and carbon starvation conditions. Meanwhile, under optimum growth conditions, the transgenic plants grew faster, bolted earlier, produced larger primary and axillary inflorescences, eventually produced more seeds than the wild-type. In average, the yield was improved by 12.9%. We conclude that GmATG8c may serve as an excellent candidate for breeding crops with enhanced NUE and better yield. PMID:22629371

High yield cell-free production of integral membrane proteins without refolding or detergents.

PubMed

Wuu, Jessica J; Swartz, James R

2008-05-01

Integral membrane proteins act as critical cellular components and are important drug targets. However, difficulties in producing membrane proteins have hampered investigations of structure and function. In vivo production systems are often limited by cell toxicity, and previous in vitro approaches have required unnatural folding pathways using detergents or lipid solutions. To overcome these limitations, we present an improved cell-free expression system which produces high yields of integral membrane proteins without the use of detergents or refolding steps. Our cell-free reaction activates an Escherichia coli-derived cell extract for transcription and translation. Purified E. coli inner membrane vesicles supply membrane-bound components and the lipid environment required for insertion and folding. Using this system, we demonstrated successful synthesis of two complex integral membrane transporters, the tetracycline pump (TetA) and mannitol permease (MtlA), in yields of 570+/-50 microg/mL and 130+/-30 microg/mL of vesicle-associated protein, respectively. These yields are up to 400 times typical in vivo concentrations. Insertion and folding of these proteins are verified by sucrose flotation, protease digestion, and activity assays. Whereas TetA incorporates efficiently into vesicle membranes with over two-thirds of the synthesized protein being inserted, MtlA yields appear to be limited by insufficient concentrations of a membrane-associated chaperone.

The Promposal: Youth Expressions of Identity and "Love" in the Digital Age

ERIC Educational Resources Information Center

Richardson, John M.

2017-01-01

The "promposal" is a growing, North American high school ritual in which one graduating senior asks another to the prom in a creative, witty, public performance that is recorded and posted online. A "YouTube" search for "promposal" yields over 49,000 hits, with videos receiving up to 8,000,000 views. What does the…

Farnesylated and methylated KRAS4b: high yield production of protein suitable for biophysical studies of prenylated protein-lipid interactions.

PubMed

Gillette, William K; Esposito, Dominic; Abreu Blanco, Maria; Alexander, Patrick; Bindu, Lakshman; Bittner, Cammi; Chertov, Oleg; Frank, Peter H; Grose, Carissa; Jones, Jane E; Meng, Zhaojing; Perkins, Shelley; Van, Que; Ghirlando, Rodolfo; Fivash, Matthew; Nissley, Dwight V; McCormick, Frank; Holderfield, Matthew; Stephen, Andrew G

2015-11-02

Prenylated proteins play key roles in several human diseases including cancer, atherosclerosis and Alzheimer's disease. KRAS4b, which is frequently mutated in pancreatic, colon and lung cancers, is processed by farnesylation, proteolytic cleavage and carboxymethylation at the C-terminus. Plasma membrane localization of KRAS4b requires this processing as does KRAS4b-dependent RAF kinase activation. Previous attempts to produce modified KRAS have relied on protein engineering approaches or in vitro farnesylation of bacterially expressed KRAS protein. The proteins produced by these methods do not accurately replicate the mature KRAS protein found in mammalian cells and the protein yield is typically low. We describe a protocol that yields 5-10 mg/L highly purified, farnesylated, and methylated KRAS4b from insect cells. Farnesylated and methylated KRAS4b is fully active in hydrolyzing GTP, binds RAF-RBD on lipid Nanodiscs and interacts with the known farnesyl-binding protein PDEδ.

Farnesylated and methylated KRAS4b: high yield production of protein suitable for biophysical studies of prenylated protein-lipid interactions

PubMed Central

Gillette, William K.; Esposito, Dominic; Abreu Blanco, Maria; Alexander, Patrick; Bindu, Lakshman; Bittner, Cammi; Chertov, Oleg; Frank, Peter H.; Grose, Carissa; Jones, Jane E.; Meng, Zhaojing; Perkins, Shelley; Van, Que; Ghirlando, Rodolfo; Fivash, Matthew; Nissley, Dwight V.; McCormick, Frank; Holderfield, Matthew; Stephen, Andrew G.

2015-01-01

Prenylated proteins play key roles in several human diseases including cancer, atherosclerosis and Alzheimer’s disease. KRAS4b, which is frequently mutated in pancreatic, colon and lung cancers, is processed by farnesylation, proteolytic cleavage and carboxymethylation at the C-terminus. Plasma membrane localization of KRAS4b requires this processing as does KRAS4b-dependent RAF kinase activation. Previous attempts to produce modified KRAS have relied on protein engineering approaches or in vitro farnesylation of bacterially expressed KRAS protein. The proteins produced by these methods do not accurately replicate the mature KRAS protein found in mammalian cells and the protein yield is typically low. We describe a protocol that yields 5–10 mg/L highly purified, farnesylated, and methylated KRAS4b from insect cells. Farnesylated and methylated KRAS4b is fully active in hydrolyzing GTP, binds RAF-RBD on lipid Nanodiscs and interacts with the known farnesyl-binding protein PDEδ. PMID:26522388

Refolding of proteins from inclusion bodies: rational design and recipes.

PubMed

Basu, Anindya; Li, Xiang; Leong, Susanna Su Jan

2011-10-01

The need to develop protein biomanufacturing platforms that can deliver proteins quickly and cost-effectively is ever more pressing. The rapid rate at which genomes can now be sequenced demands efficient protein production platforms for gene function identification. There is a continued need for the biotech industry to deliver new and more effective protein-based drugs to address new diseases. Bacterial production platforms have the advantage of high expression yields, but insoluble expression of many proteins necessitates the development of diverse and optimised refolding-based processes. Strategies employed to eliminate insoluble expression are reviewed, where it is concluded that inclusion bodies are difficult to eliminate for various reasons. Rational design of refolding systems and recipes are therefore needed to expedite production of recombinant proteins. This review article discusses efforts towards rational design of refolding systems and recipes, which can be guided by the development of refolding screening platforms that yield both qualitative and quantitative information on the progression of a given refolding process. The new opportunities presented by light scattering technologies for developing rational protein refolding buffer systems which in turn can be used to develop new process designs armed with better monitoring and controlling functionalities are discussed. The coupling of dynamic and static light scattering methodologies for incorporation into future bioprocess designs to ensure delivery of high-quality refolded proteins at faster rates is also discussed.

Using Peptide-Level Proteomics Data for Detecting Differentially Expressed Proteins.

PubMed

Suomi, Tomi; Corthals, Garry L; Nevalainen, Olli S; Elo, Laura L

2015-11-06

The expression of proteins can be quantified in high-throughput means using different types of mass spectrometers. In recent years, there have emerged label-free methods for determining protein abundance. Although the expression is initially measured at the peptide level, a common approach is to combine the peptide-level measurements into protein-level values before differential expression analysis. However, this simple combination is prone to inconsistencies between peptides and may lose valuable information. To this end, we introduce here a method for detecting differentially expressed proteins by combining peptide-level expression-change statistics. Using controlled spike-in experiments, we show that the approach of averaging peptide-level expression changes yields more accurate lists of differentially expressed proteins than does the conventional protein-level approach. This is particularly true when there are only few replicate samples or the differences between the sample groups are small. The proposed technique is implemented in the Bioconductor package PECA, and it can be downloaded from http://www.bioconductor.org.

Seasonal and latitudinal acclimatization of cardiac transcriptome responses to thermal stress in porcelain crabs, Petrolisthes cinctipes.

PubMed

Stillman, Jonathon H; Tagmount, Abderrahmane

2009-10-01

Central predictions of climate warming models include increased climate variability and increased severity of heat waves. Physiological acclimatization in populations across large-scale ecological gradients in habitat temperature fluctuation is an important factor to consider in detecting responses to climate change related increases in thermal fluctuation. We measured in vivo cardiac thermal maxima and used microarrays to profile transcriptome heat and cold stress responses in cardiac tissue of intertidal zone porcelain crabs across biogeographic and seasonal gradients in habitat temperature fluctuation. We observed acclimatization dependent induction of heat shock proteins, as well as unknown genes with heat shock protein-like expression profiles. Thermal acclimatization had the largest effect on heat stress responses of extensin-like, beta tubulin, and unknown genes. For these genes, crabs acclimatized to thermally variable sites had higher constitutive expression than specimens from low variability sites, but heat stress dramatically induced expression in specimens from low variability sites and repressed expression in specimens from highly variable sites. Our application of ecological transcriptomics has yielded new biomarkers that may represent sensitive indicators of acclimatization to habitat temperature fluctuation. Our study also has identified novel genes whose further description may yield novel understanding of cellular responses to thermal acclimatization or thermal stress.

SACE_5599, a putative regulatory protein, is involved in morphological differentiation and erythromycin production in Saccharopolyspora erythraea.

PubMed

Kirm, Benjamin; Magdevska, Vasilka; Tome, Miha; Horvat, Marinka; Karničar, Katarina; Petek, Marko; Vidmar, Robert; Baebler, Spela; Jamnik, Polona; Fujs, Štefan; Horvat, Jaka; Fonovič, Marko; Turk, Boris; Gruden, Kristina; Petković, Hrvoje; Kosec, Gregor

2013-12-17

Erythromycin is a medically important antibiotic, biosynthesized by the actinomycete Saccharopolyspora erythraea. Genes encoding erythromycin biosynthesis are organized in a gene cluster, spanning over 60 kbp of DNA. Most often, gene clusters encoding biosynthesis of secondary metabolites contain regulatory genes. In contrast, the erythromycin gene cluster does not contain regulatory genes and regulation of its biosynthesis has therefore remained poorly understood, which has for a long time limited genetic engineering approaches for erythromycin yield improvement. We used a comparative proteomic approach to screen for potential regulatory proteins involved in erythromycin biosynthesis. We have identified a putative regulatory protein SACE_5599 which shows significantly higher levels of expression in an erythromycin high-producing strain, compared to the wild type S. erythraea strain. SACE_5599 is a member of an uncharacterized family of putative regulatory genes, located in several actinomycete biosynthetic gene clusters. Importantly, increased expression of SACE_5599 was observed in the complex fermentation medium and at controlled bioprocess conditions, simulating a high-yield industrial fermentation process in the bioreactor. Inactivation of SACE_5599 in the high-producing strain significantly reduced erythromycin yield, in addition to drastically decreasing sporulation intensity of the SACE_5599-inactivated strains when cultivated on ABSM4 agar medium. In contrast, constitutive overexpression of SACE_5599 in the wild type NRRL23338 strain resulted in an increase of erythromycin yield by 32%. Similar yield increase was also observed when we overexpressed the bldD gene, a previously identified regulator of erythromycin biosynthesis, thereby for the first time revealing its potential for improving erythromycin biosynthesis. SACE_5599 is the second putative regulatory gene to be identified in S. erythraea which has positive influence on erythromycin yield. Like bldD, SACE_5599 is involved in morphological development of S. erythraea, suggesting a very close relationship between secondary metabolite biosynthesis and morphological differentiation in this organism. While the mode of action of SACE_5599 remains to be elucidated, the manipulation of this gene clearly shows potential for improvement of erythromycin production in S. erythraea in industrial setting. We have also demonstrated the applicability of the comparative proteomics approach for identifying new regulatory elements involved in biosynthesis of secondary metabolites in industrial conditions.

SACE_5599, a putative regulatory protein, is involved in morphological differentiation and erythromycin production in Saccharopolyspora erythraea

PubMed Central

2013-01-01

Background Erythromycin is a medically important antibiotic, biosynthesized by the actinomycete Saccharopolyspora erythraea. Genes encoding erythromycin biosynthesis are organized in a gene cluster, spanning over 60 kbp of DNA. Most often, gene clusters encoding biosynthesis of secondary metabolites contain regulatory genes. In contrast, the erythromycin gene cluster does not contain regulatory genes and regulation of its biosynthesis has therefore remained poorly understood, which has for a long time limited genetic engineering approaches for erythromycin yield improvement. Results We used a comparative proteomic approach to screen for potential regulatory proteins involved in erythromycin biosynthesis. We have identified a putative regulatory protein SACE_5599 which shows significantly higher levels of expression in an erythromycin high-producing strain, compared to the wild type S. erythraea strain. SACE_5599 is a member of an uncharacterized family of putative regulatory genes, located in several actinomycete biosynthetic gene clusters. Importantly, increased expression of SACE_5599 was observed in the complex fermentation medium and at controlled bioprocess conditions, simulating a high-yield industrial fermentation process in the bioreactor. Inactivation of SACE_5599 in the high-producing strain significantly reduced erythromycin yield, in addition to drastically decreasing sporulation intensity of the SACE_5599-inactivated strains when cultivated on ABSM4 agar medium. In contrast, constitutive overexpression of SACE_5599 in the wild type NRRL23338 strain resulted in an increase of erythromycin yield by 32%. Similar yield increase was also observed when we overexpressed the bldD gene, a previously identified regulator of erythromycin biosynthesis, thereby for the first time revealing its potential for improving erythromycin biosynthesis. Conclusions SACE_5599 is the second putative regulatory gene to be identified in S. erythraea which has positive influence on erythromycin yield. Like bldD, SACE_5599 is involved in morphological development of S. erythraea, suggesting a very close relationship between secondary metabolite biosynthesis and morphological differentiation in this organism. While the mode of action of SACE_5599 remains to be elucidated, the manipulation of this gene clearly shows potential for improvement of erythromycin production in S. erythraea in industrial setting. We have also demonstrated the applicability of the comparative proteomics approach for identifying new regulatory elements involved in biosynthesis of secondary metabolites in industrial conditions. PMID:24341557

Manganese peroxidase from the white-rot fungus Phanerochaete chrysosporium is enzymatically active and accumulates to high levels in transgenic maize seed.

PubMed

Clough, Richard C; Pappu, Kameshwari; Thompson, Kevin; Beifuss, Katherine; Lane, Jeff; Delaney, Donna E; Harkey, Robin; Drees, Carol; Howard, John A; Hood, Elizabeth E

2006-01-01

Manganese peroxidase (MnP) has been implicated in lignin degradation and thus has potential applications in pulp and paper bleaching, enzymatic remediation and the textile industry. Transgenic plants are an emerging protein expression platform that offer many advantages over traditional systems, in particular their potential for large-scale industrial enzyme production. Several plant expression vectors were created to evaluate the accumulation of MnP from the wood-rot fungus Phanerochaete chrysosporium in maize seed. We showed that cell wall targeting yielded full-length MnP, whereas cytoplasmic localization resulted in multiple truncated peroxidase polypeptides as detected by immunoblot analysis. In addition, the use of a seed-preferred promoter dramatically increased the expression levels and reduced the negative effects on plant health. Multiple independent transgenic lines were backcrossed with elite inbred corn lines for several generations with the maintenance of high-level expression, indicating genetic stability of the transgene.

Suppression of hepatic stellate cell activation by microRNA-29b

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sekiya, Yumiko; Ogawa, Tomohiro; Liver Research Center, Graduate School of Medicine, Osaka City University, Osaka

Highlights: {yields} Expression of miR-29b was found to be down-regulated during the activation of hepatic stellate cells in primary culture. {yields} Transfection of a miR-29b precursor markedly attenuated the expression of Col1a1 and Col1a2 mRNAs. {yields} It blunted the increased expression of {alpha}-SMA, DDR2, FN1, ITGB1, and PDGFR-b mRNAs essential for stellate cell activation. {yields} miR-29b overexpression led stellate cells to remain in a quiescent state, as evidenced by their star-like morphology. {yields} miR-29b overexpression suppressed the expression of c-fos mRNA. -- Abstract: MicroRNAs (miRNAs) participate in the regulation of cellular functions including proliferation, apoptosis, and migration. It has beenmore » previously shown that the miR-29 family is involved in regulating type I collagen expression by interacting with the 3'UTR of its mRNA. Here, we investigated the roles of miR-29b in the activation of mouse primary-cultured hepatic stellate cells (HSCs), a principal collagen-producing cell in the liver. Expression of miR-29b was found to be down-regulated during HSC activation in primary culture. Transfection of a miR-29b precursor markedly attenuated the expression of Col1a1 and Col1a2 mRNAs and additionally blunted the increased expression of {alpha}-SMA, DDR2, FN1, ITGB1, and PDGFR-{beta}, which are key genes involved in the activation of HSCs. Further, overexpression of miR-29b led HSCs to remain in a quiescent state, as evidenced by their quiescent star-like cell morphology. Although phosphorylation of FAK, ERK, and Akt, and the mRNA expression of c-jun was unaffected, miR-29b overexpression suppressed the expression of c-fos mRNA. These results suggested that miR-29b is involved in the activation of HSCs and could be a candidate molecule for suppressing their activation and consequent liver fibrosis.« less

Ibandronate promotes osteogenic differentiation of periodontal ligament stem cells by regulating the expression of microRNAs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou, Qiang; Zhao, Zhi-Ning; Cheng, Jing-Tao

2011-01-07

Research highlights: {yields} Ibandronate significantly promote the proliferation of PDLSC cells. {yields} Ibandronate enhanced the expression of ALP, COL-1, OPG, OCN, Runx2. {yields} The expression of a class of miRNAs, e.g., miR-18a, miR-133a, miR-141 and miR-19a, was significantly modified in PDLSC cells cultured with ibandronate. {yields} Ibandronate regulates the expression of diverse bone formation-related genes via miRNAs in PDLSCs. {yields} Ibandronate can suppress the activity of osteoclast while promoting the proliferation of osteoblast by regulating the expression of microRNAs. -- Abstract: Bisphosphonates (BPs) have a profound effect on bone resorption and are widely used to treat osteoclast-mediated bone diseases. Theymore » suppress bone resorption by inhibiting the activity of mature osteoclasts and/or the formation of new osteoclasts. Osteoblasts may be an alternative target for BPs. Periodontal ligament stem cells (PDLSCs) exhibit osteoblast-like features and are capable of differentiating into osteoblasts or cementoblasts. This study aimed to determine the effects of ibandronate, a nitrogen-containing BP, on the proliferation and the differentiation of PDLSCs and to identify the microRNAs (miRNAs) that mediate these effects. The PDLSCs were treated with ibandronate, and cell proliferation was measured using the MTT (3-dimethylthiazol-2,5-diphenyltetrazolium bromide) assay. The expression of genes and miRNAs involved in osteoblastic differentiation was assayed using quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). Ibandronate promoted the proliferation of PDLSCs and enhanced the expression of alkaline phosphatase (ALP), type I collagen (COL-1), osteoprotegerin (OPG), osteocalcin (OCN), and Runx2. The expression of miRNAs, including miR-18a, miR-133a, miR-141 and miR-19a, was significantly altered in the PDLSCs cultured with ibandronate. In PDLSCs, ibandronate regulates the expression of diverse bone formation-related genes via miRNAs. The exact mechanism underlying the role of ibandronate in osteoblasts has not been completely understood. Ibandronate may suppress the activity of osteoclasts while promoting the proliferation of osteoblasts by regulating the expression of miRNAs.« less

Ecological engineering helps maximize function in algal oil production.

PubMed

Jackrel, Sara L; Narwani, Anita; Bentlage, Bastian; Levine, Robert B; Hietala, David C; Savage, Phillip E; Oakley, Todd H; Denef, Vincent J; Cardinale, Bradley J

2018-05-18

Algal biofuels have the potential to curb emissions of greenhouse gases from fossil fuels, but current growing methods fail to produce fuels that meet the multiple standards necessary for economical industrial use. For example, algae grown as monocultures for biofuel production have not simultaneously and economically achieved high yields of the high-quality, lipid-rich biomass desired for the industrial-scale production of bio-oil. Decades of study in the field of ecology have demonstrated that simultaneous increases in multiple functions, such as the quantity and quality of biomass, can occur in natural ecosystems by increasing biological diversity. Here we show that species consortia of algae can improve the production of bio-oil, which benefits from both high biomass yield and high quality of biomass rich in fatty acids. We explain the underlying causes of increased quantity and quality of algal biomass among species consortia by showing that, relative to monocultures, species consortia can differentially regulate lipid metabolism genes while growing to higher levels of biomass, in part due to greater utilization of nutrient resources. We identify multiple genes involved in lipid biosynthesis that are frequently upregulated in bicultures, and further show that these elevated levels of gene expression are highly predictive of the elevated levels in biculture relative to monoculture of multiple quality metrics of algal biomass. These results show that interactions between species can alter the expression of lipid metabolism genes, and further demonstrate that our understanding of diversity-function relationships from natural ecosystems can be harnessed to improve production of bio-oil. Importance section: Algal biofuels are one of the more promising forms of renewable energy. In our study, we investigate whether ecological interactions between species of microalgae regulate two important factors in cultivation - the biomass of the crop produced and quality of the biomass that is produced. We find that species interactions often improved production yields, especially the fatty acid content of the algal biomass, and that differentially expressed genes involved in fatty acid metabolism are predictive of improved quality metrics of bio-oil. Other studies have found that diversity often improves productivity and stability in agricultural and natural ecosystems. Our results provide further evidence that growing multi-species crops of microalgae may improve the production of high-quality biomass for bio-oil. Copyright © 2018 American Society for Microbiology.

Gene expression profiling in multipotent DFAT cells derived from mature adipocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ono, Hiromasa; Database Center for Life Science; Oki, Yoshinao

2011-04-15

Highlights: {yields} Adipocyte dedifferentiation is evident in a significant decrease in typical genes. {yields} Cell proliferation is strongly related to adipocyte dedifferentiation. {yields} Dedifferentiated adipocytes express several lineage-specific genes. {yields} Comparative analyses using publicly available datasets boost the interpretation. -- Abstract: Cellular dedifferentiation signifies the withdrawal of cells from a specific differentiated state to a stem cell-like undifferentiated state. However, the mechanism of dedifferentiation remains obscure. Here we performed comparative transcriptome analyses during dedifferentiation in mature adipocytes (MAs) to identify the transcriptional signatures of multipotent dedifferentiated fat (DFAT) cells derived from MAs. Using microarray systems, we explored similarly expressed asmore » well as significantly differentially expressed genes in MAs during dedifferentiation. This analysis revealed significant changes in gene expression during this process, including a significant reduction in expression of genes for lipid metabolism concomitantly with a significant increase in expression of genes for cell movement, cell migration, tissue developmental processes, cell growth, cell proliferation, cell morphogenesis, altered cell shape, and cell differentiation. Our observations indicate that the transcriptional signatures of DFAT cells derived from MAs are summarized in terms of a significant decrease in functional phenotype-related genes and a parallel increase in cell proliferation, altered cell morphology, and regulation of the differentiation of related genes. A better understanding of the mechanisms involved in dedifferentiation may enable scientists to control and possibly alter the plasticity of the differentiated state, which may lead to benefits not only in stem cell research but also in regenerative medicine.« less

Employing epigenetics to augment the expression of therapeutic proteins in mammalian cells.

PubMed

Kwaks, Ted H J; Otte, Arie P

2006-03-01

Recombinant proteins form an increasingly large part of the portfolio of biopharmaceutical companies. Production of these often complex transgenic proteins is achieved predominantly in mammalian cell lines but the process is hampered by low yields and unstable expression. Some of these problems are caused by gene silencing at the level of chromatin - so-called epigenetic gene silencing. Here, we describe approaches, which have emerged during the past few years, designed to interfere with epigenetic gene silencing with the aim of enhancing and stabilizing transgene expression. These include targeting histones, the inclusion of specific DNA elements and targeting sites of high gene-expression. We conclude that employing epigenetic gene regulation tools, in combination with further process optimization, might represent the next step forward in the production of therapeutic proteins.

Increased bacterial cell density and recombinant protein yield using a commercial microbial cultivation system.

PubMed

Peck, Grantley R; Bowden, Timothy R; Shiell, Brian J; Michalski, Wojtek P

2014-01-01

EnBase (BioSilta, Finland) is a microbial cultivation system that replicates fed-batch systems through sustained release of glucose by enzymatic degradation of a polymeric substrate. Achievable bacterial cell densities and recombinant capripoxvirus protein expression levels, solubility, and antigenicity using the EnBase system were assessed. BL21-AI Escherichia coli expressing capripoxvirus proteins achieved up to eightfold higher cell densities when grown in EnBase media compared with standard media. Greater yields of capripoxvirus proteins were attained using EnBase media, either through increases in the amount of expressed protein per cell in conjunction with higher cell density or through the increase in cell density alone. Addition of EnBase booster enhanced protein yield for one of the proteins tested but reduced yield for the other. However, the amount of soluble forms of the capripoxvirus proteins tested was not different from that observed from cultures grown under standard conditions. Purified capripoxvirus proteins expressed using EnBase or standard media were assessed for their performance by enzyme-linked immunosorbent assay (ELISA) and were shown to be equally capable of specifically binding capripoxvirus antibodies.

Cloning and characterization of a 9-lipoxygenase gene induced by pathogen attack from Nicotiana benthamiana for biotechnological application

PubMed Central

2011-01-01

Background Plant lipoxygenases (LOXs) have been proposed to form biologically active compounds both during normal developmental stages such as germination or growth as well as during responses to environmental stress such as wounding or pathogen attack. In our previous study, we found that enzyme activity of endogenous 9-LOX in Nicotiana benthamiana was highly induced by agroinfiltration using a tobacco mosaic virus (TMV) based vector system. Results A LOX gene which is expressed after treatment of the viral vectors was isolated from Nicotiana benthamiana. As the encoded LOX has a high amino acid identity to other 9-LOX proteins, the gene was named as Nb-9-LOX. It was heterologously expressed in yeast cells and its enzymatic activity was characterized. The yeast cells expressed large quantities of stable 9-LOX (0.9 U ml-1 cell cultures) which can oxygenate linoleic acid resulting in high yields (18 μmol ml-1 cell cultures) of hydroperoxy fatty acid. The product specificity of Nb-9-LOX was examined by incubation of linoleic acid and Nb-9-LOX in combination with a 13-hydroperoxide lyase from watermelon (Cl-13-HPL) or a 9/13-hydroperoxide lyase from melon (Cm-9/13-HPL) and by LC-MS analysis. The result showed that Nb-9-LOX possesses both 9- and 13-LOX specificity, with high predominance for the 9-LOX function. The combination of recombinant Nb-9-LOX and recombinant Cm-9/13-HPL produced large amounts of C9-aldehydes (3.3 μmol mg-1 crude protein). The yield of C9-aldehydes from linoleic acid was 64%. Conclusion The yeast expressed Nb-9-LOX can be used to produce C9-aldehydes on a large scale in combination with a HPL gene with 9-HPL function, or to effectively produce 9-hydroxy-10(E),12(Z)-octadecadienoic acid in a biocatalytic process in combination with cysteine as a mild reducing agent. PMID:21450085

Characterization of zebrafish dysferlin by morpholino knockdown

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kawahara, Genri; Serafini, Peter R.; Myers, Jennifer A.

2011-09-23

Highlights: {yields} cDNAs of zebrafish dysferlin were cloned (6.3 kb). {yields} The dysferlin expression was detected in skeletal muscle, heart and eye. {yields} Injection of antisense morpholinos to dysferlin caused marked muscle disorganization. {yields} Zebrafish dysferlin expression may be involved in stabilizing muscle structures. -- Abstract: Mutations in the gene encoding dysferlin cause two distinct muscular dystrophy phenotypes: limb-girdle muscular dystrophy type 2B (LGMD-2B) and Miyoshi myopathy (MM). Dysferlin is a large transmembrane protein involved in myoblast fusion and membrane resealing. Zebrafish represent an ideal animal model to use for studying muscle disease including abnormalities of dysferlin. cDNAs of zebrafishmore » dysferlin were cloned (6.3 kb) and the predicted amino acid sequences, showed 68% similarity to predicted amino acid sequences of mammalian dysferlin. The expression of dysferlin was mainly in skeletal muscle, heart and eye, and the expression could be detected as early as 11 h post fertilization (hpf). Three different antisense oligonucleotide morpholinos were targeted to inhibit translation of this dysferlin mRNA and the morpholino-injected fish showed marked muscle disorganization which could be detected by birefringence assay. Western blot analysis using dysferlin antibodies showed that the expression of dysferlin was reduced in each of the three morphants. Dysferlin expression was shown to be reduced at the myosepta of zebrafish muscle using immunohistochemistry, although the expression of other muscle membrane components, dystrophin, laminin, {beta}-dystroglycan were detected normally. Our data suggest that zebrafish dysferlin expression is involved in stabilizing muscle structures and its downregulation causes muscle disorganization.« less

THE EFFECT OF ADVANCE IN LACTATION AND GESTATION ON MAMMARY ACTIVITY.

PubMed

Gaines, W L; Davidson, F A

1926-01-20

The rate of milk secretion in farrow cows may be expressed as See PDF for Equation, in which y = yield and t = time from calving. Pregnancy causes a decrease in yield which may be expressed as See PDF for Equation, in which i = inhibition or decrease in yield and p = time from conception. The constant K appears to be the same for various groups but b is roughly proportional to a. The decrease in yield associated with pregnancy is interpreted as due to a hormone. The hormone hypothesis also affords an interpretation of the increasing rate of milk secretion which occurs for a short time following parturition.

High Contrast PET Imaging of GRPR Expression in Prostate Cancer Using Cobalt-Labeled Bombesin Antagonist RM26

PubMed Central

Thisgaard, Helge; Rosenström, Ulrika; Dam, Johan Hygum; Larhed, Mats

2017-01-01

High gastrin releasing peptide receptor (GRPR) expression is associated with numerous cancers including prostate and breast cancer. The aim of the current study was to develop a 55Co-labeled PET agent based on GRPR antagonist RM26 for visualization of GRPR-expressing tumors. Labeling with 57Co and 55Co, stability, binding specificity, and in vitro and in vivo characteristics of 57Co-NOTA-PEG2-RM26 were studied. NOTA-PEG2-RM26 was successfully radiolabeled with 57Co and 55Co with high yields and demonstrated high stability. The radiopeptide showed retained binding specificity to GRPR in vitro and in vivo. 57Co-NOTA-PEG2-RM26 biodistribution in mice was characterized by rapid clearance of radioactivity from blood and normal non-GRPR-expressing organs and low hepatic uptake. The clearance was predominantly renal with a low degree of radioactivity reabsorption. Tumor-to-blood ratios were approximately 200 (3 h pi) and 1000 (24 h pi). The favorable biodistribution of cobalt-labeled NOTA-PEG2-RM26 translated into high contrast preclinical PET/CT (using 55Co) and SPECT/CT (using 57Co) images of PC-3 xenografts. The initial biological results suggest that 55Co-NOTA-PEG2-RM26 is a promising tracer for PET visualization of GRPR-expressing tumors. PMID:29097932

Hydrophobin fusion of an influenza virus hemagglutinin allows high transient expression in Nicotiana benthamiana, easy purification and immune response with neutralizing activity.

PubMed

Jacquet, Nicolas; Navarre, Catherine; Desmecht, Daniel; Boutry, Marc

2014-01-01

The expression of recombinant hemagglutinin in plants is a promising alternative to the current egg-based production system for the influenza vaccines. Protein-stabilizing fusion partners have been developed to overcome the low production yields and the high downstream process costs associated with the plant expression system. In this context, we tested the fusion of hydrophobin I to the hemagglutinin ectodomain of the influenza A (H1N1)pdm09 virus controlled by the hybrid En2PMA4 transcriptional promoter to rapidly produce high levels of recombinant antigen by transient expression in agro-infiltrated Nicotiana benthamiana leaves. The fusion increased the expression level by a factor of ∼ 2.5 compared to the unfused protein allowing a high accumulation level of 8.6% of the total soluble proteins. Hemagglutinin was located in ER-derived protein bodies and was successfully purified by combining an aqueous-two phase partition system and a salting out step. Hydrophobin interactions allowed the formation of high molecular weight hemagglutinin structures, while unfused proteins were produced as monomers. Purified protein was shown to be biologically active and to induce neutralizing antibodies after mice immunization. Hydrophobin fusion to influenza hemagglutinin might therefore be a promising approach for rapid, easy, and low cost production of seasonal or pandemic influenza vaccines in plants.

Gastrointestinal Spatiotemporal mRNA Expression of Ghrelin vs Growth Hormone Receptor and New Growth Yield Machine Learning Model Based on Perturbation Theory.

PubMed

Ran, Tao; Liu, Yong; Li, Hengzhi; Tang, Shaoxun; He, Zhixiong; Munteanu, Cristian R; González-Díaz, Humberto; Tan, Zhiliang; Zhou, Chuanshe

2016-07-27

The management of ruminant growth yield has economic importance. The current work presents a study of the spatiotemporal dynamic expression of Ghrelin and GHR at mRNA levels throughout the gastrointestinal tract (GIT) of kid goats under housing and grazing systems. The experiments show that the feeding system and age affected the expression of either Ghrelin or GHR with different mechanisms. Furthermore, the experimental data are used to build new Machine Learning models based on the Perturbation Theory, which can predict the effects of perturbations of Ghrelin and GHR mRNA expression on the growth yield. The models consider eight longitudinal GIT segments (rumen, abomasum, duodenum, jejunum, ileum, cecum, colon and rectum), seven time points (0, 7, 14, 28, 42, 56 and 70 d) and two feeding systems (Supplemental and Grazing feeding) as perturbations from the expected values of the growth yield. The best regression model was obtained using Random Forest, with the coefficient of determination R(2) of 0.781 for the test subset. The current results indicate that the non-linear regression model can accurately predict the growth yield and the key nodes during gastrointestinal development, which is helpful to optimize the feeding management strategies in ruminant production system.

Nesprin-2 epsilon: A novel nesprin isoform expressed in human ovary and Ntera-2 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lam, Le Thanh; Boehm, Sabrina V.; Roberts, Roland G.

2011-08-26

Highlights: {yields} A novel epsilon isoform of nesprin-2 has been discovered. {yields} This 120 kDa protein was predicted by bioinformatic analysis, but has not previously been observed. {yields} It is the main isoform expressed in a teratocarcinoma cell line and is also found in ovary. {yields} Like other nesprins, it is located at the nuclear envelope. {yields} We suggest it may have a role in very early development or in some ovary-specific function. -- Abstract: The nuclear envelope-associated cytoskeletal protein, nesprin-2, is encoded by a large gene containing several internal promoters that produce shorter isoforms. In a study of Ntera-2more » teratocarcinoma cells, a novel isoform, nesprin-2-epsilon, was found to be the major mRNA and protein product of the nesprin-2 gene. Its existence was predicted by bioinformatic analysis, but this is the first direct demonstration of both the mRNA and the 120 kDa protein which is located at the nuclear envelope. In a panel of 21 adult and foetal human tissues, the nesprin-2-epsilon mRNA was strongly expressed in ovary but was a minor isoform elsewhere. The expression pattern suggests a possible link with very early development and a likely physiological role in ovary.« less

Gastrointestinal Spatiotemporal mRNA Expression of Ghrelin vs Growth Hormone Receptor and New Growth Yield Machine Learning Model Based on Perturbation Theory

PubMed Central

Ran, Tao; Liu, Yong; Li, Hengzhi; Tang, Shaoxun; He, Zhixiong; Munteanu, Cristian R.; González-Díaz, Humberto; Tan, Zhiliang; Zhou, Chuanshe

2016-01-01

The management of ruminant growth yield has economic importance. The current work presents a study of the spatiotemporal dynamic expression of Ghrelin and GHR at mRNA levels throughout the gastrointestinal tract (GIT) of kid goats under housing and grazing systems. The experiments show that the feeding system and age affected the expression of either Ghrelin or GHR with different mechanisms. Furthermore, the experimental data are used to build new Machine Learning models based on the Perturbation Theory, which can predict the effects of perturbations of Ghrelin and GHR mRNA expression on the growth yield. The models consider eight longitudinal GIT segments (rumen, abomasum, duodenum, jejunum, ileum, cecum, colon and rectum), seven time points (0, 7, 14, 28, 42, 56 and 70 d) and two feeding systems (Supplemental and Grazing feeding) as perturbations from the expected values of the growth yield. The best regression model was obtained using Random Forest, with the coefficient of determination R2 of 0.781 for the test subset. The current results indicate that the non-linear regression model can accurately predict the growth yield and the key nodes during gastrointestinal development, which is helpful to optimize the feeding management strategies in ruminant production system. PMID:27460882

Production of recombinant albumin by a herd of cloned transgenic cattle.

PubMed

Echelard, Yann; Williams, Jennifer L; Destrempes, Margaret M; Koster, Julie A; Overton, Susan A; Pollock, Daniel P; Rapiejko, Karen T; Behboodi, Esmail; Masiello, Nicholas C; Gavin, William G; Pommer, Jerry; Van Patten, Scott M; Faber, David C; Cibelli, Jose B; Meade, Harry M

2009-06-01

Purified plasma derived human albumin has been available as a therapeutic product since World War II. However, cost effective recombinant production of albumin has been challenging due to the amount needed and the complex folding pattern of the protein. In an effort to provide an abundant source of recombinant albumin, a herd of transgenic cows expressing high levels of rhA in their milk was generated. Expression cassettes efficiently targeting the secretion of human albumin to the lactating mammary gland were obtained and tested in transgenic mice. A high expressing transgene was transfected in primary bovine cell lines to produce karyoplasts for use in a somatic cell nuclear transfer program. Founder transgenic cows were produced from four independent cell lines. Expression levels varying from 1-2 g/l to more than 40 g/l of correctly folded albumin were observed. The animals expressing the highest levels of rhA exhibited shortened lactation whereas cows yielding 1-2 g/l had normal milk production. This herd of transgenic cattle is an easily scalable and well characterized source of rhA for biomedical uses.

OsGRF4 controls grain shape, panicle length and seed shattering in rice.

PubMed

Sun, Pingyong; Zhang, Wuhan; Wang, Yihua; He, Qiang; Shu, Fu; Liu, Hai; Wang, Jie; Wang, Jianmin; Yuan, Longping; Deng, Huafeng

2016-10-01

Traits such as grain shape, panicle length and seed shattering, play important roles in grain yield and harvest. In this study, the cloning and functional analysis of PANICLE TRAITS 2 (PT2), a novel gene from the Indica rice Chuandali (CDL), is reported. PT2 is synonymous with Growth-Regulating Factor 4 (OsGRF4), which encodes a growth-regulating factor that positively regulates grain shape and panicle length and negatively regulates seed shattering. Higher expression of OsGRF4 is correlated with larger grain, longer panicle and lower seed shattering. A unique OsGRF4 mutation, which occurs at the OsmiRNA396 target site of OsGRF4, seems to be associated with high levels of OsGRF4 expression, and results in phenotypic difference. Further research showed that OsGRF4 regulated two cytokinin dehydrogenase precursor genes (CKX5 and CKX1) resulting in increased cytokinin levels, which might affect the panicle traits. High storage capacity and moderate seed shattering of OsGRF4 may be useful in high-yield breeding and mechanized harvesting of rice. Our findings provide additional insight into the molecular basis of panicle growth. © 2016 The Authors. Journal of Integrative Plant Biology Published by John Wiley & Sons Australia, Ltd on behalf of Institute of Botany, Chinese Academy of Sciences.

Fto colocalizes with a satiety mediator oxytocin in the brain and upregulates oxytocin gene expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Olszewski, Pawel K., E-mail: olsze005@umn.edu; Minnesota Obesity Center, Saint Paul, MN 55108; Fredriksson, Robert

2011-05-13

Highlights: {yields} The majority of neurons synthesizing a satiety mediator, oxytocin, coexpress Fto. {yields} The level of colocalization is similar in the male and female brain. {yields} Fto overexpression in hypothalamic neurons increases oxytocin mRNA levels by 50%. {yields} Oxytocin does not affect Fto expression through negative feedback mechanisms. -- Abstract: Single nucleotide polymorphisms in the fat mass and obesity-associated (FTO) gene have been associated with obesity in humans. Alterations in Fto expression in transgenic animals affect body weight, energy expenditure and food intake. Fto, a nuclear protein and proposed transcription co-factor, has been speculated to affect energy balance throughmore » a functional relationship with specific genes encoding feeding-related peptides. Herein, we employed double immunohistochemistry and showed that the majority of neurons synthesizing a satiety mediator, oxytocin, coexpress Fto in the brain of male and female mice. We then overexpressed Fto in a murine hypothalamic cell line and, using qPCR, detected a 50% increase in the level of oxytocin mRNA. Expression levels of several other feeding-related genes, including neuropeptide Y (NPY) and Agouti-related protein (AgRP), were unaffected by the FTO transfection. Addition of 10 and 100 nmol oxytocin to the cell culture medium did not affect Fto expression in hypothalamic cells. We conclude that Fto, a proposed transcription co-factor, influences expression of the gene encoding a satiety mediator, oxytocin.« less

PARP-1 regulates the expression of caspase-11

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yoo, Lang; Hong, Seokheon; Shin, Ki Soon

2011-05-13

Highlights: {yields} Knockdown of PARP-1 suppresses the LPS-induced expression of caspase-11. {yields} Knockdown of PARP-1 suppresses the caspase-11 promoter activity following LPS stimulation. {yields} PARP-1 is recruited to the caspase-11 promoter region containing NF-{kappa}B-binding sites following LPS stimulation. {yields} PARP-1 inhibitors cannot suppress the caspase-11 induction. {yields} PARP-1 does not suppress IFN-{gamma}-induced expression of caspase-11. -- Abstract: Poly(ADP-ribose) polymerase-1 (PARP-1) is a multifunctional enzyme that regulates DNA repair, cell death and transcription of inflammatory proteins. In the present study, we present evidence that PARP-1 regulates the expression of caspase-11 following lipopolysaccharide (LPS) stimulation. Knockdown of PARP-1 suppressed the LPS-induced expressionmore » of caspase-11 at both mRNA and protein levels as well as caspase-11 promoter activity. Importantly, PARP-1 was recruited to the caspase-11 promoter region containing predicted nuclear factor (NF)-{kappa}B-binding sites when examined by chromatin immunoprecipitation assay. However, knockdown of PARP-1 did not suppress the expression of caspase-11 induced by interferon-{gamma} that activates signal transducer and activator of transcription 1 but not NF-{kappa}B. PARP-1 enzymatic activity was not required for the caspase-11 upregulation since pharmacological inhibitors of PARP-1 did not suppress the induction of caspase-11. Our results suggest that PARP-1, as a transcriptional cofactor for NF-{kappa}B, regulates the induction of caspase-11 at a transcriptional level.« less

Mechanism of protein splicing of the Pyrococcus abyssi lon protease intein

DOE Office of Scientific and Technical Information (OSTI.GOV)

O'Brien, Kevin M.; Schufreider, Ann K.; McGill, Melissa A.

2010-12-17

Research highlights: {yields} The Pyrococcus abyssi lon protease intein promotes efficient protein splicing. {yields} Inteins with mutations that interfere with individual steps of splicing do not promote unproductive side reactions. {yields} The intein splices with Lys in place of the highly conserved penultimate His. {yields} The intein is flanked by a Gly-rich region at its C terminus that may increase the efficiency of the third step of splicing, Asn cyclization coupled to peptide bond cleavage. -- Abstract: Protein splicing is a post-translational process by which an intervening polypeptide, the intein, excises itself from the flanking polypeptides, the exteins, coupled tomore » ligation of the exteins. The lon protease of Pyrococcus abyssi (Pab) is interrupted by an intein. When over-expressed as a fusion protein in Escherichia coli, the Pab lon protease intein can promote efficient protein splicing. Mutations that block individual steps of splicing generally do not lead to unproductive side reactions, suggesting that the intein tightly coordinates the splicing process. The intein can splice, although it has Lys in place of the highly conserved penultimate His, and mutants of the intein in the C-terminal region lead to the accumulation of stable branched-ester intermediate.« less

Test battery for measuring the perception and recognition of facial expressions of emotion

PubMed Central

Wilhelm, Oliver; Hildebrandt, Andrea; Manske, Karsten; Schacht, Annekathrin; Sommer, Werner

2014-01-01

Despite the importance of perceiving and recognizing facial expressions in everyday life, there is no comprehensive test battery for the multivariate assessment of these abilities. As a first step toward such a compilation, we present 16 tasks that measure the perception and recognition of facial emotion expressions, and data illustrating each task's difficulty and reliability. The scoring of these tasks focuses on either the speed or accuracy of performance. A sample of 269 healthy young adults completed all tasks. In general, accuracy and reaction time measures for emotion-general scores showed acceptable and high estimates of internal consistency and factor reliability. Emotion-specific scores yielded lower reliabilities, yet high enough to encourage further studies with such measures. Analyses of task difficulty revealed that all tasks are suitable for measuring emotion perception and emotion recognition related abilities in normal populations. PMID:24860528

High-level expression of the Penicillium notatum glucose oxidase gene in Pichia pastoris using codon optimization.

PubMed

Gao, Zhaowei; Li, Zhuofu; Zhang, Yuhong; Huang, Huoqing; Li, Mu; Zhou, Liwei; Tang, Yunming; Yao, Bin; Zhang, Wei

2012-03-01

The glucose oxidase (GOD) gene from Penicillium notatum was expressed in Pichia pastoris. The 1,815 bp gene, god-w, encodes 604 amino acids. Recombinant GOD-w had optimal activity at 35-40°C and pH 6.2 and was stable, from pH 3 to 7 maintaining >75% maximum activity after incubation at 50°C for 1 h. GOD-w worked as well as commercial GODs to improve bread making. To achieve high-level expression of recombinant GOD in P. pastoris, 272 nucleotides involving 228 residues were mutated, consistent with the codon bias of P. pastoris. The optimized recombinant GOD-m yielded 615 U ml(-1) (2.5 g protein l(-1)) in a 3 l fermentor--410% higher than GOD-w (148 U ml(-1)), and thus is a low-cost alternative for the bread baking industry.

Collisional excitation of the highly excited hydrogen atoms in the dipole form of the semiclassical impact parameter and Born approximations

NASA Technical Reports Server (NTRS)

Omidvar, K.

1971-01-01

Expressions for the excitation cross section of the highly excited states of the hydrogenlike atoms by fast charged particles have been derived in the dipole approximation of the semiclassical impact parameter and the Born approximations, making use of a formula for the asymptotic expansion of the oscillator strength of the hydrogenlike atoms given by Menzel. When only the leading term in the asymptotic expansion is retained, the expression for the cross section becomes identical to the expression obtained by the method of the classical collision and correspondence principle given by Percival and Richards. Comparisons are made between the Bethe coefficients obtained here and the Bethe coefficients of the Born approximation for transitions where the Born calculation is available. Satisfactory agreement is obtained only for n yields n + 1 transitions, with n the principal quantum number of the excited state.

A Protein Chimera Strategy Supports Production of a Model "Difficult-to-Express" Recombinant Target.

PubMed

Hussain, Hirra; Fisher, David I; Roth, Robert G; Abbott, W Mark; Carballo-Amador, Manuel Alejandro; Warwicker, Jim; Dickson, Alan J

2018-06-22

Due in part to the needs of the biopharmaceutical industry, there has been an increased drive to generate high quality recombinant proteins in large amounts. However, achieving high yields can be a challenge as the novelty and increased complexity of new targets often makes them 'difficult-to-express'. This study aimed to define the molecular features that restrict the production of a model 'difficult-to-express' recombinant protein, Tissue Inhibitor Metalloproteinase-3 (TIMP-3). Building from experimental data, computational approaches were used to rationalise the re-design of this recombinant target to generate a chimera with enhanced secretion. The results highlight the importance of early identification of unfavourable sequence attributes, enabling the generation of engineered protein forms that bypass 'secretory' bottlenecks and result in efficient recombinant protein production. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

High- and low-LET induced chromosome damage in human lymphocytes: a time-course of aberrations in metaphase and interphase

NASA Technical Reports Server (NTRS)

George, K.; Wu, H.; Willingham, V.; Furusawa, Y.; Kawata, T.; Cucinotta, F. A.; Dicello, J. F. (Principal Investigator)

2001-01-01

PURPOSE: To investigate how cell-cycle delays in human peripheral lymphocytes affect the expression of complex chromosome damage in metaphase following high- and low-LET radiation exposure. MATERIALS AND METHODS: Whole blood was irradiated in vitro with a low and a high dose of 1 GeV u(-1) iron particles, 400MeV u(-1) neon particles or y-rays. Lymphocytes were cultured and metaphase cells were collected at different time points after 48-84h in culture. Interphase chromosomes were prematurely condensed using calyculin-A, either 48 or 72 h after exposure to iron particles or gamma-rays. Cells in first division were analysed using a combination of FISH whole-chromosome painting and DAPI/ Hoechst 33258 harlequin staining. RESULTS: There was a delay in expression of chromosome damage in metaphase that was LET- and dose-dependant. This delay was mostly related to the late emergence of complex-type damage into metaphase. Yields of damage in PCC collected 48 h after irradiation with iron particles were similar to values obtained from cells undergoing mitosis after prolonged incubation. CONCLUSION: The yield of high-LET radiation-induced complex chromosome damage could be underestimated when analysing metaphase cells collected at one time point after irradiation. Chemically induced PCC is a more accurate technique since problems with complicated cell-cycle delays are avoided.

High- and low-LET induced chromosome damage in human lymphocytes: a time-course of aberrations in metaphase and interphase.

PubMed

George, K; Wu, H; Willingham, V; Furusawa, Y; Kawata, T; Cucinotta, F A

2001-02-01

To investigate how cell-cycle delays in human peripheral lymphocytes affect the expression of complex chromosome damage in metaphase following high- and low-LET radiation exposure. Whole blood was irradiated in vitro with a low and a high dose of 1 GeV u(-1) iron particles, 400MeV u(-1) neon particles or y-rays. Lymphocytes were cultured and metaphase cells were collected at different time points after 48-84h in culture. Interphase chromosomes were prematurely condensed using calyculin-A, either 48 or 72 h after exposure to iron particles or gamma-rays. Cells in first division were analysed using a combination of FISH whole-chromosome painting and DAPI/ Hoechst 33258 harlequin staining. There was a delay in expression of chromosome damage in metaphase that was LET- and dose-dependant. This delay was mostly related to the late emergence of complex-type damage into metaphase. Yields of damage in PCC collected 48 h after irradiation with iron particles were similar to values obtained from cells undergoing mitosis after prolonged incubation. The yield of high-LET radiation-induced complex chromosome damage could be underestimated when analysing metaphase cells collected at one time point after irradiation. Chemically induced PCC is a more accurate technique since problems with complicated cell-cycle delays are avoided.

Comparison of commercial RNA extraction kits and qPCR master mixes for studying gene expression in small biopsy tissue samples from the equine gastric epithelium.

PubMed

Tesena, Parichart; Korchunjit, Wasamon; Taylor, Jane; Wongtawan, Tuempong

2017-01-01

Gastric tissue biopsy and gene expression analysis are important tools for disease diagnosis and study of the physiology of the equine stomach. However, RNA extraction from gastric biopsy samples is a complex procedure because the samples contain low quantities of RNA and are contaminated with mucous protein and bacterial flora. The objectives of these studies were to compare the performance of RNA extraction methods and to investigate the sensitivity of commercial qPCR master mixes for gene expression analysis of gastric biopsy samples. Three commercial RNA extraction methods (TRIzol ™ , GENEzol ™ and MiniPrep ™ ) and four qPCR master mixes with SYBR ® green (qPCRBIO, KAPA, QuantiNova, and PerfeCTa) were compared. RNA qualification and quantitation were compared. Real-time PCR was used to compare qPCR master mixes. The results revealed that TRIzol and GENEzol obtained significantly higher yield of RNA (P<0.01) but that TRIzol had the highest contamination of protein and DNA (P<0.05). Conversely, MiniPrep resulting in a significantly higher purification of RNA (P<0.05) but provided the lowest yield of RNA (P<0.01). For PCR master mixes, KAPA was significantly (P<0.05) more sensitive than other qPCR kits for all amounts of DNA template, particularly at the lowest amount of cDNA. In conclusion, GENEzol is the best method to obtain a high RNA yield and purification and it is more cost-effective than the others as well. Regarding the qPCR master mixes, KAPA SYBR qPCR Master Mix (2x) Universal is superior to the other tested master mixes for studying gene expression in equine gastric biopsies.

Overexpression of pyruvate decarboxylase in the yeast Hansenula polymorpha results in increased ethanol yield in high-temperature fermentation of xylose.

PubMed

Ishchuk, Olena P; Voronovsky, Andriy Y; Stasyk, Oleh V; Gayda, Galina Z; Gonchar, Mykhailo V; Abbas, Charles A; Sibirny, Andriy A

2008-11-01

Improvement of xylose fermentation is of great importance to the fuel ethanol industry. The nonconventional thermotolerant yeast Hansenula polymorpha naturally ferments xylose to ethanol at high temperatures (48-50 degrees C). Introduction of a mutation that impairs ethanol reutilization in H. polymorpha led to an increase in ethanol yield from xylose. The native and heterologous (Kluyveromyces lactis) PDC1 genes coding for pyruvate decarboxylase were expressed at high levels in H. polymorpha under the control of the strong constitutive promoter of the glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH). This resulted in increased pyruvate decarboxylase activity and improved ethanol production from xylose. The introduction of multiple copies of the H. polymorpha PDC1 gene driven by the strong constitutive promoter led to a 20-fold increase in pyruvate decarboxylase activity and up to a threefold elevation of ethanol production.

One-step extraction of functional recombinant aquaporin Z from inclusion bodies with optimal detergent.

PubMed

Wang, Lili; Zhou, Hu; Li, Zhengjun; Lim, Teck Kwang; Lim, Xin Shan; Lin, Qingsong

2015-11-01

Aquaporins are integral membrane channel proteins found in all kingdoms of life. The Escherichia coli aquaporin Z (AqpZ) has been shown to solely conduct water at high permeability. Functional AqpZ is generally purified from the membrane fraction. However, the quantity of the purified protein is limited. In this study, a new method is developed to achieve high yield of bioactive AqpZ protein. A mild detergent n-dodecyl-β-D-maltopyranoside (DDM) was used to solubilize the over-expressed insoluble AqpZ from inclusion bodies without a refolding process. The recovered AqpZ protein showed high water permeability comparable with AqpZ obtained from the membrane fraction. In this way, the total yield of bioactive AqpZ has been increased greatly, which will facilitate the structural and functional characterization and future applications of AqpZ. Copyright © 2015 Elsevier Inc. All rights reserved.

Physiological and Proteomic Analysis of Escherichia coli Iron-Limited Chemostat Growth

PubMed Central

Folsom, James Patrick; Parker, Albert E.

2014-01-01

Iron bioavailability is a major limiter of bacterial growth in mammalian host tissue and thus represents an important area of study. Escherichia coli K-12 metabolism was studied at four levels of iron limitation in chemostats using physiological and proteomic analyses. The data documented an E. coli acclimation gradient where progressively more severe iron scarcity resulted in a larger percentage of substrate carbon being directed into an overflow metabolism accompanied by a decrease in biomass yield on glucose. Acetate was the primary secreted organic by-product for moderate levels of iron limitation, but as stress increased, the metabolism shifted to secrete primarily lactate (∼70% of catabolized glucose carbon). Proteomic analysis reinforced the physiological data and quantified relative increases in glycolysis enzyme abundance and decreases in tricarboxylic acid (TCA) cycle enzyme abundance with increasing iron limitation stress. The combined data indicated that E. coli responds to limiting iron by investing the scarce resource in essential enzymes, at the cost of catabolic efficiency (i.e., downregulating high-ATP-yielding pathways containing enzymes with large iron requirements, like the TCA cycle). Acclimation to iron-limited growth was contrasted experimentally with acclimation to glucose-limited growth to identify both general and nutrient-specific acclimation strategies. While the iron-limited cultures maximized biomass yields on iron and increased expression of iron acquisition strategies, the glucose-limited cultures maximized biomass yields on glucose and increased expression of carbon acquisition strategies. This study quantified ecologically competitive acclimations to nutrient limitations, yielding knowledge essential for understanding medically relevant bacterial responses to host and to developing intervention strategies. PMID:24837288

High-Yield Site-Specific Conjugation of Fibroblast Growth Factor 1 with Monomethylauristatin E via Cysteine Flanked by Basic Residues.

PubMed

Lobocki, Michal; Zakrzewska, Malgorzata; Szlachcic, Anna; Krzyscik, Mateusz A; Sokolowska-Wedzina, Aleksandra; Otlewski, Jacek

2017-07-19

Site-specific conjugation is a leading trend in the development of protein conjugates, including antibody-drug conjugates (ADCs), suitable for targeted cancer therapy. Here, we present a very efficient strategy for specific attachment of a cytotoxic drug to fibroblast growth factor 1 (FGF1), a natural ligand of FGF receptors (FGFRs), which are over-expressed in several types of lung, breast, and gastric cancers and are therefore an attractive molecular target. Recently, we showed that FGF1 fused to monomethylauristatin E (vcMMAE) was highly cytotoxic to cells presenting FGFRs on their surface and could be used as a targeting agent alternative to an antibody. Unfortunately, conjugation via maleimide chemistry to endogenous FGF1 cysteines or a cysteine introduced at the N-terminus proceeded with low yield and led to nonhomogeneous products. To improve the conjugation, we introduced a novel Lys-Cys-Lys motif at either FGF1 terminus, which increased cysteine reactivity and allowed us to obtain an FGF1 conjugate with a defined site of conjugation and a yield exceeding 95%. Using FGFR-expressing cancer lines, we confirmed specific cytotoxity of the obtained C-terminal FGF1-vcMMAE conjugate and its selective endocytososis as compared with FGFR1-negative cells. This simple and powerful approach relying on the introduction of a short sequence containing cysteine and positively charged amino acids could be used universally to improve the efficiency of the site-specific chemical modification of other proteins.

Seed-Specific Expression of OsDWF4, a Rate-Limiting Gene Involved in Brassinosteroids Biosynthesis, Improves Both Grain Yield and Quality in Rice.

PubMed

Li, Qian-Feng; Yu, Jia-Wen; Lu, Jun; Fei, Hong-Yuan; Luo, Ming; Cao, Bu-Wei; Huang, Li-Chun; Zhang, Chang-Quan; Liu, Qiao-Quan

2018-04-18

Brassinosteroids (BRs) are essential plant-specific steroidal hormones that regulate diverse growth and developmental processes in plants. We evaluated the effects of OsDWF4, a gene that encodes a rate-limiting enzyme in BR biosynthesis, on both rice yield and quality when driven by the Gt1 or Ubi promoter, which correspond to seed-specific or constitutive expression, respectively. Generally, transgenic plants expressing OsDWF4 showed increased grain yield with more tillers and longer and heavier seeds. Moreover, the starch physicochemical properties of the transgenic rice were also improved. Interestingly, OsDWF4 was found to exert different effects on either rice yield or quality when driven by the different promoters. The overall performance of the pGt1::OsDWF4 lines was better than that of the pUbi::OsDWF4 lines. Our data not only demonstrate the effects of OsDWF4 overexpression on both rice yield and quality but also suggest that a seed-specific promoter is a good choice in BR-mediated rice breeding programs.

Nandrolone reduces activation of Notch signaling in denervated muscle associated with increased Numb expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Xin-Hua; Department of Medicine, Mount Sinai School of Medicine, New York, NY 10029; Yao, Shen

2011-10-14

Highlights: {yields} Nerve transection increased Notch signaling in paralyzed muscle. {yields} Nandrolone prevented denervation-induced Notch signaling. {yields} Nandrolone induced the expression of an inhibitor of the Notch signaling, Numb. {yields} Reduction of denervation-induced Notch signaling by nandrolone is likely through upregulation of Numb. -- Abstract: Nandrolone, an anabolic steroid, slows denervation-atrophy in rat muscle. The molecular mechanisms responsible for this effect are not well understood. Androgens and anabolic steroids activate Notch signaling in animal models of aging and thereby mitigate sarcopenia. To explore the molecular mechanisms by which nandrolone prevents denervation-atrophy, we investigated the effects of nandrolone on Notch signalingmore » in denervated rat gastrocnemius muscle. Denervation significantly increased Notch activity reflected by elevated levels of nuclear Notch intracellular domain (NICD) and expression of Hey1 (a Notch target gene). Activation was greatest at 7 and 35 days after denervation but remained present at 56 days after denervation. Activation of Notch in denervated muscle was prevented by nandrolone associated with upregulated expression of Numb mRNA and protein. These data demonstrate that denervation activates Notch signaling, and that nandrolone abrogates this response associated with increased expression of Numb, suggesting a potential mechanism by which nandrolone reduces denervation-atrophy.« less

A Recombinant Human Anti-Platelet scFv Antibody Produced in Pichia pastoris for Atheroma Targeting

PubMed Central

Vallet-Courbin, Amelie; Larivière, Mélusine; Hocquellet, Agnès; Hemadou, Audrey; Parimala, Sarjapura-Nagaraja; Laroche-Traineau, Jeanny; Santarelli, Xavier; Clofent-Sanchez, Gisèle; Jacobin-Valat, Marie-Josée; Noubhani, Abdelmajid

2017-01-01

Cells of the innate and adaptive immune system are key factors in the progression of atherosclerotic plaque, leading to plaque instability and rupture, potentially resulting in acute atherothrombotic events such as coronary artery disease, cerebrovascular disease and peripheral arterial disease. Here, we describe the cloning, expression, purification, and immunoreactivity assessment of a recombinant single-chain variable fragment (scFv) derived from a human anti-αIIbβ3 antibody (HuAb) selected to target atheromatous lesions for the presence of platelets. Indeed, platelets within atheroma plaques have been shown to play a role in inflammation, in platelet-leucocyte aggregates and in thrombi formation and might thus be considered relevant biomarkers of atherosclerotic progression. The DNA sequence that encodes the anti-αIIbβ3 TEG4 scFv previously obtained from a phage-display selection on activated platelets, was inserted into the eukaryote vector (pPICZαA) in fusion with a tag sequence encoding 2 cysteines useable for specific probes grafting experiments. The recombinant protein was expressed at high yields in Pichia pastoris (30 mg/L culture). The advantage of P. pastoris as an expression system is the production and secretion of recombinant proteins in the supernatant, ruling out the difficulties encountered when scFv are produced in the cytoplasm of bacteria (low yield, low solubility and reduced affinity). The improved conditions allowed for the recovery of highly purified and biologically active scFv fragments ready to be grafted in a site-directed way to nanoparticles for the imaging of atherosclerotic plaques involving inflammatory processes and thus at high risk of instability. PMID:28125612

Stable 293 T and CHO cell lines expressing cleaved, stable HIV-1 envelope glycoprotein trimers for structural and vaccine studies.

PubMed

Chung, Nancy P Y; Matthews, Katie; Kim, Helen J; Ketas, Thomas J; Golabek, Michael; de Los Reyes, Kevin; Korzun, Jacob; Yasmeen, Anila; Sanders, Rogier W; Klasse, Per Johan; Wilson, Ian A; Ward, Andrew B; Marozsan, Andre J; Moore, John P; Cupo, Albert

2014-04-25

Recombinant soluble, cleaved HIV-1 envelope glycoprotein SOSIP.664 gp140 trimers based on the subtype A BG505 sequence are being studied structurally and tested as immunogens in animals. For these trimers to become a vaccine candidate for human trials, they would need to be made in appropriate amounts at an acceptable quality. Accomplishing such tasks by transient transfection is likely to be challenging. The traditional way to express recombinant proteins in large amounts is via a permanent cell line, usually of mammalian origin. Making cell lines that produce BG505 SOSIP.664 trimers requires the co-expression of the Furin protease to ensure that the cleavage site between the gp120 and gp41 subunits is fully utilized. We designed a vector capable of expressing Env and Furin, and used it to create Stable 293 T and CHO Flp-In™ cell lines through site-specific recombination. Both lines produce high quality, cleaved trimers at yields of up to 12-15 mg per 1 × 109 cells. Trimer expression at such levels was maintained for up to 30 days (10 passages) after initial seeding and was consistently superior to what could be achieved by transient transfection. Electron microscopy studies confirm that the purified trimers have the same native-like appearance as those derived by transient transfection and used to generate high-resolution structures. They also have appropriate antigenic properties, including the presentation of the quaternary epitope for the broadly neutralizing antibody PGT145. The BG505 SOSIP.664 trimer-expressing cell lines yield proteins of an appropriate quality for structural studies and animal immunogenicity experiments. The methodology is suitable for making similar lines under Good Manufacturing Practice conditions, to produce trimers for human clinical trials. Moreover, any env gene can be incorporated into this vector system, allowing the manufacture of SOSIP trimers from multiple genotypes, either by transient transfection or from stable cell lines.

Biophysical Constraints Arising from Compositional Context in Synthetic Gene Networks.

PubMed

Yeung, Enoch; Dy, Aaron J; Martin, Kyle B; Ng, Andrew H; Del Vecchio, Domitilla; Beck, James L; Collins, James J; Murray, Richard M

2017-07-26

Synthetic gene expression is highly sensitive to intragenic compositional context (promoter structure, spacing regions between promoter and coding sequences, and ribosome binding sites). However, much less is known about the effects of intergenic compositional context (spatial arrangement and orientation of entire genes on DNA) on expression levels in synthetic gene networks. We compare expression of induced genes arranged in convergent, divergent, or tandem orientations. Induction of convergent genes yielded up to 400% higher expression, greater ultrasensitivity, and dynamic range than divergent- or tandem-oriented genes. Orientation affects gene expression whether one or both genes are induced. We postulate that transcriptional interference in divergent and tandem genes, mediated by supercoiling, can explain differences in expression and validate this hypothesis through modeling and in vitro supercoiling relaxation experiments. Treatment with gyrase abrogated intergenic context effects, bringing expression levels within 30% of each other. We rebuilt the toggle switch with convergent genes, taking advantage of supercoiling effects to improve threshold detection and switch stability. Copyright © 2017 Elsevier Inc. All rights reserved.

Transient, Inducible, Placenta-Specific Gene Expression in Mice

PubMed Central

Fan, Xiujun; Petitt, Matthew; Gamboa, Matthew; Huang, Mei; Dhal, Sabita; Druzin, Maurice L.; Wu, Joseph C.

2012-01-01

Molecular understanding of placental functions and pregnancy disorders is limited by the absence of methods for placenta-specific gene manipulation. Although persistent placenta-specific gene expression has been achieved by lentivirus-based gene delivery methods, developmentally and physiologically important placental genes have highly stage-specific functions, requiring controllable, transient expression systems for functional analysis. Here, we describe an inducible, placenta-specific gene expression system that enables high-level, transient transgene expression and monitoring of gene expression by live bioluminescence imaging in mouse placenta at different stages of pregnancy. We used the third generation tetracycline-responsive tranactivator protein Tet-On 3G, with 10- to 100-fold increased sensitivity to doxycycline (Dox) compared with previous versions, enabling unusually sensitive on-off control of gene expression in vivo. Transgenic mice expressing Tet-On 3G were created using a new integrase-based, site-specific approach, yielding high-level transgene expression driven by a ubiquitous promoter. Blastocysts from these mice were transduced with the Tet-On 3G-response element promoter-driving firefly luciferase using lentivirus-mediated placenta-specific gene delivery and transferred into wild-type pseudopregnant recipients for placenta-specific, Dox-inducible gene expression. Systemic Dox administration at various time points during pregnancy led to transient, placenta-specific firefly luciferase expression as early as d 5 of pregnancy in a Dox dose-dependent manner. This system enables, for the first time, reliable pregnancy stage-specific induction of gene expression in the placenta and live monitoring of gene expression during pregnancy. It will be widely applicable to studies of both placental development and pregnancy, and the site-specific Tet-On G3 mouse will be valuable for studies in a broad range of tissues. PMID:23011919

Comparison of laccase production levels in Pichia pastoris and Cryptococcus sp. S-2.

PubMed

Nishibori, Nahoko; Masaki, Kazuo; Tsuchioka, Hiroaki; Fujii, Tsutomu; Iefuji, Haruyuki

2013-04-01

The heterologous expression of the laccase gene from Trametes versicolor and Gaeumannomyces graminis was evaluated in the yeasts Pichia pastoris and Cryptococcus sp. S-2. The expression levels of both laccase genes in Cryptococcus sp. S-2 were considerably higher than those in P. pastoris. The codon usage of Cryptococcus sp. S-2 as well as the GC content were similar to those of T. versicolor and G. graminis. These results suggest that using a host with a similar codon usage for the expressed gene may improve protein expression. The use of Cryptococcus sp. S-2 as a host may be advantageous for the heterologous expression of genes with high GC content. Moreover, this yeast provides the same advantages as P. pastoris for the production of recombinant proteins, such as growth on minimal medium, capacity for high-density growth during fermentation, and capability for post-translational modifications. Therefore, we propose that Cryptococcus sp. S-2 be used as an expression host to improve enzyme production levels when other hosts have not yielded good results. Copyright © 2012 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

A mutant sumo facilitates quick plasmid construction for expressing proteins with native N-termini after fusion tag removal

USDA-ARS?s Scientific Manuscript database

Sumo is one of the fusion tags commonly used to enhance the solubility and yield of recombinant proteins. One advantage of using sumo is that the removal of the sumo tag is highly specific because its recognition by the ULP sumo protease is determined by its structural characteristics, instead of th...

Direct multiplexed measurement of gene expression with color-coded probe pairs.

PubMed

Geiss, Gary K; Bumgarner, Roger E; Birditt, Brian; Dahl, Timothy; Dowidar, Naeem; Dunaway, Dwayne L; Fell, H Perry; Ferree, Sean; George, Renee D; Grogan, Tammy; James, Jeffrey J; Maysuria, Malini; Mitton, Jeffrey D; Oliveri, Paola; Osborn, Jennifer L; Peng, Tao; Ratcliffe, Amber L; Webster, Philippa J; Davidson, Eric H; Hood, Leroy; Dimitrov, Krassen

2008-03-01

We describe a technology, the NanoString nCounter gene expression system, which captures and counts individual mRNA transcripts. Advantages over existing platforms include direct measurement of mRNA expression levels without enzymatic reactions or bias, sensitivity coupled with high multiplex capability, and digital readout. Experiments performed on 509 human genes yielded a replicate correlation coefficient of 0.999, a detection limit between 0.1 fM and 0.5 fM, and a linear dynamic range of over 500-fold. Comparison of the NanoString nCounter gene expression system with microarrays and TaqMan PCR demonstrated that the nCounter system is more sensitive than microarrays and similar in sensitivity to real-time PCR. Finally, a comparison of transcript levels for 21 genes across seven samples measured by the nCounter system and SYBR Green real-time PCR demonstrated similar patterns of gene expression at all transcript levels.

A toy model for the yield of a tamped fission bomb

NASA Astrophysics Data System (ADS)

Reed, B. Cameron

2018-02-01

A simple expression is developed for estimating the yield of a tamped fission bomb, that is, a basic nuclear weapon comprising a fissile core jacketed by a surrounding neutron-reflecting tamper. This expression is based on modeling the nuclear chain reaction as a geometric progression in combination with a previously published expression for the threshold-criticality condition for such a core. The derivation is especially straightforward, as it requires no knowledge of diffusion theory and should be accessible to students of both physics and policy. The calculation can be set up as a single page spreadsheet. Application to the Little Boy and Fat Man bombs of World War II gives results in reasonable accord with published yield estimates for these weapons.

High-yield expression in Escherichia coli, purification and application of budding yeast K2 killer protein.

PubMed

Podoliankaitė, Monika; Lukša, Juliana; Vyšniauskis, Gintautas; Sereikaitė, Jolanta; Melvydas, Vytautas; Serva, Saulius; Servienė, Elena

2014-07-01

Saccharomyces cerevisiae K2 toxin is a highly active extracellular protein, important as a biocontrol agent for biotechnological applications in the wine industry. This protein is produced at negligible levels in yeast, making difficult to isolate it in amounts sufficient for investigation and generation of analysis tools. In this work, we demonstrate the use of a bacterial system for expression of the recombinant K2 protein, suitable for generation of antibodies specific for toxin of the yeast origin. Synthesis of the full-length S. cerevisiae K2 preprotoxin in Escherichia coli was found to be toxic to the host cell, resulting in diminished growth. Such effect was abolished by the introduction of the C-terminal truncation into K2 protein, directing it into non-toxic inclusion body fraction. The obtained protein is of limited solubility thus, facilitating the purification by simple and efficient chromatography-free procedure. The protein aggregates were successfully refolded into a soluble form yielding sufficient amounts of a tag-less truncated K2 protein suitable for polyclonal antibody production. Antibodies were raised in rabbit and found to be specific for detection of both antigen and native S. cerevisiae K2 toxin.

Using Haloarcula marismortui Bacteriorhodopsin as a Fusion Tag for Enhancing and Visible Expression of Integral Membrane Proteins in Escherichia coli

PubMed Central

Hsu, Min-Feng; Yu, Tsung-Fu; Chou, Chia-Cheng; Fu, Hsu-Yuan; Yang, Chii-Shen; Wang, Andrew H. J.

2013-01-01

Membrane proteins are key targets for pharmacological intervention because of their vital functions. Structural and functional studies of membrane proteins have been severely hampered because of the difficulties in producing sufficient quantities of properly folded and biologically active proteins. Here we generate a high-level expression system of integral membrane proteins in Escherichia coli by using a mutated bacteriorhodopsin (BR) from Haloarcula marismortui (HmBRI/D94N) as a fusion partner. A purification strategy was designed by incorporating a His-tag on the target membrane protein for affinity purification and an appropriate protease cleavage site to generate the final products. The fusion system can be used to detect the intended target membrane proteins during overexpression and purification either with the naked eye or by directly monitoring their characteristic optical absorption. In this study, we applied this approach to produce two functional integral membrane proteins, undecaprenyl pyrophosphate phosphatase and carnitine/butyrobetaine antiporter with significant yield enhancement. This technology could facilitate the development of a high-throughput strategy to screen for conditions that improve the yield of correctly folded target membrane proteins. Other robust BRs can also be incorporated in this system. PMID:23457558

Generation of high-yield insulin producing cells from human bone marrow mesenchymal stem cells.

PubMed

Jafarian, Arefeh; Taghikhani, Mohammad; Abroun, Saeid; Pourpak, Zahra; Allahverdi, Amir; Soleimani, Masoud

2014-07-01

Allogenic islet transplantation is a most efficient approach for treatment of diabetes mellitus. However, the scarcity of islets and long term need for an immunosuppressant limits its application. Recently, cell replacement therapies that generate of unlimited sources of β cells have been developed to overcome these limitations. In this study we have described a stage specific differentiation protocol for the generation of insulin producing islet-like clusters from human bone marrow mesenchymal stem cells (hBM-MSCs). This specific stepwise protocol induced differentiation of hMSCs into definitive endoderm, pancreatic endoderm and pancreatic endocrine cells that expressed of sox17, foxa2, pdx1, ngn3, nkx2.2, insulin, glucagon, somatostatin, pancreatic polypeptide, and glut2 transcripts respectively. In addition, immunocytochemical analysis confirmed protein expression of the above mentioned genes. Western blot analysis discriminated insulin from proinsulin in the final differentiated cells. In derived insulin producing cells (IPCs), secreted insulin and C-peptide was in a glucose dependent manner. We have developed a protocol that generates effective high-yield human IPCs from hBM-MSCs in vitro. These finding suggest that functional IPCs generated by this procedure can be used as a cell-based approach for insulin dependent diabetes mellitus.

OsGRF4 controls grain shape, panicle length and seed shattering in rice

PubMed Central

Sun, Pingyong; Zhang, Wuhan; Wang, Yihua; He, Qiang; Shu, Fu; Liu, Hai; Wang, Jie; Wang, Jianmin; Yuan, Longping

2016-01-01

Abstract Traits such as grain shape, panicle length and seed shattering, play important roles in grain yield and harvest. In this study, the cloning and functional analysis of PANICLE TRAITS 2 (PT2), a novel gene from the Indica rice Chuandali (CDL), is reported. PT2 is synonymous with Growth‐Regulating Factor 4 (OsGRF4), which encodes a growth‐regulating factor that positively regulates grain shape and panicle length and negatively regulates seed shattering. Higher expression of OsGRF4 is correlated with larger grain, longer panicle and lower seed shattering. A unique OsGRF4 mutation, which occurs at the OsmiRNA396 target site of OsGRF4, seems to be associated with high levels of OsGRF4 expression, and results in phenotypic difference. Further research showed that OsGRF4 regulated two cytokinin dehydrogenase precursor genes (CKX5 and CKX1) resulting in increased cytokinin levels, which might affect the panicle traits. High storage capacity and moderate seed shattering of OsGRF4 may be useful in high‐yield breeding and mechanized harvesting of rice. Our findings provide additional insight into the molecular basis of panicle growth. PMID:26936408

PPAR{alpha} deficiency augments a ketogenic diet-induced circadian PAI-1 expression possibly through PPAR{gamma} activation in the liver

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oishi, Katsutaka, E-mail: k-ooishi@aist.go.jp; Uchida, Daisuke; Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki

Research highlights: {yields} PPAR{alpha} deficiency augments a ketogenic diet-induced circadian PAI-1 expression. {yields} Hepatic expressions of PPAR{gamma} and PCG-1{alpha} are induced by a ketogenic diet. {yields} PPAR{gamma} antagonist attenuates a ketogenic diet-induced PAI-1 expression. {yields} Ketogenic diet advances the phase of circadian clock in a PPAR{alpha}-independent manner. -- Abstract: An increased level of plasminogen activator inhibitor-1 (PAI-1) is considered a risk factor for cardiovascular diseases, and PAI-1 gene expression is under the control of molecular circadian clocks in mammals. We recently showed that PAI-1 expression is augmented in a phase-advanced circadian manner in mice fed with a ketogenic diet (KD).more » To determine whether peroxisome proliferator-activated receptor {alpha} (PPAR{alpha}) is involved in hypofibrinolytic status induced by a KD, we examined the expression profiles of PAI-1 and circadian clock genes in PPAR{alpha}-null KD mice. Chronic administration of bezafibrate induced the PAI-1 gene expression in a PPAR{alpha}-dependent manner. Feeding with a KD augmented the circadian expression of PAI-1 mRNA in the hearts and livers of wild-type (WT) mice as previously described. The KD-induced mRNA expression of typical PPAR{alpha} target genes such as Cyp4A10 and FGF21 was damped in PPAR{alpha}-null mice. However, plasma PAI-1 concentrations were significantly more elevated in PPAR{alpha}-null KD mice in accordance with hepatic mRNA levels. These observations suggest that PPAR{alpha} activation is dispensable for KD-induced PAI-1 expression. We also found that hyperlipidemia, fatty liver, and the hepatic expressions of PPAR{gamma} and its coactivator PCG-1{alpha} were more effectively induced in PPAR{alpha}-null, than in WT mice on a KD. Furthermore, KD-induced hepatic PAI-1 expression was significantly suppressed by supplementation with bisphenol A diglycidyl ether, a PPAR{gamma} antagonist, in both WT and PPAR{alpha}-null mice. PPAR{gamma} activation seems to be involved in KD-induced hypofibrinolysis by augmenting PAI-1 gene expression in the fatty liver.« less

Engineering a Saccharomyces cerevisiae wine yeast that exhibits reduced ethanol production during fermentation under controlled microoxygenation conditions.

PubMed

Heux, Stéphanie; Sablayrolles, Jean-Marie; Cachon, Rémy; Dequin, Sylvie

2006-09-01

We recently showed that expressing an H(2)O-NADH oxidase in Saccharomyces cerevisiae drastically reduces the intracellular NADH concentration and substantially alters the distribution of metabolic fluxes in the cell. Although the engineered strain produces a reduced amount of ethanol, a high level of acetaldehyde accumulates early in the process (1 g/liter), impairing growth and fermentation performance. To overcome these undesirable effects, we carried out a comprehensive analysis of the impact of oxygen on the metabolic network of the same NADH oxidase-expressing strain. While reducing the oxygen transfer rate led to a gradual recovery of the growth and fermentation performance, its impact on the ethanol yield was negligible. In contrast, supplying oxygen only during the stationary phase resulted in a 7% reduction in the ethanol yield, but without affecting growth and fermentation. This approach thus represents an effective strategy for producing wine with reduced levels of alcohol. Importantly, our data also point to a significant role for NAD(+) reoxidation in controlling the glycolytic flux, indicating that engineered yeast strains expressing an NADH oxidase can be used as a powerful tool for gaining insight into redox metabolism in yeast.

Cloning, optimization of induction conditions and purification of Mycobacterium tuberculosis Rv1733c protein expressed in Escherichia coli

PubMed Central

Ashayeri-Panah, Mitra; Eftekhar, Fereshteh; Kazemi, Bahram; Joseph, Joan

2017-01-01

Background and Objectives: Rv1733c is a latency antigen from Mycobacterium tuberculosis, a probable integral-membrane protein with promiscuous T-cell and B-cell epitopes, making it a potential vaccine candidate against tuberculosis. This study aimed to clone and optimize the expression of recombinant Rv1733c in Escherichia coli for purification. Materials and Methods: Chemically synthesized rv1733c coding sequence was cloned in pET-23a(+) followed by transforming E. coli BL21 (DE3) cells. To evaluate the induction conditions for optimized expression, factorial design of experiments was employed using four different media as well as four levels of isopropyl-b-D-thiogalactopyranosid [IPTG] concentration and duration of induction. The recombinant protein was then purified using a His-tag purification kit and detected through western blotting. Results: Recombinant Rv1733c (> 24 kDa) was expressed and accumulated in the cytoplasm of the E. coli cells. Medium composition showed the most significant effect on the yield of the recombinant protein (P = 0.000). The highest yield of recombinant Rv1733c occurred in the presence of 0.4 mM of IPTG in Terrific Broth medium (containing 1.2% tryptone, 2.4% yeast extract, 72 mM K 2 HPO 4 , 17 mM KH 2 PO 4 and 0.4% glycerol) after 10 h at 37°C. Under these conditions, the expression level was around 0.5 g/L of culture medium. Purified Rv1733c was detected by an anti-polyhistidine antibody and a tuberculosis patient’s serum. Systematic optimization of induction conditions gave us high yield of recombinant polyhistidine-tagged Rv1733c in E. coli which was successfuly purified. Conclusion: We believe that the purified Rv1733c recombinant protein from M. tuberculosis might be a good candidate for vaccine production against tuberculosis. PMID:29213997

Yield and turnover of illicit indoor cannabis (Cannabis spp.) plantations in Belgium.

PubMed

Vanhove, Wouter; Surmont, Tim; Van Damme, Patrick; De Ruyver, Brice

2012-07-10

In prosecution, Belgian judiciary currently uses outdated yield figures (28.1g per plant, sold at € 3/g at grower level) for fining illicit indoor cannabis plantations. Using state-of-the-art cultivation techniques, our growth experiments showed that yield is better expressed in g/m(2) cultivated surface area rather than in g per plant, and that yield varies significantly between different cannabis strains. It was found that the lower-bound of the one-sided 95% confidence interval of the yield of an indoor cannabis plantation can be set at 575 g/m(2). Prices and pricing mechanisms were investigated using interviews with respondents selected through snowball sampling. Results reveal that (i) the Belgian cannabis market chain is highly complex; (ii) unit prices are predominantly determined by transaction sizes; but also (iii) a set of product- and socially-related price-fixing mechanisms have an equally important role. At grower level, respondents reported prices for 1 g of dry cannabis buds to range € 3.00-4.25. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

The antifibrotic effects of TGF-{beta}1 siRNA on hepatic fibrosis in rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lang, Qing; Liu, Qi; Xu, Ning

2011-06-10

Highlights: {yields} We constructed CCL4 induced liver fibrosis model successfully. {yields} We proofed that the TGF-{beta}1 siRNA had a definite therapy effect to CCL4 induced liver fibrosis. {yields} The therapy effect of TGF-{beta}1 siRNA had dose-dependent. -- Abstract: Background/aims: Hepatic fibrosis results from the excessive secretion of matrix proteins by hepatic stellate cells (HSCs), which proliferate during fibrotic liver injury. Transforming growth factor (TGF)-{beta}1 is the dominant stimulus for extracellular matrix (ECM) production by stellate cells. Our study was designed to investigate the antifibrotic effects of using short interference RNA (siRNA) to target TGF-{beta}1 in hepatic fibrosis and its mechanismmore » in rats exposed to a high-fat diet and carbon tetrachloride (CCL4). Methods: A total of 40 healthy, male SD (Sprague-Dawley) rats were randomly divided into five even groups containing of eight rats each: normal group, model group, TGF-{beta}1 siRNA 0.125 mg/kg treatment group, TGF-{beta}1 siRNA 0.25 mg/kg treatment group and TGF-{beta}1 siRNA negative control group (0.25 mg/kg). CCL4 and a high-fat diet were used for 8 weeks to induce hepatic fibrosis. All the rats were then sacrificed to collect liver tissue samples. A portion of the liver samples were soaked in formalin for Hematoxylin-Eosin staining, classifying the degree of liver fibrosis, and detecting the expression of type I and III collagen and TGF-{beta}1; the remaining liver samples were stored in liquid nitrogen to be used for detecting TGF-{beta}1 by Western blotting and for measuring the mRNA expression of type I and III collagen and TGF-{beta}1 by quantitative real-time polymerase chain reaction. Results: Comparing the TGF-{beta}1 siRNA 0.25 mg/kg treatment group to the model group, the TGF-{beta}1 siRNA negative control group and the TGF-{beta}1 siRNA 0.125 mg/kg treatment group showed significantly reduced levels of pathological changes, protein expression and the mRNA expression of TGF-{beta}1, type I collagen and type III collagen (P < 0.01). Conclusions: Using siRNA to target TGF-{beta}1 can inhibit the expression of TGF-{beta}1 and attenuate rat hepatic fibrosis induced by a high-fat diet and CCL4. A possible mechanism is through the down-regulation of TGF-{beta}1 expression, which could inhibit HSC activation, as well as the proliferation and collagen production of collagen reducing, so that collagen deposition in the liver is reduced.« less

Medium-throughput processing of whole mount in situ hybridisation experiments into gene expression domains.

PubMed

Crombach, Anton; Cicin-Sain, Damjan; Wotton, Karl R; Jaeger, Johannes

2012-01-01

Understanding the function and evolution of developmental regulatory networks requires the characterisation and quantification of spatio-temporal gene expression patterns across a range of systems and species. However, most high-throughput methods to measure the dynamics of gene expression do not preserve the detailed spatial information needed in this context. For this reason, quantification methods based on image bioinformatics have become increasingly important over the past few years. Most available approaches in this field either focus on the detailed and accurate quantification of a small set of gene expression patterns, or attempt high-throughput analysis of spatial expression through binary pattern extraction and large-scale analysis of the resulting datasets. Here we present a robust, "medium-throughput" pipeline to process in situ hybridisation patterns from embryos of different species of flies. It bridges the gap between high-resolution, and high-throughput image processing methods, enabling us to quantify graded expression patterns along the antero-posterior axis of the embryo in an efficient and straightforward manner. Our method is based on a robust enzymatic (colorimetric) in situ hybridisation protocol and rapid data acquisition through wide-field microscopy. Data processing consists of image segmentation, profile extraction, and determination of expression domain boundary positions using a spline approximation. It results in sets of measured boundaries sorted by gene and developmental time point, which are analysed in terms of expression variability or spatio-temporal dynamics. Our method yields integrated time series of spatial gene expression, which can be used to reverse-engineer developmental gene regulatory networks across species. It is easily adaptable to other processes and species, enabling the in silico reconstitution of gene regulatory networks in a wide range of developmental contexts.

Non-growing Rhodopseudomonas palustris Increases the Hydrogen Gas Yield from Acetate by Shifting from the Glyoxylate Shunt to the Tricarboxylic Acid Cycle*

PubMed Central

McKinlay, James B.; Oda, Yasuhiro; Rühl, Martin; Posto, Amanda L.; Sauer, Uwe; Harwood, Caroline S.

2014-01-01

When starved for nitrogen, non-growing cells of the photosynthetic bacterium Rhodopseudomonas palustris continue to metabolize acetate and produce H2, an important industrial chemical and potential biofuel. The enzyme nitrogenase catalyzes H2 formation. The highest H2 yields are obtained when cells are deprived of N2 and thus use available electrons to synthesize H2 as the exclusive product of nitrogenase. To understand how R. palustris responds metabolically to increase H2 yields when it is starved for N2, and thus not growing, we tracked changes in biomass composition and global transcript levels. In addition to a 3.5-fold higher H2 yield by non-growing cells we also observed an accumulation of polyhydroxybutyrate to over 30% of the dry cell weight. The transcriptome of R. palustris showed down-regulation of biosynthetic processes and up-regulation of nitrogen scavenging mechanisms in response to N2 starvation but gene expression changes did not point to metabolic activities that could generate the reductant necessary to explain the high H2 yield. We therefore tracked 13C-labeled acetate through central metabolic pathways. We found that non-growing cells shifted their metabolism to use the tricarboxylic acid cycle to metabolize acetate in contrast to growing cells, which used the glyoxylate cycle exclusively. This shift enabled cells to more fully oxidize acetate, providing the necessary reducing power to explain the high H2 yield. PMID:24302724

Selected wild strains of Agaricus bisporus produce high yields of mushrooms at 25°C.

PubMed

Navarro, Pilar; Savoie, Jean-Michel

2015-01-01

To cultivate the button mushroom Agaricus bisporus in warm countries or during summer in temperate countries, while saving energy, is a challenge that could be addressed by using the biological diversity of the species. The objective was to evaluate the yield potential of eight wild strains previously selected in small scale experiments for their ability to produce mature fruiting bodies at 25°C and above. Culture units of 8 kg of compost were used. The yield expressed as weight or number per surface unit and earliness of fruiting were recorded during cultivation in climatic rooms at 17, 25 or 30°C. Only strains of A. bisporus var. burnettii were able to fruit at 30°C. At 25°C they produced the highest yields (27 kg m(-2)) and had best earliness. The yields at 25°C for the strains of A. bisporus var. bisporus ranged from 12 to 16 kg m(-2). The yield ratios 25°C/17°C ranged from 0.8 to 1.2. The variety burnettii originated in the Sonoran Desert in California showed adaptation for quickly producing fruiting bodies at high temperature when humidity conditions were favorable. Strains of the variety bisporus showed interesting potentials for their ability to produce mature fruiting bodies at higher temperature than present cultivars and might be used in breeding programs. Copyright © 2012 Revista Iberoamericana de Micología. Published by Elsevier Espana. All rights reserved.

Heterologous expression of proteins from Plasmodium falciparum: results from 1000 genes.

PubMed

Mehlin, Christopher; Boni, Erica; Buckner, Frederick S; Engel, Linnea; Feist, Tiffany; Gelb, Michael H; Haji, Lutfiyah; Kim, David; Liu, Colleen; Mueller, Natascha; Myler, Peter J; Reddy, J T; Sampson, Joshua N; Subramanian, E; Van Voorhis, Wesley C; Worthey, Elizabeth; Zucker, Frank; Hol, Wim G J

2006-08-01

As part of a structural genomics initiative, 1000 open reading frames from Plasmodium falciparum, the causative agent of the most deadly form of malaria, were tested in an E. coli protein expression system. Three hundred and thirty-seven of these targets were observed to express, although typically the protein was insoluble. Sixty-three of the targets provided soluble protein in yields ranging from 0.9 to 406.6 mg from one liter of rich media. Higher molecular weight, greater protein disorder (segmental analysis, SEG), more basic isoelectric point (pI), and a lack of homology to E. coli proteins were all highly and independently correlated with difficulties in expression. Surprisingly, codon usage and the percentage of adenosines and thymidines (%AT) did not appear to play a significant role. Of those proteins which expressed, high pI and a hypothetical annotation were both strongly and independently correlated with insolubility. The overwhelmingly important role of pI in both expression and solubility appears to be a surprising and fundamental issue in the heterologous expression of P. falciparum proteins in E. coli. Twelve targets which did not express in E. coli from the native gene sequence were codon-optimized through whole gene synthesis, resulting in the (insoluble) expression of three of these proteins. Seventeen targets which were expressed insolubly in E. coli were moved into a baculovirus/Sf-21 system, resulting in the soluble expression of one protein at a high level and six others at a low level. A variety of factors conspire to make the heterologous expression of P. falciparum proteins challenging, and these observations lay the groundwork for a rational approach to prioritizing and, ultimately, eliminating these impediments.

High-Throughput Microfluidic Labyrinth for the Label-free Isolation of Circulating Tumor Cells.

PubMed

Lin, Eric; Rivera-Báez, Lianette; Fouladdel, Shamileh; Yoon, Hyeun Joong; Guthrie, Stephanie; Wieger, Jacob; Deol, Yadwinder; Keller, Evan; Sahai, Vaibhav; Simeone, Diane M; Burness, Monika L; Azizi, Ebrahim; Wicha, Max S; Nagrath, Sunitha

2017-09-27

We present "Labyrinth," a label-free microfluidic device to isolate circulating tumor cells (CTCs) using the combination of long loops and sharp corners to focus both CTCs and white blood cells (WBCs) at a high throughput of 2.5 mL/min. The high yield (>90%) and purity (600 WBCs/mL) of Labyrinth enabled us to profile gene expression in CTCs. As proof of principle, we used previously established cancer stem cell gene signatures to profile single cells isolated from the blood of breast cancer patients. We observed heterogeneous subpopulations of CTCs expressing genes for stem cells, epithelial cells, mesenchymal cells, and cells transitioning between epithelial and mesenchymal. Labyrinth offers a cell-surface marker-independent single-cell isolation platform to study heterogeneous CTC subpopulations. Copyright © 2017 Elsevier Inc. All rights reserved.

Quercetin-induced downregulation of phospholipase D1 inhibits proliferation and invasion in U87 glioma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Mi Hee; Min, Do Sik, E-mail: minds@pusan.ac.kr

Highlights: {yields} Quercetin, a bioactive flavonoid, suppresses expression and enzymatic activity of phospholipase D1. {yields} Quercetin abolishes NFkB-induced phospholipase D1 expression via inhibition of NFkB transactivation. {yields} Quercetin-induced suppression of phospholipase D1 inhibits invasion and proliferation of human glioma cells. -- Abstract: Phospholipase D (PLD) has been recognized as a regulator of cell proliferation and tumorigenesis, but little is known about the molecules regulating PLD expression. Thus, the identification of small molecules inhibiting PLD expression would be an important advance in PLD-mediated physiology. Quercetin, a ubiquitous bioactive flavonoid, is known to inhibit proliferation and induce apoptosis in a variety ofmore » cancer cells. In the present study, we examined the effect of quercetin on the expression of PLD in U87 glioma cells. Quercetin significantly suppressed the expression of PLD1 at the transcriptional level. Moreover, quercetin abolished the protein expression of PLD1 in a time and dose-dependent manner, as well as inhibited PLD activity. Quercetin suppressed NF{kappa}B-induced PLD1 expression via inhibition of NFkB transactivation. Furthermore, quercetin inhibited activation and invasion of metalloproteinase-2 (MMP-2), a key modulator of glioma cell invasion, induced by phosphatidic acid (PA), a product of PLD activity. Taken together these data demonstrate that quercetin abolishes PLD1 expression and subsequently inhibits invasion and proliferation of glioma cells.« less

Optimization and revision of the production process of the Necator americanus glutathione S-transferase 1 (Na-GST-1), the lead hookworm vaccine recombinant protein candidate

PubMed Central

Curti, Elena; Seid, Christopher A; Hudspeth, Elissa; Center, Lori; Rezende, Wanderson; Pollet, Jeroen; Kwityn, Cliff; Hammond, Molly; Matsunami, Rise K; Engler, David A; Hotez, Peter J; Elena Bottazzi, Maria

2014-01-01

Infection by the human hookworm Necator americanus is a leading cause of anemia and disability in the developing countries of Africa, Asia, and the Americas. In order to prevent childhood hookworm disease in resource poor settings, a recombinant vaccine is under development by the Sabin Vaccine Institute and Texas Children’s Hospital Center for Vaccine Development, a Product Development Partnership (PDP). Previously, we reported on the expression and purification of a highly promising hookworm vaccine candidate, Na-GST-1, an N. americanus glutathione s-transferase expressed in Pichia pastoris (yeast), which led to production of 1.5 g of 95% pure recombinant protein at a 20L scale.1, 2, 3 This yield and purity of Na-GST-1 was sufficient for early pilot manufacturing and initial phase 1 clinical testing. However, based on the number of doses which would be required to allow mass vaccination and a potential goal to deliver a vaccine as inexpensively as possible, a higher yield of expression of the recombinant antigen at the lowest possible cost is highly desirable. Here we report on modifications to the fermentation (upstream process) of the antigen expressed in P. pastoris, and to the purification (downstream process) of the recombinant protein that allowed for a 2–3-fold improvement in the final yield of Na-GST-1 purified protein. The major improvements included upstream process changes such as the addition of a sorbitol pulse and co-feed during methanol induction as well as an extension of the induction stage to approximately 96 hours; downstream process changes included modifying the UFDF to flat sheet with a 10 kDa Molecular Weight cut-off (MWCO), adjusting the capacity of an ion-exchange chromatography step utilizing a gradient elution as opposed to the original step elution, and altering the hydrophobic interaction chromatography conditions. The full process, as well as the purity and stability profiles of the target Na-GST-1, and its formulation on Alhydrogel®, is described. PMID:25424799

High yield expression and purification of equilibrative nucleoside transporter 7 (ENT7) from Arabidopsis thaliana.

PubMed

Girke, Christopher; Arutyunova, Elena; Syed, Maria; Traub, Michaela; Möhlmann, Torsten; Lemieux, M Joanne

2015-09-01

Equilibrative nucleoside transporters (ENTs) facilitate the import of nucleosides and their analogs into cells in a bidirectional, non-concentrative manner. However, in contrast to their name, most characterized plant ENTs act in a concentrative manner. A direct characterization of any ENT protein has been hindered due to difficulties in overexpression and obtaining pure recombinant protein. The equilibrative nucleoside transporter 7 from Arabidopsis thaliana (AtENT7) was expressed in Xenopus laevis oocytes to assess mechanism of substrate uptake. Recombinant protein fused to enhanced green fluorescent protein (eGFP) was expressed in Pichia pastoris to characterize its oligomeric state by gel filtration and substrate binding by microscale thermophoresis (MST). AtENT7 expressed in X. laevis oocytes works as a classic equilibrative transporter. The expression of AtENT7-eGFP in the P. pastoris system yielded milligram amounts of pure protein that exists as stable homodimers. The concentration dependent binding of purine and pyrimidine nucleosides to the purified recombinant protein, assessed by MST, confirmed that AtENT7-eGFP is properly folded. For the first time the binding of nucleobases was observed for AtENT7. The availability of pure recombinant AtENT7 will permit detailed kinetic and structural studies of this unique member of the ENT family and, given the functional similarity to mammalian ENTs, will serve as a good model for understanding the structural basis of translocation mechanism for the family. Copyright © 2015 Elsevier B.V. All rights reserved.

Engineering growth factors for regenerative medicine applications.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mitchell, Aaron C.; Briquez, Priscilla S.; Hubbell, Jeffrey A.

Growth factors are important morphogenetic proteins that instruct cell behavior and guide tissue repair and renewal. Although their therapeutic potential holds great promise in regenerative medicine applications, translation of growth factors into clinical treatments has been hindered by limitations including poor protein stability, low recombinant expression yield, and suboptimal efficacy. This review highlights current tools, technologies, and approaches to design integrated and effective growth factor-based therapies for regenerative medicine applications. The first section describes rational and combinatorial protein engineering approaches that have been utilized to improve growth factor stability, expression yield, biodistribution, and serum half-life, or alter their cell traffickingmore » behavior or receptor binding affinity. The second section highlights elegant biomaterial-based systems, inspired by the natural extracellular matrix milieu, that have been developed for effective spatial and temporal delivery of growth factors to cell surface receptors. Although appearing distinct, these two approaches are highly complementary and involve principles of molecular design and engineering to be considered in parallel when developing optimal materials for clinical applications.« less

An improved process for the production of highly purified recombinant thaumatin tagged-variants.

PubMed

Healey, Robert D; Lebhar, Helene; Hornung, Simon; Thordarson, Pall; Marquis, Christopher P

2017-12-15

The sweetest tasting molecule known is the protein thaumatin, first isolated from the katemfe fruit, Thaumatococcus daniellii. Thaumatin is used in the food and beverage industry as a low-calorie sugar substitute. Thaumatin interacts with taste receptors in the oral cavity eliciting a persistent sweet taste and a bitter, liquorice flavor. Recombinant thaumatin was expressed in Pichia pastoris and through a co-expression strategy with a molecular chaperone, yields of one engineered thaumatin variant increased by greater than two-fold. A detailed purification strategy for thaumatin is reported resulting in a homogenous sample recovered at a yield of 42%. The recombinant thaumatins were extensively characterised using size exclusion chromatography for homogeneity, reversed-phase HPLC for purity (99%), peptide digest LC-MS/MS for sequence determination, and circular dichroism and tryptophan fluorescence spectroscopies for conformational characterisation. These new thaumatin variants are amenable for bioconjugation, providing chemical biology tools for thaumatin:taste receptor interaction studies. Copyright © 2017 Elsevier Ltd. All rights reserved.

Gene Expression Signatures Based on Variability can Robustly Predict Tumor Progression and Prognosis

PubMed Central

Dinalankara, Wikum; Bravo, Héctor Corrada

2015-01-01

Gene expression signatures are commonly used to create cancer prognosis and diagnosis methods, yet only a small number of them are successfully deployed in the clinic since many fail to replicate performance on subsequent validation. A primary reason for this lack of reproducibility is the fact that these signatures attempt to model the highly variable and unstable genomic behavior of cancer. Our group recently introduced gene expression anti-profiles as a robust methodology to derive gene expression signatures based on the observation that while gene expression measurements are highly heterogeneous across tumors of a specific cancer type relative to the normal tissue, their degree of deviation from normal tissue expression in specific genes involved in tissue differentiation is a stable tumor mark that is reproducible across experiments and cancer types. Here we show that constructing gene expression signatures based on variability and the anti-profile approach yields classifiers capable of successfully distinguishing benign growths from cancerous growths based on deviation from normal expression. We then show that this same approach generates stable and reproducible signatures that predict probability of relapse and survival based on tumor gene expression. These results suggest that using the anti-profile framework for the discovery of genomic signatures is an avenue leading to the development of reproducible signatures suitable for adoption in clinical settings. PMID:26078586

Discharge current modes of high power impulse magnetron sputtering

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Zhongzhen, E-mail: wuzz@pkusz.edu.cn; Xiao, Shu; Ma, Zhengyong

2015-09-15

Based on the production and disappearance of ions and electrons in the high power impulse magnetron sputtering plasma near the target, the expression of the discharge current is derived. Depending on the slope, six possible modes are deduced for the discharge current and the feasibility of each mode is discussed. The discharge parameters and target properties are simplified into the discharge voltage, sputtering yield, and ionization energy which mainly affect the discharge plasma. The relationship between these factors and the discharge current modes is also investigated.

Electron correlation in Hooke's law atom in the high-density limit.

PubMed

Gill, P M W; O'Neill, D P

2005-03-01

Closed-form expressions for the first three terms in the perturbation expansion of the exact energy and Hartree-Fock energy of the lowest singlet and triplet states of the Hooke's law atom are found. These yield elementary formulas for the exact correlation energies (-49.7028 and -5.807 65 mE(h)) of the two states in the high-density limit and lead to a pair of necessary conditions on the exact correlation kernel G(w) in Hartree-Fock-Wigner theory.

Cancer cell redirection biomarker discovery using a mutual information approach.

PubMed

Roche, Kimberly; Feltus, F Alex; Park, Jang Pyo; Coissieux, Marie-May; Chang, Chenyan; Chan, Vera B S; Bentires-Alj, Mohamed; Booth, Brian W

2017-01-01

Introducing tumor-derived cells into normal mammary stem cell niches at a sufficiently high ratio of normal to tumorous cells causes those tumor cells to undergo a change to normal mammary phenotype and yield normal mammary progeny. This phenomenon has been termed cancer cell redirection. We have developed an in vitro model that mimics in vivo redirection of cancer cells by the normal mammary microenvironment. Using the RNA profiling data from this cellular model, we examined high-level characteristics of the normal, redirected, and tumor transcriptomes and found the global expression profiles clearly distinguish the three expression states. To identify potential redirection biomarkers that cause the redirected state to shift toward the normal expression pattern, we used mutual information relationships between normal, redirected, and tumor cell groups. Mutual information relationship analysis reduced a dataset of over 35,000 gene expression measurements spread over 13,000 curated gene sets to a set of 20 significant molecular signatures totaling 906 unique loci. Several of these molecular signatures are hallmark drivers of the tumor state. Using differential expression as a guide, we further refined the gene set to 120 core redirection biomarker genes. The expression levels of these core biomarkers are sufficient to make the normal and redirected gene expression states indistinguishable from each other but radically different from the tumor state.

Cancer cell redirection biomarker discovery using a mutual information approach

PubMed Central

Roche, Kimberly; Feltus, F. Alex; Park, Jang Pyo; Coissieux, Marie-May; Chang, Chenyan; Chan, Vera B. S.; Bentires-Alj, Mohamed

2017-01-01

Introducing tumor-derived cells into normal mammary stem cell niches at a sufficiently high ratio of normal to tumorous cells causes those tumor cells to undergo a change to normal mammary phenotype and yield normal mammary progeny. This phenomenon has been termed cancer cell redirection. We have developed an in vitro model that mimics in vivo redirection of cancer cells by the normal mammary microenvironment. Using the RNA profiling data from this cellular model, we examined high-level characteristics of the normal, redirected, and tumor transcriptomes and found the global expression profiles clearly distinguish the three expression states. To identify potential redirection biomarkers that cause the redirected state to shift toward the normal expression pattern, we used mutual information relationships between normal, redirected, and tumor cell groups. Mutual information relationship analysis reduced a dataset of over 35,000 gene expression measurements spread over 13,000 curated gene sets to a set of 20 significant molecular signatures totaling 906 unique loci. Several of these molecular signatures are hallmark drivers of the tumor state. Using differential expression as a guide, we further refined the gene set to 120 core redirection biomarker genes. The expression levels of these core biomarkers are sufficient to make the normal and redirected gene expression states indistinguishable from each other but radically different from the tumor state. PMID:28594912

Recombinants proteins for industrial uses: utilization of Pichia pastoris expression system.

PubMed

Rabert, Claudia; Weinacker, Daniel; Pessoa, Adalberto; Farías, Jorge G

2013-01-01

The innovation in industrial process with impact in the efficient production is the major challenge for actual industry. A high numerous of enzymes are utilized in at different level of process; the search for new alternatives with better characteristic has become a field of study of great interest, the recombinant protein achievement in a different host system is an alternative widely assessed for production of this. The microorganism Pichia pastoris has been used like a successful expression system in diverse areas, improved the yield and extraction-recovery of the product expressed. The reported of diverse authors in the production of enzymes with different application in industry is varied, in this review the different industry areas and the characteristic of the enzymes produced are detailed.

Chilling Affects Phytohormone and Post-Embryonic Development Pathways during Bud Break and Fruit Set in Apple (Malus domestica Borkh.)

PubMed Central

Kumar, Gulshan; Gupta, Khushboo; Pathania, Shivalika; Swarnkar, Mohit Kumar; Rattan, Usha Kumari; Singh, Gagandeep; Sharma, Ram Kumar; Singh, Anil Kumar

2017-01-01

The availability of sufficient chilling during bud dormancy plays an important role in the subsequent yield and quality of apple fruit, whereas, insufficient chilling availability negatively impacts the apple production. The transcriptome profiling during bud dormancy release and initial fruit set under low and high chill conditions was performed using RNA-seq. The comparative high number of differentially expressed genes during bud break and fruit set under high chill condition indicates that chilling availability was associated with transcriptional reorganization. The comparative analysis reveals the differential expression of genes involved in phytohormone metabolism, particularly for Abscisic acid, gibberellic acid, ethylene, auxin and cytokinin. The expression of Dormancy Associated MADS-box, Flowering Locus C-like, Flowering Locus T-like and Terminal Flower 1-like genes was found to be modulated under differential chilling. The co-expression network analysis indentified two high chill specific modules that were found to be enriched for “post-embryonic development” GO terms. The network analysis also identified hub genes including Early flowering 7, RAF10, ZEP4 and F-box, which may be involved in regulating chilling-mediated dormancy release and fruit set. The results of transcriptome and co-expression network analysis indicate that chilling availability majorly regulates phytohormone-related pathways and post-embryonic development during bud break. PMID:28198417

Dihydroavenanthramide D inhibits human breast cancer cell invasion through suppression of MMP-9 expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Young-Rae; Noh, Eun-Mi; Oh, Hyun Ju

2011-02-25

Research highlights: {yields} MMP-9 plays a pivotal role in the invasion of MCF-7 breast cancer cells. {yields} TPA stimulates MMP-9 expression through activation of MAPK/NF-{kappa}B and MAPK/AP-1 pathways. {yields} Dihydroavenanthramide D suppresses MMP-9 expression via inhibition of TPA-induced MAPK/NF-{kappa}B and MAPK/AP-1 activations. {yields} Dihydroavenanthramide D blocks cell invasion of MCF-7 breast cancer cells. -- Abstract: Dihydroavenanthramide D (DHAvD) is a synthetic analog to naturally occurring avenanthramide, which is the active component of oat. Previous study demonstrates that DHAvD strongly inhibits activation of nuclear factor-kappa B (NF-{kappa}B), which is a major component in cancer cell invasion. The present study investigated whethermore » DHAvD can modulate MMP-9 expression and cell invasion in MCF-7 human breast cancer cells. MMP-9 expression and cell invasion in response to 12-O-tetradecanoylphorbol-13-acetate (TPA) was increased, whereas these inductions were muted by DHAvD. DHAvD also suppressed activation of mitogen-activated protein kinase (MAPK), and MAPK-mediated nuclear factor-kappa B (NF-{kappa}B) and activator protein-1 (AP-1) activations in TPA-treated MCF-7 cells. The results indicate that DHAvD-mediated inhibition of TPA-induced MMP-9 expression and cell invasion involves the suppression of the MAPK/NF-{kappa}B and MAPK/AP-1 pathways in MCF-7 cells. DHAvD may have potential value in breast cancer metastasis.« less

Overexpression of OsGATA12 regulates chlorophyll content, delays plant senescence and improves rice yield under high density planting.

PubMed

Lu, Guangwen; Casaretto, José A; Ying, Shan; Mahmood, Kashif; Liu, Fang; Bi, Yong-Mei; Rothstein, Steven J

2017-05-01

Agronomic traits controlling the formation, architecture and physiology of source and sink organs are main determinants of rice productivity. Semi-dwarf rice varieties with low tiller formation but high seed production per panicle and dark green and thick leaves with prolonged source activity are among the desirable traits to further increase the yield potential of rice. Here, we report the functional characterization of a zinc finger transcription factor, OsGATA12, whose overexpression causes increased leaf greenness, reduction of leaf and tiller number, and affects yield parameters. Reduced tillering allowed testing the transgenic plants under high density which resulted in significantly increased yield per area and higher harvest index compared to wild-type. We show that delayed senescence of transgenic plants and the corresponding longer stay-green phenotype is mainly due to increased chlorophyll and chloroplast number. Further, our work postulates that the increased greenness observed in the transgenic plants is due to more chlorophyll synthesis but most significantly to decreased chlorophyll degradation, which is supported by the reduced expression of genes involved in the chlorophyll degradation pathway. In particular we show evidence for the down-regulation of the STAY GREEN RICE gene and in vivo repression of its promoter by OsGATA12, which suggests a transcriptional repression function for a GATA transcription factor for prolonging the onset of senescence in cereals.

[Influence of over expression of CsRCA on photosynthesis of cucumber seedlings under high temperature stress.

PubMed

Bi, Huan Gai; Dong, Xu Bing; Liu, Pei Pei; Li, Qing Ming; Ai, Xi Zhen

2016-07-01

In the present work, transgenic cucumber seedlings over expressing CsRCA and wild-type cucumber seedlings '08-1'at three-leaf stage exposed to high temperature (40 ℃, PFD 600 μmol· m -2 · s -1 ) were used to study the regulatory mechanism of photosynthesis by CsRCA. The results showed that the mRNA abundance of rbcL and rbcS as well as the activities of ribulose bisphosphate carboxylic enzyme (Rubisco) and Rubisco activase (RCA) were significantly higher in CsRCA over-expressing cucumber seedlings than in wild type (WT). Following 2-h exposure to high temperature, a notable decrease was observed in photosynthetic rate (P n ), photochemical perfor-mance index based on the absorption of light energy (PI ABS ), activities of Rubisco and RCA as well as the relative expression of rbcL, rbcS and CsRCA in both wild-type cucumber seedlings and transgenic cucumber seedlings. It was found that high temperature stress led to higher W k , a parameter of chlorophyll (Chl) a fluorescence OJIP curve. Furthermore, high temperature greatly reduced the efficiency of electron transfer along the electron transport chain beyond Q A (ψ 0 ) and the quantum yield for electron transport (φ E0 ), indicating that PSII oxygen complexes (OEC) and electron transport chain downstream Q A were inhibited by high temperature. However, the inhibition could be alleviated by over expressing CsRCA in cucumber seedlings. Taken together, our data suggested that over expressing CsRCA improves photosynthesis in cucumber seedlings under high temperature stress by enhancing activities of the Rubisco and RCA, and maintaining the number of active reaction centers.

Susceptibility to viral infection is enhanced by stable expression of 3A or 3AB proteins from foot-and-mouth disease virus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rosas, Maria F.; Vieira, Yuri A.; Postigo, Raul

2008-10-10

The foot-and-mouth disease virus (FMDV) 3A protein is involved in virulence and host range. A distinguishing feature of FMDV 3B among picornaviruses is that three non-identical copies are encoded in the viral RNA and required for optimal replication in cell culture. Here, we have studied the involvement of the 3AB region on viral infection using constitutive and transient expression systems. BHK-21 stably transformed clones expressed low levels of FMDV 3A or 3A(B) proteins in the cell cytoplasm. Transformed cells stably expressing these proteins did not exhibit inner cellular rearrangements detectable by electron microscope analysis. Upon FMDV infection, clones expressing eithermore » 3A alone or 3A(B) proteins showed a significant increase in the percentage of infected cells, the number of plaque forming units and the virus yield. The 3A-enhancing effect was specific for FMDV as no increase in viral multiplication was observed in transformed clones infected with another picornavirus, encephalomyocarditis virus, or the negative-strand RNA virus vesicular stomatitis virus. A potential role of 3A protein in viral RNA translation was discarded by the lack of effect on FMDV IRES-dependent translation. Increased viral susceptibility was not caused by a released factor; neither the supernatant of transformed clones nor the addition of purified 3A protein to the infection medium was responsible for this effect. Unlike stable expression, high levels of 3A or 3A(B) protein transient expression led to unspecific inhibition of viral infection. Therefore, the effect observed on viral yield, which inversely correlated with the intracellular levels of 3A protein, suggests a transacting role operating on the FMDV multiplication cycle.« less

The relative expression levels of insulin-like growth factor 1 and myostatin mRNA in the asynchronous development of skeletal muscle in ducks during early development.

PubMed

Hu, Yan; Liu, Hongxiang; Shan, Yanju; Ji, Gaige; Xu, Wenjuan; Shu, Jingting; Li, Huifang

2015-08-10

Genetic selection is a powerful tool for modifying poultry muscle yield. Insulin-like growth factor I (IGF-I) and myostatin (MSTN) are important regulators of muscle growth, especially in the myogenesis stage. This study compared the developmental pattern of the pectoralis major (PM) and lateral gastrocnemius (LM) muscles, mRNA expression characterization of IGF-I and MSTN-A and their correlation between 14 days in ovo and 1 week post-hatch in two Chinese local duck breeds. During early development, the growth of duck PM and LM followed an asynchronous pattern. Variations in PM growth rate observed with development followed the relative variations of MSTN and IGF-I expression; however, the same behavior was not observed in LM. Moreover, the profile of IGF-I expression in duck skeletal muscles indicated that genetic selection for high meat-yield poultry has altered the temporal expression of IGF-I and affected cellular characteristics and mass by hatch in a PM-specific manner. The MSTN-A expression profile showed synchronization with the growth of skeletal muscle and peaks of myofiber proliferation. The expression patterns of IGF-I and MSTN suggest that duck pectoralis fibers are prioritized for proliferation in embryogenesis. The IGF-1/MSTN-A mRNA ratios in PM and LM presented very similar trends in the changes of myofiber characteristics, and differences in the IGF-1/MSTN-A mRNA ratio in PM between the two breeds corresponded to the timing of differences in PM mass between the varieties. Our results support the hypothesis that relative levels of IGF-I and MSTN mRNA may participate in ordering muscle growth rates with selected development. Copyright © 2015 Elsevier B.V. All rights reserved.

Expression, purification, and characterization of the Necator americanus aspartic protease-1 (Na-APR-1 (M74)) antigen, a component of the bivalent human hookworm vaccine.

PubMed

Seid, Christopher A; Curti, Elena; Jones, R Mark; Hudspeth, Elissa; Rezende, Wanderson; Pollet, Jeroen; Center, Lori; Versteeg, Leroy; Pritchard, Sonya; Musiychuk, Konstantin; Yusibov, Vidadi; Hotez, Peter J; Bottazzi, Maria Elena

2015-01-01

Over 400 million people living in the world's poorest developing nations are infected with hookworms, mostly of the genus Necator americanus. A bivalent human hookworm vaccine composed of the Necator americanus Glutathione S-Transferase-1 (Na-GST-1) and the Necator americanus Aspartic Protease-1 (Na-APR-1 (M74)) is currently under development by the Sabin Vaccine Institute Product Development Partnership (Sabin PDP). Both monovalent vaccines are currently in Phase 1 trials. Both Na-GST-1 and Na-APR-1 antigens are expressed as recombinant proteins. While Na-GST-1 was found to express with high yields in Pichia pastoris, the level of expression of Na-APR-1 in this host was too low to be suitable for a manufacturing process. When the tobacco plant Nicotiana benthamiana was evaluated as an expression system, acceptable levels of solubility, yield, and stability were attained. Observed expression levels of Na-APR-1 (M74) using this system are ∼300 mg/kg. Here we describe the achievements and obstacles encountered during process development as well as characterization and stability of the purified Na-APR-1 (M74) protein and formulated vaccine. The expression, purification and analysis of purified Na-APR-1 (M74) protein obtained from representative 5 kg reproducibility runs performed to qualify the Na-APR-1 (M74) production process is also presented. This process has been successfully transferred to a pilot plant and a 50 kg scale manufacturing campaign under current Good Manufacturing Practice (cGMP) has been performed. The 50 kg run has provided a sufficient amount of protein to support the ongoing hookworm vaccine development program of the Sabin PDP.

In type 1 diabetics, high-dose biotin may compensate for low hepatic insulin exposure, promoting a more normal expression of glycolytic and gluconeogenic enyzymes and thereby aiding glycemic control.

PubMed

McCarty, Mark F

2016-10-01

In type 1 diabetics, hepatic exposure to insulin is chronically subnormal even in the context of insulin therapy; as a result, expression of glycolytic enzymes is decreased, and that of gluconeogenic enzymes is enhanced, resulting in a physiologically inappropriate elevation of hepatic glucose output. Subnormal expression of glucokinase (GK) is of particular importance in this regard. Possible strategies for correcting this perturbation of hepatic enzyme expression include administration of small molecule allosteric activators of GK, as well as a procedure known as chronic intermittent intravenous insulin therapy (CIIIT); however, side effects accompany the use of GK activators, and CIIIT is time and labor intensive. Alternatively, administration of high-dose biotin has potential for modulating hepatic enzyme expression in a favorable way. Studies in rodents and in cultured hepatocytes demonstrate that, in the context of low insulin exposure, supra-physiological levels of biotin induce increased expression of GK while suppressing that of the key gluconeogenic enzyme phosphoenolpyruvate carboxykinase. These effects may be a downstream consequence of the fact that biotin down-regulates mRNA expression of FOXO1; insulin's antagonism of the activity of this transcription factor is largely responsible for its modulatory impact on hepatic glycolysis and gluconeogenesis. Hence, high-dose biotin may compensate for subnormal insulin exposure by suppressing FOXO1 levels. High-dose biotin also has the potential to oppose hepatic steatosis by down-regulating SREBP-1 expression. Two pilot trials of high-dose biotin (16 or 2mg per day) in type 1 diabetics have yielded promising results. There is also some reason to suspect that high-dose biotin could aid control of diabetic neuropathy and nephropathy via its stimulatory effect on cGMP production. Owing to the safety, good tolerance, moderate expense, and current availability of high-dose biotin, this strategy merits more extensive evaluation in type 1 diabetes. Copyright © 2016 Elsevier Ltd. All rights reserved.

Biometry and diversity of Arabica coffee genotypes cultivated in a high density plant system.

PubMed

Rodrigues, W N; Tomaz, M A; Ferrão, M A G; Martins, L D; Colodetti, T V; Brinate, S V B; Amaral, J F T; Sobreira, F M; Apostólico, M A

2016-02-11

The present study was developed to respond to the need for an increase in crop yield in the mountain region of Caparaó (southern Espírito Santo State, Brazil), an area of traditional coffee production. This study aimed to analyze the diversity and characterize the crop yield of genotypes of Coffea arabica L. with potential for cultivation in high plant density systems. In addition, it also aimed to quantify the expression of agronomic traits in this cultivation system and provide information on the genotypes with the highest cultivation potential in the studied region. The experiment followed a randomized block design with 16 genotypes, four repetitions, and six plants per experimental plot. Plant spacing was 2.00 x 0.60 m, with a total of 8333 plants per hectare, representing a high-density cultivation system. Coffee plants were cultivated until the start of their reproductive phenological cycles and were evaluated along four complete reproductive cycles. Genotypes with high crop yield and beverage quality, short canopy, and rust resistance were selected. C. arabica genotypes showed variability in almost all characteristics. It was possible to identify different responses among genotypes grown in a high plant density cultivation system. Although the chlorophyll a content was similar among genotypes, the genotypes Acauã, Araponga MG1, Sacramento MG1, Tupi, and Catuaí IAC 44 showed a higher chlorophyll b content than the other genotypes. Among these, Sacramento MG1 also showed high leafiness and growth of vegetative structures, whereas Araponga MG1, Pau-Brasil MG1, and Tupi showed high fruit production. In addition, Araponga MG1 had also a higher and more stable crop yield over the years.

Retroviral vectors encoding ADA regulatory locus control region provide enhanced T-cell-specific transgene expression

PubMed Central

2009-01-01

Background Murine retroviral vectors have been used in several hundred gene therapy clinical trials, but have fallen out of favor for a number of reasons. One issue is that gene expression from viral or internal promoters is highly variable and essentially unregulated. Moreover, with retroviral vectors, gene expression is usually silenced over time. Mammalian genes, in contrast, are characterized by highly regulated, precise levels of expression in both a temporal and a cell-specific manner. To ascertain if recapitulation of endogenous adenosine deaminase (ADA) expression can be achieved in a vector construct we created a new series of Moloney murine leukemia virus (MuLV) based retroviral vector that carry human regulatory elements including combinations of the ADA promoter, the ADA locus control region (LCR), ADA introns and human polyadenylation sequences in a self-inactivating vector backbone. Methods A MuLV-based retroviral vector with a self-inactivating (SIN) backbone, the phosphoglycerate kinase promoter (PGK) and the enhanced green fluorescent protein (eGFP), as a reporter gene, was generated. Subsequent vectors were constructed from this basic vector by deletion or addition of certain elements. The added elements that were assessed are the human ADA promoter, human ADA locus control region (LCR), introns 7, 8, and 11 from the human ADA gene, and human growth hormone polyadenylation signal. Retroviral vector particles were produced by transient three-plasmid transfection of 293T cells. Retroviral vectors encoding eGFP were titered by transducing 293A cells, and then the proportion of GFP-positive cells was determined using fluorescence-activated cell sorting (FACS). Non T-cell and T-cell lines were transduced at a multiplicity of infection (MOI) of 0.1 and the yield of eGFP transgene expression was evaluated by FACS analysis using mean fluorescent intensity (MFI) detection. Results Vectors that contained the ADA LCR were preferentially expressed in T-cell lines. Further improvements in T-cell specific gene expression were observed with the incorporation of additional cis-regulatory elements, such as a human polyadenylation signal and intron 7 from the human ADA gene. Conclusion These studies suggest that the combination of an authentically regulated ADA gene in a murine retroviral vector, together with additional locus-specific regulatory refinements, will yield a vector with a safer profile and greater efficacy in terms of high-level, therapeutic, regulated gene expression for the treatment of ADA-deficient severe combined immunodeficiency. PMID:20042112

High Energy Laser Beam Propagation in the Atmosphere: The Integral Invariants of the Nonlinear Parabolic Equation and the Method of Moments

NASA Technical Reports Server (NTRS)

Manning, Robert M.

2012-01-01

The method of moments is used to define and derive expressions for laser beam deflection and beam radius broadening for high-energy propagation through the Earth s atmosphere. These expressions are augmented with the integral invariants of the corresponding nonlinear parabolic equation that describes the electric field of high-energy laser beam to propagation to yield universal equations for the aforementioned quantities; the beam deflection is a linear function of the propagation distance whereas the beam broadening is a quadratic function of distance. The coefficients of these expressions are then derived from a thin screen approximation solution of the nonlinear parabolic equation to give corresponding analytical expressions for a target located outside the Earth s atmospheric layer. These equations, which are graphically presented for a host of propagation scenarios, as well as the thin screen model, are easily amenable to the phase expansions of the wave front for the specification and design of adaptive optics algorithms to correct for the inherent phase aberrations. This work finds application in, for example, the analysis of beamed energy propulsion for space-based vehicles.

High yield bacterial expression, purification and characterisation of bioactive Human Tousled-like Kinase 1B involved in cancer.

PubMed

Bhoir, Siddhant; Shaik, Althaf; Thiruvenkatam, Vijay; Kirubakaran, Sivapriya

2018-03-19

Human Tousled-like kinases (TLKs) are highly conserved serine/threonine protein kinases responsible for cell proliferation, DNA repair, and genome surveillance. Their possible involvement in cancer via efficient DNA repair mechanisms have made them clinically relevant molecular targets for anticancer therapy. Innovative approaches in chemical biology have played a key role in validating the importance of kinases as molecular targets. However, the detailed understanding of the protein structure and the mechanisms of protein-drug interaction through biochemical and biophysical techniques demands a method for the production of an active protein of exceptional stability and purity on a large scale. We have designed a bacterial expression system to express and purify biologically active, wild-type Human Tousled-like Kinase 1B (hTLK1B) by co-expression with the protein phosphatase from bacteriophage λ. We have obtained remarkably high amounts of the soluble and homogeneously dephosphorylated form of biologically active hTLK1B with our unique, custom-built vector design strategy. The recombinant hTLK1B can be used for the structural studies and may further facilitate the development of new TLK inhibitors for anti-cancer therapy using a structure-based drug design approach.

A novel system for the production of high levels of functional human therapeutic proteins in stable cells with a Semliki Forest virus noncytopathic vector.

PubMed

Casales, Erkuden; Aranda, Alejandro; Quetglas, Jose I; Ruiz-Guillen, Marta; Rodriguez-Madoz, Juan R; Prieto, Jesus; Smerdou, Cristian

2010-05-31

Semliki Forest virus (SFV) vectors lead to high protein expression in mammalian cells, but expression is transient due to vector cytopathic effects, inhibition of host cell proteins and RNA-based expression. We have used a noncytopathic SFV mutant (ncSFV) RNA vector to generate stable cell lines expressing two human therapeutic proteins: insulin-like growth factor I (IGF-I) and cardiotrophin-1 (CT-1). Therapeutic genes were fused at the carboxy-terminal end of Puromycin N-acetyl-transferase gene by using as a linker the sequence coding for foot-and-mouth disease virus (FMDV) 2A autoprotease. These cassettes were cloned into the ncSFV vector. Recombinant ncSFV vectors allowed rapid and efficient selection of stable BHK cell lines with puromycin. These cells expressed IGF-I and CT-1 in supernatants at levels reaching 1.4 and 8.6 microg/10(6)cells/24 hours, respectively. Two cell lines generated with each vector were passaged ten times during 30 days, showing constant levels of protein expression. Recombinant proteins expressed at different passages were functional by in vitro signaling assays. Stability at RNA level was unexpectedly high, showing a very low mutation rate in the CT-1 sequence, which did not increase at high passages. CT-1 was efficiently purified from supernatants of ncSFV cell lines, obtaining a yield of approximately 2mg/L/24 hours. These results indicate that the ncSFV vector has a great potential for the production of recombinant proteins in mammalian cells. 2010 Elsevier B.V. All rights reserved.

Hijacked then lost in translation: the plight of the recombinant host cell in membrane protein structural biology projects.

PubMed

Bill, Roslyn M; von der Haar, Tobias

2015-06-01

Membrane protein structural biology is critically dependent upon the supply of high-quality protein. Over the last few years, the value of crystallising biochemically characterised, recombinant targets that incorporate stabilising mutations has been established. Nonetheless, obtaining sufficient yields of many recombinant membrane proteins is still a major challenge. Solutions are now emerging based on an improved understanding of recombinant host cells; as a 'cell factory' each cell is tasked with managing limited resources to simultaneously balance its own growth demands with those imposed by an expression plasmid. This review examines emerging insights into the role of translation and protein folding in defining high-yielding recombinant membrane protein production in a range of host cells. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

[Effect of gene optimization on the expression and purification of HDV small antigen produced by genetic engineering].

PubMed

Ding, Jun-Ying; Meng, Qing-Ling; Guo, Min-Zhuo; Yi, Yao; Su, Qiu-Dong; Lu, Xue-Xin; Qiu, Feng; Bi, Sheng-Li

2012-10-01

To study the effect of gene optimization on the expression and purification of HDV small antigen produced by genetic engineering. Based on the colon preference of E. coli, the HDV small antigen original gene from GenBank was optimized. Both the original gene and the optimized gene expressed in prokaryotic cells, SDS-PAGE was made to analyze the protein expression yield and to decide which protein expression style was more proportion than the other. Furthermore, two antigens were purified by chromatography in order to compare the purity by SDS-PAGE and Image Lab software. SDS-PAGE indicated that the molecular weight of target proteins from two groups were the same as we expected. Gene optimization resulted in the higher yield and it could make the product more soluble. After chromatography, the purity of target protein from optimized gene was up to 96.3%, obviously purer than that from original gene. Gene optimization could increase the protein expression yield and solubility of genetic engineering HDV small antigen. In addition, the product from the optimized gene group was easier to be purified for diagnosis usage.

Influence of cosubstrate concentration on xylose conversion by recombinant, XYL1-expressing Saccharomyces cerevisiae: a comparison of different sugars and ethanol as cosubstrates.

PubMed Central

Meinander, N Q; Hahn-Hägerdal, B

1997-01-01

Conversion of xylose to xylitol by recombinant Saccharomyces cerevisiae expressing the XYL1 gene, encoding xylose reductase, was investigated by using different cosubstrates as generators of reduced cofactors. The effect of a pulse addition of the cosubstrate on xylose conversion in cosubstrate-limited fed-batch cultivation was studied. Glucose, mannose, and fructose, which are transported with high affinity by the same transport system as is xylose, inhibited xylose conversion by 99, 77, and 78%, respectively, reflecting competitive inhibition of xylose transport. Pulse addition of maltose, which is transported by a specific transport system, did not inhibit xylose conversion. Pulse addition of galactose, which is also transported by a specific transporter, inhibited xylose conversion by 51%, in accordance with noncompetitive inhibition between the galactose and glucose/ xylose transport systems. Pulse addition of ethanol inhibited xylose conversion by 15%, explained by inhibition of xylose transport through interference with the hydrophobic regions of the cell membrane. The xylitol yields on the different cosubstrates varied widely. Galactose gave the highest xylitol yield, 5.6 times higher than that for glucose. The difference in redox metabolism of glucose and galactose was suggested to enhance the availability of reduced cofactors for xylose reduction with galactose. The differences in xylitol yield observed between some of the other sugars may also reflect differences in redox metabolism. With all cosubstrates, the xylitol yield was higher under cosubstrate limitation than with cosubstrate excess. PMID:9143128

What is shared, what is different? Core relational themes and expressive displays of eight positive emotions.

PubMed

Campos, Belinda; Shiota, Michelle N; Keltner, Dacher; Gonzaga, Gian C; Goetz, Jennifer L

2013-01-01

Understanding positive emotions' shared and differentiating features can yield valuable insight into the structure of positive emotion space and identify emotion states, or aspects of emotion states, that are most relevant for particular psychological processes and outcomes. We report two studies that examined core relational themes (Study 1) and expressive displays (Study 2) for eight positive emotion constructs--amusement, awe, contentment, gratitude, interest, joy, love, and pride. Across studies, all eight emotions shared one quality: high positive valence. Distinctive core relational theme and expressive display patterns were found for four emotions--amusement, awe, interest, and pride. Gratitude was associated with a distinct core relational theme but not an expressive display. Joy and love were each associated with a distinct expressive display but their core relational themes also characterised pride and gratitude, respectively. Contentment was associated with a distinct expressive display but not a core relational theme. The implications of this work for the study of positive emotion are discussed.

Constitutive expression, purification and characterisation of pectin methylesterase from Aspergillus niger in Pichia pastoris for potential application in the fruit juice industry.

PubMed

Jiang, Xiuping; Chen, Peng; Yin, Maolu; Yang, Qing

2013-01-01

Pectin methylesterase (PME) catalyses the hydrolysis of the methyl ester of pectin, yielding free carboxyl groups and methanol. PME is widely used in the food, cosmetic and pharmaceutical industries. PME from Aspergillus niger was constitutively expressed to a high level in the yeast Pichia pastoris. The recombinant PME was purified by a combination of ammonium sulfate fractionation and ion exchange chromatography, giving an overall yield of 28.0%. It appeared as a single band in sodium dodecyl sulfate polyacrylamide gel electrophoresis, with a molecular mass of about 45 kDa. Optimal activity of the enzyme occurred at a temperature of 50 °C and a pH of 4.7. The K(m), V(max) and k(cat) values of the enzyme with respect to pectin were 8.6 mmol L⁻¹ [Formula: See Text], 1.376 mmol min⁻¹ mg⁻¹ and 8.26 × 10² s⁻¹ respectively. Cations such as K⁺, Mg²⁺, Ni²⁺, Mn²⁺ and Co²⁺ slightly inhibited its activity, whereas Na⁺ had no effect. PME from A. niger was constitutively expressed to a high level in P. pastoris without methanol induction. The recombinant PME was purified and characterised and shown to be a good candidate for potential application in the fruit juice industry. Copyright © 2012 Society of Chemical Industry.

Over-accumulation of nuclear IGF-1 receptor in tumor cells requires elevated expression of the receptor and the SUMO-conjugating enzyme Ubc9

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deng, Hua; Lin, Yingbo; Badin, Margherita

2011-01-14

Research highlights: {yields} SUMOylation mediates nuclear translocation of IGF-1R which activates transcription. {yields} Here we show that nuclear IGF-1R over-accumulates in tumor cells. {yields} This requires overexpression of the receptor that is a common feature in tumor cells. {yields} An increased expression of the SUMO ligase Ubc9 seems to be an involved mechanism too. -- Abstract: The insulin-like growth factor 1 receptor (IGF-1R) plays crucial roles in tumor cell growth and is overexpressed in many cancers. IGF-1R's trans-membrane kinase signaling pathways have been well characterized. Very recently, we showed that SUMOylation mediates nuclear translocation of the IGF-1R, and that nuclearmore » IGF-1R (nIGF-1R) binds to enhancer regions and activates transcription. We identified three lysine residues in the {beta}-subunit of the receptor and that mutation of these blocks nuclear translocation and gene activation. Furthermore, accumulation of nIGF-1R was proven strongly dependent on the specific SUMO-conjugating enzyme Ubc9. Here we show that nIGF-1R originates solely from the cell membrane and that phosphorylation of the core tyrosine residues of the receptor kinase is crucial for nuclear accumulation. We also compared the levels of nIGF-1R, measured as nuclear/membrane ratios, in tumor and normal cells. We found that the breast cancer cell line MCF-7 has 13-fold higher amounts of nIGF-1R than breast epithelial cells (IME) which showed only a small amount of nIGF-1R. In comparison, the total expression of IGF-1R was only 3.7- higher in MCF-7. Comparison of several other tumor and normal cell lines showed similar tumor cell over-accumulation of nIGF-1R, exceeding the total receptor expression substantially. Ectopic overexpression (>10-fold) of the receptor increased nIGF-1R in IME cells but not to that high level as in wild type MCF-7. The levels of Ubc9 were higher in all tumor cell lines, compared to the normal cells, and this probably contributes to over-accumulation of nIGF-1R. Over-accumulation of nIGF-1R may contribute to deregulated gene expression and therewith play a pathophysiological role in cancer cells.« less

MicroRNAs expression in ox-LDL treated HUVECs: MiR-365 modulates apoptosis and Bcl-2 expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qin, Bing; Xiao, Bo; Liang, Desheng

Highlights: {yields} We evaluated the role of miRNAs in ox-LDL induced apoptosis in ECs. {yields} We found 4 up-regulated and 11 down-regulated miRNAs in apoptotic ECs. {yields} Target genes of the dysregulated miRNAs regulate ECs apoptosis and atherosclerosis. {yields} MiR-365 promotes ECs apoptosis via suppressing Bcl-2 expression. {yields} MiR-365 inhibitor alleviates ECs apoptosis induced by ox-LDL. -- Abstract: Endothelial cells (ECs) apoptosis induced by oxidized low-density lipoprotein (ox-LDL) is thought to play a critical role in atherosclerosis. MicroRNAs (miRNAs) are a class of noncoding RNAs that posttranscriptionally regulate the expression of genes involved in diverse cell functions, including differentiation, growth,more » proliferation, and apoptosis. However, whether miRNAs are associated with ox-LDL induced apoptosis and their effect on ECs is still unknown. Therefore, this study evaluated potential miRNAs and their involvement in ECs apoptosis in response to ox-LDL stimulation. Microarray and qRT-PCR analysis performed on human umbilical vein endothelial cells (HUVECs) exposed to ox-LDL identified 15 differentially expressed (4 up- and 11 down-regulated) miRNAs. Web-based query tools were utilized to predict the target genes of the differentially expressed miRNAs, and the potential target genes were classified into different function categories with the gene ontology (GO) term and KEGG pathway annotation. In particular, bioinformatics analysis suggested that anti-apoptotic protein B-cell CLL/lymphoma 2 (Bcl-2) is a target gene of miR-365, an apoptomir up-regulated by ox-LDL stimulation in HUVECs. We further showed that transfection of miR-365 inhibitor partly restored Bcl-2 expression at both mRNA and protein levels, leading to a reduction of ox-LDL-mediated apoptosis in HUVECs. Taken together, our findings indicate that miRNAs participate in ox-LDL-mediated apoptosis in HUVECs. MiR-365 potentiates ox-LDL-induced ECs apoptosis by regulating the expression of Bcl-2, suggesting potential novel therapeutic targets for atherosclerosis.« less

Rate constants for the thermal decomposition of ethanol and its bimolecular reactions with OH and D : reflected shock tube and theoretical studies.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sivaramakrishnan, R.; Su, M.-C.; Michael, J. V.

2010-09-09

The thermal decomposition of ethanol and its reactions with OH and D have been studied with both shock tube experiments and ab initio transition state theory-based master equation calculations. Dissociation rate constants for ethanol have been measured at high T in reflected shock waves using OH optical absorption and high-sensitivity H-atom ARAS detection. The three dissociation processes that are dominant at high T are: C{sub 2}H{sub 5}OH {yields} C{sub 2}H{sub 4} + H{sub 2}O; C{sub 2}H{sub 5}OH {yields} CH{sub 3} + CH{sub 2}OH; C{sub 2}H{sub 5}OH {yields} C{sub 2}H{sub 5} + OH. The rate coefficient for reaction C was measuredmore » directly with high sensitivity at 308 nm using a multipass optical White cell. Meanwhile, H-atom ARAS measurements yield the overall rate coefficient and that for the sum of reactions B and C, since H-atoms are instantaneously formed from the decompositions of CH{sub 2}OH and C{sub 2}H{sub 5} into CH{sub 2}O + H and C{sub 2}H{sub 4} + H, respectively. By difference, rate constants for reaction 1 could be obtained. One potential complication is the scavenging of OH by unreacted ethanol in the OH experiments, and therefore, rate constants for OH + C{sub 2}H{sub 5}OH {yields} products were measured using tert-butyl hydroperoxide (tBH) as the thermal source for OH. The present experiments can be represented by the Arrhenius expression k = (2.5 {+-} 0.43) x 10{sup -11} exp(- 911 {+-} 191 K/T) cm{sup 3} molecule{sup -1} s{sup -1} over the T range 857-1297 K. For completeness, we have also measured the rate coefficient for the reaction of D atoms with ethanol D + C{sub 2}H{sub 5}OH {yields} products whose H analogue is another key reaction in the combustion of ethanol. Over the T range 1054-1359 K, the rate constants from the present experiments can be represented by the Arrhenius expression, k = (3.98 {+-} 0.76) x 10{sup -10} exp(- 4494 {+-} 235 K/T) cm{sup 3} molecule{sup -1} s{sup -1}. The high-pressure rate coefficients for reactions B and C were studied with variable reaction coordinate transition state theory employing directly determined CASPT2/cc-pvdz interaction energies. Reactions A, D, and E were studied with conventional transition state theory employing QCISD(T)/CBS energies. For the saddle point in reaction A, additional high-level corrections are evaluated. The predicted reaction exo- and endothermicities are in good agreement with the current Active Thermochemical Tables values. The transition state theory predictions for the microcanonical rate coefficients in ethanol decomposition are incorporated in master equation calculations to yield predictions for the temperature and pressure dependences of reactions A-C. With modest adjustments (<1 kcal/mol) to a few key barrier heights, the present experimental and adjusted theoretical results yield a consistent description of both the decomposition (1-3) and abstraction kinetics (4 and 5). The present results are compared with earlier experimental and theoretical work.« less

Genome-Wide Transcriptome Analysis During Anthesis Reveals New Insights into the Molecular Basis of Heat Stress Responses in Tolerant and Sensitive Rice Varieties.

PubMed

González-Schain, Nahuel; Dreni, Ludovico; Lawas, Lovely M F; Galbiati, Massimo; Colombo, Lucia; Heuer, Sigrid; Jagadish, Krishna S V; Kater, Martin M

2016-01-01

Rice is one of the main food crops in the world. In the near future, yield is expected to be under pressure due to unfavorable climatic conditions, such as increasing temperatures. Therefore, improving rice germplasm in order to guarantee rice production under harsh environmental conditions is of top priority. Although many physiological studies have contributed to understanding heat responses during anthesis, the most heat-sensitive stage, molecular data are still largely lacking. In this study, an RNA-sequencing approach of heat- and control-treated reproductive tissues during anthesis was carried out using N22, one of the most heat-tolerant rice cultivars known to date. This analysis revealed that expression of genes encoding a number of transcription factor families, together with signal transduction and metabolic pathway genes, is repressed. On the other hand, expression of genes encoding heat shock factors and heat shock proteins was highly activated. Many of these genes are predominantly expressed at late stages of anther development. Further physiological experiments using heat-tolerant N22 and two sensitive cultivars suggest that reduced yield in heat-sensitive plants may be associated with poor pollen development or production in anthers prior to anthesis. In parallel, induction levels of a set of heat-responsive genes in these tissues correlated well with heat tolerance. Altogether, these findings suggest that proper expression of protective chaperones in anthers is needed before anthesis to overcome stress damage and to ensure fertilization. Genes putatively controlling this process were identified and are valuable candidates to consider for molecular breeding of highly productive heat-tolerant cultivars. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Genomics and relative expression analysis identifies key genes associated with high female to male flower ratio in Jatropha curcas L.

PubMed

Gangwar, Manali; Sood, Hemant; Chauhan, Rajinder Singh

2016-04-01

Jatropha curcas, has been projected as a major source of biodiesel due to high seed oil content (42 %). A major roadblock for commercialization of Jatropha-based biodiesel is low seed yield per inflorescence, which is affected by low female to male flower ratio (1:25-30). Molecular dissection of female flower development by analyzing genes involved in phase transitions and floral organ development is, therefore, crucial for increasing seed yield. Expression analysis of 42 genes implicated in floral organ development and sex determination was done at six floral developmental stages of a J. curcas genotype (IC561235) with inherently higher female to male flower ratio (1:8-10). Relative expression analysis of these genes was done on low ratio genotype. Genes TFL1, SUP, AP1, CRY2, CUC2, CKX1, TAA1 and PIN1 were associated with reproductive phase transition. Further, genes CUC2, TAA1, CKX1 and PIN1 were associated with female flowering while SUP and CRY2 in female flower transition. Relative expression of these genes with respect to low female flower ratio genotype showed up to ~7 folds increase in transcript abundance of SUP, TAA1, CRY2 and CKX1 genes in intermediate buds but not a significant increase (~1.25 folds) in female flowers, thereby suggesting that these genes possibly play a significant role in increased transition towards female flowering by promoting abortion of male flower primordia. The outcome of study has implications in feedstock improvement of J. curcas through functional validation and eventual utilization of key genes associated with female flowering.

A strategy for high-level expression of a single-chain variable fragment against TNFα by subcloning antibody variable regions from the phage display vector pCANTAB 5E into pBV220.

PubMed

Yang, Tao; Yang, Lijun; Chai, Weiran; Li, Renke; Xie, Jun; Niu, Bo

2011-03-01

A phage display single-chain variable fragment (scFv) library against TNFα was constructed using a recombinant phage antibody system (RPAS). The cloned scFv gene was introduced into the phage display vector pCANTAB 5E and expressed in Escherichia coli (E. coli) with a yield of up to 0.15 mg/l of total protein. With the attempt to improve the expression level of TNF-scFv, a strategy was established for subcloning the scFv gene from pCANTAB 5E into the plasmid pBV220. Under the control of a highly efficient tandem P(R)P(L) promoter system, scFv production was increased to 30% of total protein as inclusion bodies. After extraction from the cell pellet by sonication, the inclusion bodies were solubilized and denatured in the presence of 8M urea. Purification of denatured scFv was performed using nickel column chromatography followed by renaturation. The purity and activity of the refolded scFv were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting and by an enzyme-linked immunoabsorbent assay (ELISA). The results reveal that the overall yield of bioactive TNF-scFv from E. coli flask cultures was more than 45 mg/l culture medium and 15 mg/g wet weight cells. The renatured scFv exhibited binding activity similarly to soluble scFv. In conclusion we developed a method to over-express TNF-scFv, which have biological function after purification and renaturation. Copyright © 2010 Elsevier Inc. All rights reserved.

Complementarity determining regions and frameworks contribute to the disulfide bond independent folding of intrinsically stable scFv

PubMed Central

Gąciarz, Anna

2017-01-01

CyDisCo is a system facilitating disulfide bond formation in recombinant proteins in the cytoplasm of Escherichia coli. Previously we screened for soluble expression of single chain antibody fragments (scFv) in the cytoplasm of E. coli in the presence and absence of CyDisCo, with >90% being solubly expressed. Two scFv, those derived from natalizumab and trastuzumab, were solubly produced in high amounts even in the absence of folding catalysts i.e. disulfide bond formation is not critical for their folding. Here we investigate the contribution of the framework and the complementarity determining regions (CDRs) of scFv to the disulfide-independence of folding. We swapped CDRs between four scFv that have different properties, including two scFv that can efficiently fold independently from disulfide bonds and two more disulfide-dependent scFv. To confirm disulfide-independence we generated cysteine to alanine mutants of the disulfide-independent scFv. All of the scFv were tested for soluble expression in the cytoplasm of E. coli in the presence and absence of the oxidative folding catalysts Erv1p and PDI. Eight of the hybrid scFv were solubly produced in the presence of CyDisCo, while seven were solubly produced in the absence of CyDisCo, though the yields were often much lower when CyDisCo was absent. Soluble expression was also observed for scFv natalizumab and trastuzumab containing no cysteines. We compared yields, thermal stability and secondary structure of solubly produced scFv and undertook binding studies by western blotting, dot blotting or surface plasmon resonance of those produced in good yields. Our results indicate that both the CDRs and the framework contribute to the disulfide-dependence of soluble production of scFv, with the CDRs having the largest effect. In addition, there was no correlation between thermal stability and disulfide-dependence of folding and only a weak correlation between the yield of protein and the thermal stability of the protein. PMID:29253024

Complementarity determining regions and frameworks contribute to the disulfide bond independent folding of intrinsically stable scFv.

PubMed

Gąciarz, Anna; Ruddock, Lloyd W

2017-01-01

CyDisCo is a system facilitating disulfide bond formation in recombinant proteins in the cytoplasm of Escherichia coli. Previously we screened for soluble expression of single chain antibody fragments (scFv) in the cytoplasm of E. coli in the presence and absence of CyDisCo, with >90% being solubly expressed. Two scFv, those derived from natalizumab and trastuzumab, were solubly produced in high amounts even in the absence of folding catalysts i.e. disulfide bond formation is not critical for their folding. Here we investigate the contribution of the framework and the complementarity determining regions (CDRs) of scFv to the disulfide-independence of folding. We swapped CDRs between four scFv that have different properties, including two scFv that can efficiently fold independently from disulfide bonds and two more disulfide-dependent scFv. To confirm disulfide-independence we generated cysteine to alanine mutants of the disulfide-independent scFv. All of the scFv were tested for soluble expression in the cytoplasm of E. coli in the presence and absence of the oxidative folding catalysts Erv1p and PDI. Eight of the hybrid scFv were solubly produced in the presence of CyDisCo, while seven were solubly produced in the absence of CyDisCo, though the yields were often much lower when CyDisCo was absent. Soluble expression was also observed for scFv natalizumab and trastuzumab containing no cysteines. We compared yields, thermal stability and secondary structure of solubly produced scFv and undertook binding studies by western blotting, dot blotting or surface plasmon resonance of those produced in good yields. Our results indicate that both the CDRs and the framework contribute to the disulfide-dependence of soluble production of scFv, with the CDRs having the largest effect. In addition, there was no correlation between thermal stability and disulfide-dependence of folding and only a weak correlation between the yield of protein and the thermal stability of the protein.

Hepatic steatosis inhibits autophagic proteolysis via impairment of autophagosomal acidification and cathepsin expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Inami, Yoshihiro; Yamashina, Shunhei, E-mail: syamashi@juntendo.ac.jp; Izumi, Kousuke

2011-09-09

Highlights: {yields} Acidification of autophagosome was blunted in steatotic hepatocytes. {yields} Hepatic steatosis did not disturb fusion of isolated autophagosome and lysosome. {yields} Proteinase activity of cathepsin B and L in autolysosomes was inhibited by steatosis. {yields} Hepatic expression of cathepsin B and L was suppressed by steatosis. -- Abstract: Autophagy, one of protein degradation system, contributes to maintain cellular homeostasis and cell defense. Recently, some evidences indicated that autophagy and lipid metabolism are interrelated. Here, we demonstrate that hepatic steatosis impairs autophagic proteolysis. Though accumulation of autophagosome is observed in hepatocytes from ob/ob mice, expression of p62 was augmentedmore » in liver from ob/ob mice more than control mice. Moreover, degradation of the long-lived protein leucine was significantly suppressed in hepatocytes isolated from ob/ob mice. More than 80% of autophagosomes were stained by LysoTracker Red (LTR) in hepatocytes from control mice; however, rate of LTR-stained autophagosomes in hepatocytes were suppressed in ob/ob mice. On the other hand, clearance of autolysosomes loaded with LTR was blunted in hepatocytes from ob/ob mice. Although fusion of isolated autophagosome and lysosome was not disturbed, proteinase activity of cathepsin B and L in autolysosomes and cathepsin B and L expression of liver were suppressed in ob/ob mice. These results indicate that lipid accumulation blunts autophagic proteolysis via impairment of autophagosomal acidification and cathepsin expression.« less

High-level expression of thermostable cellulolytic enzymes in tobacco transplastomic plants and their use in hydrolysis of an industrially pretreated Arundo donax L. biomass.

PubMed

Castiglia, Daniela; Sannino, Lorenza; Marcolongo, Loredana; Ionata, Elena; Tamburino, Rachele; De Stradis, Angelo; Cobucci-Ponzano, Beatrice; Moracci, Marco; La Cara, Francesco; Scotti, Nunzia

2016-01-01

Biofuels production from plant biomasses is a complex multi-step process with important economic burdens. Several biotechnological approaches have been pursued to reduce biofuels production costs. The aim of the present study was to explore the production in tobacco plastome of three genes encoding (hemi)cellulolytic enzymes from thermophilic and hyperthermophilic bacterium and Archaea, respectively, and test their application in the bioconversion of an important industrially pretreated biomass feedstock (A. donax) for production of second-generation biofuels. The selected enzymes, endoglucanase, endo-β-1,4-xylanase and β-glucosidase, were expressed in tobacco plastome with a protein yield range from 2 % to more than 75 % of total soluble proteins (TSP). The accumulation of endoglucanase (up to 2 % TSP) gave altered plant phenotypes whose severity was directly linked to the enzyme yield. The most severe seedling-lethal phenotype was due to the impairment of plastid development associated to the binding of endoglucanase protein to thylakoids. Endo-β-1,4-xylanase and β-glucosidase, produced at very high level without detrimental effects on plant development, were enriched (fourfold) by heat treatment (105.4 and 255.4 U/mg, respectively). Both plastid-derived biocatalysts retained the main features of the native or recombinantly expressed enzymes with interesting differences. Plastid-derived xylanase and β-glucosidase resulted more thermophilic than the E. coli recombinant and native counterpart, respectively. Bioconversion experiments, carried out at 50 and 60 °C, demonstrated that plastid-derived enzymes were able to hydrolyse an industrially pretreated giant reed biomass. In particular, the replacement of commercial enzyme with plastid-derived xylanase, at 60 °C, produced an increase of both xylose recovery and hydrolysis rate; whereas the replacement of both xylanase and β-glucosidase produced glucose levels similar to those observed with the commercial cocktails, and xylose yields always higher in the whole 24-72 h range. The very high production level of thermophilic and hyperthermophilic enzymes, their stability and bioconversion efficiencies described in this study demonstrate that plastid transformation represents a real cost-effective production platform for cellulolytic enzymes.

Nanoparticle-mediated intracellular lipid accumulation during C2C12 cell differentiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tsukahara, Tamotsu, E-mail: ttamotsu@shinshu-u.ac.jp; Haniu, Hisao, E-mail: hhaniu@shinshu-u.ac.jp

2011-03-25

Research highlights: {yields} HTT2800 has a significant effect on intracellular lipid accumulation. {yields} HTT2800 reduced muscle-specific genes and led to the emergence of adipocyte-related genes. {yields} HT2800 converts the differentiation pathway of C2C12 myoblasts to that of adipoblast-like cells. -- Abstract: In this report, we sought to elucidate whether multiwall carbon nanotubes are involved in the modulation of the proliferation and differentiation of the skeletal muscle cell line C2C12. Skeletal muscle is a major mass peripheral tissue that accounts for 40% of total body weight and 50% of energy consumption. We focused on the differentiation pathway of myoblasts after exposuremore » to a vapor-grown carbon fiber, HTT2800, which is one of the most highly purified carbon nanotubes. This treatment leads in parallel to the expression of a typical adipose differentiation program. We found that HTT2800 stimulated intracellular lipid accumulation in C2C12 cells. We have also shown by quantified PCR analysis that the expression of adipose-related genes was markedly upregulated during HTT2800 exposure. Taken together, these results suggest that HTT2800 specifically converts the differentiation pathway of C2C12 myoblasts to that of adipoblast-like cells.« less

Insulin-like growth factor binding protein-1 levels are increased in patients with IgA nephropathy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tokunaga, Koki; Uto, Hirofumi, E-mail: hirouto@m2.kufm.kagoshima-u.ac.jp; Takami, Yoichiro

2010-08-20

Research highlights: {yields} IGFBP-1 mRNA over express in kidneys obtained from mice model of IgA nephropathy. {yields} Serum IGFBP-1 levels are high in patients with IgA nephropathy. {yields} Serum IGFBP-1 levels correlate with renal function and the severity of renal injury. -- Abstract: The mechanisms underlying the pathogenesis of immunoglobulin A (IgA) nephropathy (IgAN) are not well understood. In this study, we examined gene expression profiles in kidneys obtained from mice with high serum IgA levels (HIGA mice), which exhibit features of human IgAN. Female inbred HIGA, established from the ddY line, were used in these experiments. Serum IgA levels,more » renal IgA deposition, mesangial proliferation, and glomerulosclerosis were increased in 32-week-old HIGA mice in comparison to ddY animals. By microarray analysis, five genes were observed to be increased by more than 2.5-fold in 32-week-old HIGA in comparison to 16-week-old HIGA; these same five genes were decreased more than 2.5-fold in 32-week-old ddY in comparison to 16-week-old ddY mice. Of these five genes, insulin-like growth factor (IGF) binding protein (IGFBP)-1 exhibited differential expression between these mouse lines, as confirmed by quantitative RT-PCR. In addition, serum IGFBP-1 levels were significantly higher in patients with IgAN than in healthy controls. In patients with IgAN, these levels correlated with measures of renal function, such as estimated glomerular filtration rate (eGFR), but not with sex, age, serum IgA, C3 levels, or IGF-1 levels. Pathologically, serum IGFBP-1 levels were significantly associated with the severity of renal injury, as assessed by mesangial cell proliferation and interstitial fibrosis. These results suggest that increased IGFBP-1 levels are associated with the severity of renal pathology in patients with IgAN.« less

Probabilistic pathway construction.

PubMed

Yousofshahi, Mona; Lee, Kyongbum; Hassoun, Soha

2011-07-01

Expression of novel synthesis pathways in host organisms amenable to genetic manipulations has emerged as an attractive metabolic engineering strategy to overproduce natural products, biofuels, biopolymers and other commercially useful metabolites. We present a pathway construction algorithm for identifying viable synthesis pathways compatible with balanced cell growth. Rather than exhaustive exploration, we investigate probabilistic selection of reactions to construct the pathways. Three different selection schemes are investigated for the selection of reactions: high metabolite connectivity, low connectivity and uniformly random. For all case studies, which involved a diverse set of target metabolites, the uniformly random selection scheme resulted in the highest average maximum yield. When compared to an exhaustive search enumerating all possible reaction routes, our probabilistic algorithm returned nearly identical distributions of yields, while requiring far less computing time (minutes vs. years). The pathways identified by our algorithm have previously been confirmed in the literature as viable, high-yield synthesis routes. Prospectively, our algorithm could facilitate the design of novel, non-native synthesis routes by efficiently exploring the diversity of biochemical transformations in nature. Copyright © 2011 Elsevier Inc. All rights reserved.

Ethanol production from lignocellulosic hydrolysates using engineered Saccharomyces cerevisiae harboring xylose isomerase-based pathway.

PubMed

Ko, Ja Kyong; Um, Youngsoon; Woo, Han Min; Kim, Kyoung Heon; Lee, Sun-Mi

2016-06-01

The efficient co-fermentation of glucose and xylose is necessary for the economically feasible bioethanol production from lignocellulosic biomass. Even with xylose utilizing Saccharomyces cerevisiae, the efficiency of the lignocellulosic ethanol production remains suboptimal mainly due to the low conversion yield of xylose to ethanol. In this study, we evaluated the co-fermentation performances of SXA-R2P-E, a recently engineered isomerase-based xylose utilizing strain, in mixed sugars and in lignocellulosic hydrolysates. In a high-sugar fermentation with 70g/L of glucose and 40g/L of xylose, SXA-R2P-E produced 50g/L of ethanol with an yield of 0.43gethanol/gsugars at 72h. From dilute acid-pretreated hydrolysates of rice straw and hardwood (oak), the strain produced 18-21g/L of ethanol with among the highest yield of 0.43-0.46gethanol/gsugars ever reported. This study shows a highly promising potential of a xylose isomerase-expressing strain as an industrially relevant ethanol producer from lignocellulosic hydrolysates. Copyright © 2016 Elsevier Ltd. All rights reserved.

Increasing the yield of middle silk gland expression system through transgenic knock-down of endogenous sericin-1.

PubMed

Ma, Sanyuan; Xia, Xiaojuan; Li, Yufeng; Sun, Le; Liu, Yue; Liu, Yuanyuan; Wang, Xiaogang; Shi, Run; Chang, Jiasong; Zhao, Ping; Xia, Qingyou

2017-08-01

Various genetically modified bioreactor systems have been developed to meet the increasing demands of recombinant proteins. Silk gland of Bombyx mori holds great potential to be a cost-effective bioreactor for commercial-scale production of recombinant proteins. However, the actual yields of proteins obtained from the current silk gland expression systems are too low for the proteins to be dissolved and purified in a large scale. Here, we proposed a strategy that reducing endogenous sericin proteins would increase the expression yield of foreign proteins. Using transgenic RNA interference, we successfully reduced the expression of BmSer1 to 50%. A total 26 transgenic lines expressing Discosoma sp. red fluorescent protein (DsRed) in the middle silk gland (MSG) under the control of BmSer1 promoter were established to analyze the expression of recombinant. qRT-PCR and western blotting showed that in BmSer1 knock-down lines, the expression of DsRed had significantly increased both at mRNA and protein levels. We did an additional analysis of DsRed/BmSer1 distribution in cocoon and effect of DsRed protein accumulation on the silk fiber formation process. This study describes not only a novel method to enhance recombinant protein expression in MSG bioreactor, but also a strategy to optimize other bioreactor systems.

Recombinant expression of a functional myo-inositol-1-phosphate synthase (MIPS) in Mycobacterium smegmatis.

PubMed

Huang, Xinyi; Hernick, Marcy

2015-10-01

Myo-inositol-1-phosphate synthase (MIPS, E.C. 5.5.1.4) catalyzes the first step in inositol production-the conversion of glucose-6-phosphate (Glc-6P) to myo-inositol-1-phosphate. While the three dimensional structure of MIPS from Mycobacterium tuberculosis has been solved, biochemical studies examining the in vitro activity have not been reported to date. Herein we report the in vitro activity of mycobacterial MIPS expressed in E. coli and Mycobacterium smegmatis. Recombinant expression in E. coli yields a soluble protein capable of binding the NAD(+) cofactor; however, it has no significant activity with the Glc-6P substrate. In contrast, recombinant expression in M. smegmatis mc(2)4517 yields a functionally active protein. Examination of structural data suggests that MtMIPS expressed in E. coli adopts a fold that is missing a key helix containing two critical (conserved) Lys side chains, which likely explains the inability of the E. coli expressed protein to bind and turnover the Glc-6P substrate. Recombinant expression in M. smegmatis may yield a protein that adopts a fold in which this key helix is formed enabling proper positioning of important side chains, thereby allowing for Glc-6P substrate binding and turnover. Detailed mechanistic studies may be feasible following optimization of the recombinant MIPS expression protocol in M. smegmatis.

NHR-23 dependent collagen and hedgehog-related genes required for molting

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kouns, Nathaniel A.; Nakielna, Johana; Behensky, Frantisek

2011-10-07

Highlights: {yields} NHR-23 is a critical regulator of nematode development and molting. {yields} The manuscript characterizes the loss-of-function phenotype of an nhr-23 mutant. {yields} Whole genome expression analysis identifies new potential targets of NHR-23. {yields} Hedgehog-related genes are identified as NHR-23 dependent genes. {yields} New link between sterol mediated signaling and regulation by NHR-23 is found. -- Abstract: NHR-23, a conserved member of the nuclear receptor family of transcription factors, is required for normal development in Caenorhabditis elegans where it plays a critical role in growth and molting. In a search for NHR-23 dependent genes, we performed whole genome comparativemore » expression microarrays on both control and nhr-23 inhibited synchronized larvae. Genes that decreased in response to nhr-23 RNAi included several collagen genes. Unexpectedly, several hedgehog-related genes were also down-regulated after nhr-23 RNAi. A homozygous nhr-23 deletion allele was used to confirm the RNAi knockdown phenotypes and the changes in gene expression. Our results indicate that NHR-23 is a critical co-regulator of functionally linked genes involved in growth and molting and reveal evolutionary parallels among the ecdysozoa.« less

Constitutive expression of cell wall invertase genes increases grain yield and starch content in maize.

PubMed

Li, Bei; Liu, Hua; Zhang, Yue; Kang, Tao; Zhang, Li; Tong, Jianhua; Xiao, Langtao; Zhang, Hongxia

2013-12-01

Grain size, number and starch content are important determinants of grain yield and quality. One of the most important biological processes that determine these components is the carbon partitioning during the early grain filling, which requires the function of cell wall invertase. Here, we showed the constitutive expression of cell wall invertase-encoding gene from Arabidopsis, rice (Oryza sativa) or maize (Zea mays), driven by the cauliflower mosaic virus (CaMV) 35S promoter, all increased cell wall invertase activities in different tissues and organs, including leaves and developing seeds, and substantially improved grain yield up to 145.3% in transgenic maize plants as compared to the wild-type plants, an effect that was reproduced in our 2-year field trials at different locations. The dramatically increased grain yield is due to the enlarged ears with both enhanced grain size and grain number. Constitutive expression of the invertase-encoding gene also increased total starch content up to 20% in the transgenic kernels. Our results suggest that cell wall invertase gene can be genetically engineered to improve both grain yield and grain quality in crop plants. © 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Heparin-binding peptide as a novel affinity tag for purification of recombinant proteins.

PubMed

Morris, Jacqueline; Jayanthi, Srinivas; Langston, Rebekah; Daily, Anna; Kight, Alicia; McNabb, David S; Henry, Ralph; Kumar, Thallapuranam Krishnaswamy Suresh

2016-10-01

Purification of recombinant proteins constitutes a significant part of the downstream processing in biopharmaceutical industries. Major costs involved in the production of bio-therapeutics mainly depend on the number of purification steps used during the downstream process. Affinity chromatography is a widely used method for the purification of recombinant proteins expressed in different expression host platforms. Recombinant protein purification is achieved by fusing appropriate affinity tags to either N- or C- terminus of the target recombinant proteins. Currently available protein/peptide affinity tags have proved quite useful in the purification of recombinant proteins. However, these affinity tags suffer from specific limitations in their use under different conditions of purification. In this study, we have designed a novel 34-amino acid heparin-binding affinity tag (HB-tag) for the purification of recombinant proteins expressed in Escherichia coli (E. coli) cells. HB-tag fused recombinant proteins were overexpressed in E. coli in high yields. A one-step heparin-Sepharose-based affinity chromatography protocol was developed to purify HB-fused recombinant proteins to homogeneity using a simple sodium chloride step gradient elution. The HB-tag has also been shown to facilitate the purification of target recombinant proteins from their 8 M urea denatured state(s). The HB-tag has been demonstrated to be successfully released from the fusion protein by an appropriate protease treatment to obtain the recombinant target protein(s) in high yields. Results of the two-dimensional NMR spectroscopy experiments indicate that the purified recombinant target protein(s) exist in the native conformation. Polyclonal antibodies raised against the HB-peptide sequence, exhibited high binding specificity and sensitivity to the HB-fused recombinant proteins (∼10 ng) in different crude cell extracts obtained from diverse expression hosts. In our opinion, the HB-tag provides a cost-effective, rapid, and reliable avenue for the purification of recombinant proteins in heterologous hosts. Copyright © 2016 Elsevier Inc. All rights reserved.

Anti-MUC1 antibody inhibits EGF receptor signaling in cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hisatsune, Akinori, E-mail: hisatsun@kumamoto-u.ac.jp; Nakayama, Hideki; Kawasaki, Mitsuru

2011-02-18

Research highlights: {yields} We identified changes in the expression and function of EGFR by anti-MUC1 antibody. {yields} An anti-MUC1 antibody GP1.4 decreased EGFR from cell surface by internalization. {yields} GP1.4 specifically inhibited ERK signaling triggered EGF-EGFR signaling pathway. {yields} Internalization of EGFR was dependent on the presence of MUC1 on cell surface. {yields} GP1.4 significantly inhibited EGF-dependent cancer cell proliferation and migration. -- Abstract: MUC1 is a type I transmembrane glycoprotein aberrantly overexpressed in various cancer cells. High expression of MUC1 is closely associated with cancer progression and metastasis, leading to poor prognosis. We previously reported that MUC1 is internalizedmore » by the binding of the anti-MUC1 antibody, from the cell surface to the intracellular region via the macropinocytotic pathway. Since MUC1 is closely associated with ErbBs, such as EGF receptor (EGFR) in cancer cells, we examined the effect of the anti-MUC1 antibody on EGFR trafficking. Our results show that: (1) anti-MUC1 antibody GP1.4, but not another anti-MUC1 antibody C595, triggered the internalization of EGFR in pancreatic cancer cells; (2) internalization of EGFR by GP1.4 resulted in the inhibition of ERK phosphorylation by EGF stimulation, in a MUC1 dependent manner; (3) inhibition of ERK phosphorylation by GP1.4 resulted in the suppression of proliferation and migration of pancreatic cancer cells. We conclude that the internalization of EGFR by anti-MUC1 antibody GP1.4 inhibits the progression of cancer cells via the inhibition of EGFR signaling.« less

The Source of the PB1 Gene in Influenza Vaccine Reassortants Selectively Alters the Hemagglutinin Content of the Resulting Seed Virus

PubMed Central

Cobbin, Joanna C. A.; Verity, Erin E.; Gilbertson, Brad P.; Rockman, Steven P.

2013-01-01

The yields of egg-grown influenza vaccines are maximized by the production of a seed strain using a reassortment of the seasonal influenza virus isolate with a highly egg-adapted strain. The seed virus is selected based on high yields of viral hemagglutinin (HA) and expression of the surface antigens from the seasonal isolate. The remaining proteins are usually derived from the high-growth parent. However, a retrospective analysis of vaccine seeds revealed that the seasonal PB1 gene was selected in more than 50% of reassortment events. Using the model seasonal H3N2 virus A/Udorn/307/72 (Udorn) virus and the high-growth A/Puerto Rico/8/34 (PR8) virus, we assessed the influence of the source of the PB1 gene on virus growth and vaccine yield. Classical reassortment of these two strains led to the selection of viruses that predominantly had the Udorn PB1 gene. The presence of Udorn PB1 in the seed virus, however, did not result in higher yields of virus or HA compared to the yields in the corresponding seed virus with PR8 PB1. The 8-fold-fewer virions produced with the seed virus containing the Udorn PB1 were somewhat compensated for by a 4-fold increase in HA per virion. A higher HA/nucleoprotein (NP) ratio was found in past vaccine preparations when the seasonal PB1 was present, also indicative of a higher HA density in these vaccine viruses. As the HA viral RNA (vRNA) and mRNA levels in infected cells were similar, we propose that PB1 selectively alters the translation of viral mRNA. This study helps to explain the variability of vaccine seeds with respect to HA yield. PMID:23468502

Systematic evaluation of RNA quality, microarray data reliability and pathway analysis in fresh, fresh frozen and formalin-fixed paraffin-embedded tissue samples.

PubMed

Wimmer, Isabella; Tröscher, Anna R; Brunner, Florian; Rubino, Stephen J; Bien, Christian G; Weiner, Howard L; Lassmann, Hans; Bauer, Jan

2018-04-20

Formalin-fixed paraffin-embedded (FFPE) tissues are valuable resources commonly used in pathology. However, formalin fixation modifies nucleic acids challenging the isolation of high-quality RNA for genetic profiling. Here, we assessed feasibility and reliability of microarray studies analysing transcriptome data from fresh, fresh-frozen (FF) and FFPE tissues. We show that reproducible microarray data can be generated from only 2 ng FFPE-derived RNA. For RNA quality assessment, fragment size distribution (DV200) and qPCR proved most suitable. During RNA isolation, extending tissue lysis time to 10 hours reduced high-molecular-weight species, while additional incubation at 70 °C markedly increased RNA yields. Since FF- and FFPE-derived microarrays constitute different data entities, we used indirect measures to investigate gene signal variation and relative gene expression. Whole-genome analyses revealed high concordance rates, while reviewing on single-genes basis showed higher data variation in FFPE than FF arrays. Using an experimental model, gene set enrichment analysis (GSEA) of FFPE-derived microarrays and fresh tissue-derived RNA-Seq datasets yielded similarly affected pathways confirming the applicability of FFPE tissue in global gene expression analysis. Our study provides a workflow comprising RNA isolation, quality assessment and microarray profiling using minimal RNA input, thus enabling hypothesis-generating pathway analyses from limited amounts of precious, pathologically significant FFPE tissues.

Metabolic and transcriptomic signatures of rice floral organs reveal sugar starvation as a factor in reproductive failure under heat and drought stress.

PubMed

Li, Xia; Lawas, Lovely M F; Malo, Richard; Glaubitz, Ulrike; Erban, Alexander; Mauleon, Ramil; Heuer, Sigrid; Zuther, Ellen; Kopka, Joachim; Hincha, Dirk K; Jagadish, Krishna S V

2015-10-01

Heat and drought stress are projected to become major challenges to sustain rice (Oryza sativa L.) yields with global climate change. Both stresses lead to yield losses when they coincide with flowering. A significant knowledge gap exists in the mechanistic understanding of the responses of rice floral organs that determine reproductive success under stress. Our work connects the metabolomic and transcriptomic changes in anthers, pistils before pollination and pollinated pistils in a heat-tolerant (N22) and a heat-sensitive (Moroberekan) cultivar. Systematic analysis of the floral organs revealed contrasts in metabolic profiles across anthers and pistils. Constitutive metabolic markers were identified that can define reproductive success in rice under stress. Six out of nine candidate metabolites identified by intersection analysis of stressed anthers were differentially accumulated in N22 compared with Moroberekan under non-stress conditions. Sugar metabolism was identified to be the crucial metabolic and transcriptional component that differentiated floral organ tolerance or susceptibility to stress. While susceptible Moroberekan specifically showed high expression of the Carbon Starved Anthers (CSA) gene under combined heat and drought, tolerant N22 responded with high expression of genes encoding a sugar transporter (MST8) and a cell wall invertase (INV4) as markers of high sink strength. © 2015 John Wiley & Sons Ltd.

The replication of a mouse adapted SARS-CoV in a mouse cell line stably expressing the murine SARS-CoV receptor mACE2 efficiently induces the expression of proinflammatory cytokines

PubMed Central

Regla-Nava, Jose A.; Jimenez-Guardeño, Jose M.; Nieto-Torres, Jose L.; Gallagher, Thomas M.; Enjuanes, Luis; DeDiego, Marta L.

2013-01-01

Infection of conventional mice with a mouse adapted (MA15) severe acute respiratory syndrome (SARS) coronavirus (CoV) reproduces many aspects of human SARS such as pathological changes in lung, viremia, neutrophilia, and lethality. However, established mouse cell lines highly susceptible to mouse-adapted SARS-CoV infection are not available. In this work, efficiently transfectable mouse cell lines stably expressing the murine SARS-CoV receptor angiotensin converting enzyme 2 (ACE2) have been generated. These cells yielded high SARS-CoV-MA15 titers and also served as excellent tools for plaque assays. In addition, in these cell lines, SARS-CoV-MA15 induced the expression of proinflammatory cytokines and IFN-β, mimicking what has been observed in experimental animal models infected with SARS-CoV and SARS patients. These cell lines are valuable tools to perform in vitro studies in a mouse cell system that reflects the species used for in vivo studies of SARS-CoV-MA15 pathogenesis. PMID:23911968

Role of CD137 signaling in dengue virus-mediated apoptosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nagila, Amar; Department of Biochemistry, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok; Netsawang, Janjuree

Highlights: {yields} For the first time the role of CD137 in dengue virus (DENV) infection. {yields} Induction of DENV-mediated apoptosis by CD137 signaling. {yields} Sensitization to CD137-mediated apoptosis by dengue virus capsid protein (DENV C). {yields} Nuclear localization of DENV C is required for CD137-mediated apoptosis. -- Abstract: Hepatic dysfunction is a well recognized feature of dengue virus (DENV) infection. However, molecular mechanisms of hepatic injury are still poorly understood. A complex interaction between DENV and the host immune response contributes to DENV-mediated tissue injury. DENV capsid protein (DENV C) physically interacts with the human death domain-associated protein Daxx. Amore » double substitution mutation in DENV C (R85A/K86A) abrogates Daxx interaction, nuclear localization and apoptosis. Therefore we compared the expression of cell death genes between HepG2 cells expressing DENV C and DENV C (R85A/K86A) using a real-time PCR array. Expression of CD137, which is a member of the tumor necrosis factor receptor family, increased significantly in HepG2 cells expressing DENV C compared to HepG2 cells expressing DENV C (R85A/K86A). In addition, CD137-mediated apoptotic activity in HepG2 cells expressing DENV C was significantly increased by anti-CD137 antibody compared to that of HepG2 cells expressing DENV C (R85A/K86A). In DENV-infected HepG2 cells, CD137 mRNA and CD137 positive cells significantly increased and CD137-mediated apoptotic activity was increased by anti-CD137 antibody. This work is the first to demonstrate the contribution of CD137 signaling to DENV-mediated apoptosis.« less

A novel 2,6-diisopropylphenyl-docosahexaenoamide conjugate induces apoptosis in T cell acute lymphoblastic leukemia cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Altenburg, Jeffrey D.; Harvey, Kevin A.; McCray, Sharon

2011-07-29

Highlights: {yields} 2,6-Diisopropylphenyl-docosahexaenoamide conjugates (DIP-DHA) inhibits the proliferation of T-cell leukemic cell lines. {yields} DIP-DHA resulted in increased activation of caspase-3, and caspase-7. {yields} DIP-DHA significantly downregulated CXCR4 surface expression. -- Abstract: We have previously characterized the effects of 2,6-diisopropylphenyl-docosahexaenoamide (DIP-DHA) conjugates and their analogs on the proliferation and progression of breast cancer cell lines. For this study, we investigated the effects of the DIP-DHA conjugate on 2 representative T cell acute lymphoblastic leukemia (T-ALL) cell lines: CEM and Jurkat. Treatment of both cell lines with DIP-DHA resulted in significantly greater inhibition of proliferation and induction of apoptosis than thatmore » of parent compounds, 2,6-diisopropylphenol (DIP) or docosahexaenoate (DHA). Treatment of the cells with DIP-DHA resulted in increased activation of caspase-3, and caspase-7. Furthermore, induction of apoptosis in both cell lines was reversed in the presence of a caspase family inhibitor. Treatment with DIP-DHA reduced mitochondrial membrane potential. These observations suggest that the effects are driven by intrinsic apoptotic pathways. DIP-DHA treatment also downregulated surface CXCR4 expression, an important chemokine receptor involved in cancer metastasis that is highly expressed in both CEM and Jurkat cells. In conclusion, our data suggest that the DIP-DHA conjugate exhibits significantly more potent effects on CEM and Jurkat cells than that of DIP or DHA alone. These conjugates have potential use for treatment of patients with T cell acute lymphoblastic leukemia.« less

Transgenic plants with enhanced growth characteristics

DOEpatents

Unkefer, Pat J.; Anderson, Penelope S.; Knight, Thomas J.

2016-09-06

The invention relates to transgenic plants exhibiting dramatically enhanced growth rates, greater seed and fruit/pod yields, earlier and more productive flowering, more efficient nitrogen utilization, increased tolerance to high salt conditions, and increased biomass yields. In one embodiment, transgenic plants engineered to over-express both glutamine phenylpyruvate transaminase (GPT) and glutamine synthetase (GS) are provided. The GPT+GS double-transgenic plants of the invention consistently exhibit enhanced growth characteristics, with T0 generation lines showing an increase in biomass over wild type counterparts of between 50% and 300%. Generations that result from sexual crosses and/or selfing typically perform even better, with some of the double-transgenic plants achieving an astounding four-fold biomass increase over wild type plants.

Protocol: high throughput silica-based purification of RNA from Arabidopsis seedlings in a 96-well format

PubMed Central

2011-01-01

The increasing popularity of systems-based approaches to plant research has resulted in a demand for high throughput (HTP) methods to be developed. RNA extraction from multiple samples in an experiment is a significant bottleneck in performing systems-level genomic studies. Therefore we have established a high throughput method of RNA extraction from Arabidopsis thaliana to facilitate gene expression studies in this widely used plant model. We present optimised manual and automated protocols for the extraction of total RNA from 9-day-old Arabidopsis seedlings in a 96 well plate format using silica membrane-based methodology. Consistent and reproducible yields of high quality RNA are isolated averaging 8.9 μg total RNA per sample (~20 mg plant tissue). The purified RNA is suitable for subsequent qPCR analysis of the expression of over 500 genes in triplicate from each sample. Using the automated procedure, 192 samples (2 × 96 well plates) can easily be fully processed (samples homogenised, RNA purified and quantified) in less than half a day. Additionally we demonstrate that plant samples can be stored in RNAlater at -20°C (but not 4°C) for 10 months prior to extraction with no significant effect on RNA yield or quality. Additionally, disrupted samples can be stored in the lysis buffer at -20°C for at least 6 months prior to completion of the extraction procedure providing a flexible sampling and storage scheme to facilitate complex time series experiments. PMID:22136293

Protocol: high throughput silica-based purification of RNA from Arabidopsis seedlings in a 96-well format.

PubMed

Salvo-Chirnside, Eliane; Kane, Steven; Kerr, Lorraine E

2011-12-02

The increasing popularity of systems-based approaches to plant research has resulted in a demand for high throughput (HTP) methods to be developed. RNA extraction from multiple samples in an experiment is a significant bottleneck in performing systems-level genomic studies. Therefore we have established a high throughput method of RNA extraction from Arabidopsis thaliana to facilitate gene expression studies in this widely used plant model. We present optimised manual and automated protocols for the extraction of total RNA from 9-day-old Arabidopsis seedlings in a 96 well plate format using silica membrane-based methodology. Consistent and reproducible yields of high quality RNA are isolated averaging 8.9 μg total RNA per sample (~20 mg plant tissue). The purified RNA is suitable for subsequent qPCR analysis of the expression of over 500 genes in triplicate from each sample. Using the automated procedure, 192 samples (2 × 96 well plates) can easily be fully processed (samples homogenised, RNA purified and quantified) in less than half a day. Additionally we demonstrate that plant samples can be stored in RNAlater at -20°C (but not 4°C) for 10 months prior to extraction with no significant effect on RNA yield or quality. Additionally, disrupted samples can be stored in the lysis buffer at -20°C for at least 6 months prior to completion of the extraction procedure providing a flexible sampling and storage scheme to facilitate complex time series experiments.

[Tobacco--a highly efficient producer of vaccines].

PubMed

Budzianowski, Jaromir

2010-01-01

Along with the depreciation of tobacco as a source of nicotine-containing commercial products, the increase of its appreciation as a potential producer of recombinant therapeutical proteins can be observed. Two species of tobacco--Nicotiana tabacum L. and N. benthamiana are easily grown by well established methods of field or green-house cultivation or cell culture, yield high biomass and soluble protein content, can be easily transformed by several methods and are not food for humans or feed for animals. Expression of foreign proteins, including vaccines, can be achieved in those plants either through stable transformation of nuclear or plastid (chloroplast) genomes or by transient transformation using infection with plant virus or bacteria--Agrobacterium tumefaciens (agroinfiltration). The most advanced mode of agrofiltration termed magnifection, which combines benefits of virus and Agrobacterium and depends on using Agrobacterium with viral pro-vectors, enables high-yield and rapid expression of therapeutical proteins, even in a few days, and can be employed on an industrial scale. Expression of many antigenic proteins, which may serve as antiviral, antibacterial, antiprotozoan and anticancer vaccines, and additionally a few autoantigens designed for the treatment of autoimunogenic diseases, like diabetes, have been achieved in tobacco. To date, a vaccine against Newcastle virus disease in poultry produced by tobacco cell culture has been approved for commercial application and several other vaccines are in advanced stage of development. The possibility of a high-level production of vaccines in tobacco against pandemic influenza or anthrax and plague due to a bioterroristic attack, as well as of individualised anticancer vaccines against non-Hodgkin's lymphoma (NHL) in a much shorter period of time than by traditional methods became realistic and hence caused increased interest in tobacco as a high-efficient producer of vaccines not only of specialistic biotechnology firms but also a big pharmaceutical corporation and a department of defence.

Treatment of PCR products with exonuclease I and heat-labile alkaline phosphatase improves the visibility of combined bisulfite restriction analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Watanabe, Kousuke; Emoto, Noriko; Sunohara, Mitsuhiro

2010-08-27

Research highlights: {yields} Incubating PCR products at a high temperature causes smears in gel electrophoresis. {yields} Smears interfere with the interpretation of methylation analysis using COBRA. {yields} Treatment with exonuclease I and heat-labile alkaline phosphatase eliminates smears. {yields} The elimination of smears improves the visibility of COBRA. -- Abstract: DNA methylation plays a vital role in the regulation of gene expression. Abnormal promoter hypermethylation is an important mechanism of inactivating tumor suppressor genes in human cancers. Combined bisulfite restriction analysis (COBRA) is a widely used method for identifying the DNA methylation of specific CpG sites. Here, we report that exonucleasemore » I and heat-labile alkaline phosphatase can be used for PCR purification for COBRA, improving the visibility of gel electrophoresis after restriction digestion. This improvement is observed when restriction digestion is performed at a high temperature, such as 60 {sup o}C or 65 {sup o}C, with BstUI and TaqI, respectively. This simple method can be applied instead of DNA purification using spin columns or phenol/chloroform extraction. It can also be applied to other situations when PCR products are digested by thermophile-derived restriction enzymes, such as PCR restriction fragment length polymorphism (RFLP) analysis.« less

A transferable model for singlet-fission kinetics.

PubMed

Yost, Shane R; Lee, Jiye; Wilson, Mark W B; Wu, Tony; McMahon, David P; Parkhurst, Rebecca R; Thompson, Nicholas J; Congreve, Daniel N; Rao, Akshay; Johnson, Kerr; Sfeir, Matthew Y; Bawendi, Moungi G; Swager, Timothy M; Friend, Richard H; Baldo, Marc A; Van Voorhis, Troy

2014-06-01

Exciton fission is a process that occurs in certain organic materials whereby one singlet exciton splits into two independent triplets. In photovoltaic devices these two triplet excitons can each generate an electron, producing quantum yields per photon of >100% and potentially enabling single-junction power efficiencies above 40%. Here, we measure fission dynamics using ultrafast photoinduced absorption and present a first-principles expression that successfully reproduces the fission rate in materials with vastly different structures. Fission is non-adiabatic and Marcus-like in weakly interacting systems, becoming adiabatic and coupling-independent at larger interaction strengths. In neat films, we demonstrate fission yields near unity even when monomers are separated by >5 Å. For efficient solar cells, however, we show that fission must outcompete charge generation from the singlet exciton. This work lays the foundation for tailoring molecular properties like solubility and energy level alignment while maintaining the high fission yield required for photovoltaic applications.

Identification, Characterization, and Recombinant Expression of Epidermicin NI01, a Novel Unmodified Bacteriocin Produced by Staphylococcus epidermidis That Displays Potent Activity against Staphylococci

PubMed Central

Sandiford, Stephanie

2012-01-01

We describe the discovery, purification, characterization, and expression of an antimicrobial peptide, epidermicin NI01, which is an unmodified bacteriocin produced by Staphylococcus epidermidis strain 224. It is a highly cationic, hydrophobic, plasmid-encoded peptide that exhibits potent antimicrobial activity toward a wide range of pathogenic Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA), enterococci, and biofilm-forming S. epidermidis strains. Purification of the peptide was achieved using a combination of hydrophobic interaction, cation exchange, and high-performance liquid chromatography (HPLC). Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) analysis yielded a molecular mass of 6,074 Da, and partial sequence data of the peptide were elucidated using a combination of tandem mass spectrometry (MS/MS) and de novo sequencing. The draft genome sequence of the producing strain was obtained using 454 pyrosequencing technology, thus enabling the identification of the structural gene using the de novo peptide sequence data previously obtained. Epidermicin NI01 contains 51 residues with four tryptophan and nine lysine residues, and the sequence showed approximately 50% identity to peptides lacticin Z, lacticin Q, and aureocin A53, all of which belong to a new family of unmodified type II-like bacteriocins. The peptide is active in the nanomolar range against S. epidermidis, MRSA isolates, and vancomycin-resistant enterococci. Other unique features displayed by epidermicin include a high degree of protease stability and the ability to retain antimicrobial activity over a pH range of 2 to 10, and exposure to the peptide does not result in development of resistance in susceptible isolates. In this study we also show the structural gene alone can be cloned into Escherichia coli strain BL21(DE3), and expression yields active peptide. PMID:22155816

The regulation of tooth morphogenesis is associated with epithelial cell proliferation and the expression of Sonic hedgehog through epithelial-mesenchymal interactions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ishida, Kentaro; Murofushi, Mayumi; Nakao, Kazuhisa

2011-02-18

Research highlights: {yields} Bioengineered teeth regulated the contact area of epithelium and mesenchyme. {yields} The crown width is regulated by the contact area of the epithelium and mesenchyme. {yields} This regulation is associated with cell proliferation and Sonic hedgehog expression. {yields} The cusp number is correlated with the crown width of the bioengineered tooth. {yields} Cell proliferation and Shh expression areas regulate the tooth morphogenesis. -- Abstract: Ectodermal organs, such as the tooth, salivary gland, hair, and mammary gland, develop through reciprocal epithelial-mesenchymal interactions. Tooth morphologies are defined by the crown width and tooth length (macro-morphologies), and by the numbermore » and locations of the cusp and roots (micro-morphologies). In our current study, we report that the crown width of a bioengineered molar tooth, which was reconstructed using dissociated epithelial and mesenchymal cells via an organ germ method, can be regulated by the contact area between epithelial and mesenchymal cell layers. We further show that this is associated with cell proliferation and Sonic hedgehog (Shh) expression in the inner enamel epithelium after the germ stage has formed a secondary enamel knot. We also demonstrate that the cusp number is significantly correlated with the crown width of the bioengineered tooth. These findings suggest that the tooth micro-morphology, i.e. the cusp formation, is regulated after the tooth width, or macro-morphology, is determined. These findings also suggest that the spatiotemporal patterning of cell proliferation and the Shh expression areas in the epithelium regulate the crown width and cusp formation of the developing tooth.« less

Heterologous Expression and Isolation of Influenza A Virus Nuclear Export Protein NEP.

PubMed

Golovko, A O; Koroleva, O N; Drutsa, V L

2017-12-01

Influenza A virus nuclear export protein NEP (NS2, 14.4 kDa) plays a key role in various steps of the virus life cycle. Highly purified protein preparations are required for structural and functional studies. In this study, we designed a series of Escherichia coli plasmid constructs for highly efficient expression of the NEP gene under control of the constitutive trp promoter. An efficient method for extraction of NEP from inclusion bodies based on dodecyl sulfate treatment was developed. Preparations of purified NEP with either N- or C-terminal (His) 6 -tag were obtained using Ni-NTA agarose affinity chromatography with yield of more than 20 mg per liter of culture. According to CD data, the secondary structure of the proteins matched that of natural NEP. A high propensity of NEP to aggregate over a wide range of conditions was observed.

High-level expression in Saccharomyces cerevisiae enables isolation and spectroscopic characterization of functional human adenosine A2a receptor

PubMed Central

O’Malley, Michelle A.; Lazarova, Tzvetana; Britton, Zachary T.; Robinson, Anne S.

2007-01-01

The G-protein coupled receptors (GPCRs) are a class of membrane proteins that trigger cellular responses to external stimuli, and are believed to be targets for nearly half of all pharmaceutical drugs on the market. However, little is known regarding their folding and cellular interactions, as well as what factors are crucial for their activity. Further structural characterization of GPCRs has largely been complicated by problems with expression, purification, and preservation of activity in vitro. Previously, we have demonstrated high-level expression (~4 mg/L of culture) of functional human adenosine A2a receptor fused to a green fluorescent protein (A2aR-GFP) from Saccharomyces cerevisiae. In this work we re-engineered A2aR with a purification tag, developed an adequate purification scheme, and performed biophysical characterization on purified receptors. Milligram amounts per liter of culture of A2aR and A2aR-GFP were functionally expressed in S. cerevisiae, with a C-terminal deca-histidine tag. Lysis procedures were developed for optimal membrane protein solubilization and recovery through monitoring fluorescence of A2aR-GFP-His10. One-step purification of the protein was achieved through immobilized metal affinity chromatography. After initial solubilization in n-dodecyl-β-D-maltoside (DDM), a combination of added cholesterol hemisuccinate (CHS) in 3-(3-cholamidopropyl)-dimethylammoniopropane sulfonate (CHAPS) was required to stabilize the functional state of the protein. Isolated A2aR under these conditions was found to be largely alpha-helical, and properly incorporated into a mixed-micelle environment. The A2a-His10 receptor was purified in quantities of 6 +/− 2 mg/L of culture, with ligand-binding yields of 1 mg/L, although all protein bound to xanthine affinity resin. This represents the highest purified total and functional yields for A2aR yet achieved from any heterologous expression system. PMID:17591446

Developing a scalable model of recombinant protein yield from Pichia pastoris: the influence of culture conditions, biomass and induction regime

PubMed Central

Holmes, William J; Darby, Richard AJ; Wilks, Martin DB; Smith, Rodney; Bill, Roslyn M

2009-01-01

Background The optimisation and scale-up of process conditions leading to high yields of recombinant proteins is an enduring bottleneck in the post-genomic sciences. Typical experiments rely on varying selected parameters through repeated rounds of trial-and-error optimisation. To rationalise this, several groups have recently adopted the 'design of experiments' (DoE) approach frequently used in industry. Studies have focused on parameters such as medium composition, nutrient feed rates and induction of expression in shake flasks or bioreactors, as well as oxygen transfer rates in micro-well plates. In this study we wanted to generate a predictive model that described small-scale screens and to test its scalability to bioreactors. Results Here we demonstrate how the use of a DoE approach in a multi-well mini-bioreactor permitted the rapid establishment of high yielding production phase conditions that could be transferred to a 7 L bioreactor. Using green fluorescent protein secreted from Pichia pastoris, we derived a predictive model of protein yield as a function of the three most commonly-varied process parameters: temperature, pH and the percentage of dissolved oxygen in the culture medium. Importantly, when yield was normalised to culture volume and density, the model was scalable from mL to L working volumes. By increasing pre-induction biomass accumulation, model-predicted yields were further improved. Yield improvement was most significant, however, on varying the fed-batch induction regime to minimise methanol accumulation so that the productivity of the culture increased throughout the whole induction period. These findings suggest the importance of matching the rate of protein production with the host metabolism. Conclusion We demonstrate how a rational, stepwise approach to recombinant protein production screens can reduce process development time. PMID:19570229

Optimizing antibody expression: The nuts and bolts.

PubMed

Ayyar, B Vijayalakshmi; Arora, Sushrut; Ravi, Shiva Shankar

2017-03-01

Antibodies are extensively utilized entities in biomedical research, and in the development of diagnostics and therapeutics. Many of these applications require high amounts of antibodies. However, meeting this ever-increasing demand of antibodies in the global market is one of the outstanding challenges. The need to maintain a balance between demand and supply of antibodies has led the researchers to discover better means and methods for optimizing their expression. These strategies aim to increase the volumetric productivity of the antibodies along with the reduction of associated manufacturing costs. Recent years have witnessed major advances in recombinant protein technology, owing to the introduction of novel cloning strategies, gene manipulation techniques, and an array of cell and vector engineering techniques, together with the progress in fermentation technologies. These innovations were also highly beneficial for antibody expression. Antibody expression depends upon the complex interplay of multiple factors that may require fine tuning at diverse levels to achieve maximum yields. However, each antibody is unique and requires individual consideration and customization for optimizing the associated expression parameters. This review provides a comprehensive overview of several state-of-the-art approaches, such as host selection, strain engineering, codon optimization, gene optimization, vector modification and process optimization that are deemed suitable for enhancing antibody expression. Copyright © 2017 Elsevier Inc. All rights reserved.

Production of membrane proteins without cells or detergents.

PubMed

Rajesh, Sundaresan; Knowles, Timothy; Overduin, Michael

2011-04-30

The production of membrane proteins in cellular systems is besieged by several problems due to their hydrophobic nature which often causes misfolding, protein aggregation and cytotoxicity, resulting in poor yields of stable proteins. Cell-free expression has emerged as one of the most versatile alternatives for circumventing these obstacles by producing membrane proteins directly into designed hydrophobic environments. Efficient optimisation of expression and solubilisation conditions using a variety of detergents, membrane mimetics and lipids has yielded structurally and functionally intact membrane proteins, with yields several fold above the levels possible from cell-based systems. Here we review recently developed techniques available to produce functional membrane proteins, and discuss amphipols, nanodisc and styrene maleic acid lipid particle (SMALP) technologies that can be exploited alongside cell-free expression of membrane proteins. Copyright © 2010 Elsevier B.V. All rights reserved.

Recent advances in reconstructing microbial secondary metabolites biosynthesis in Aspergillus spp.

PubMed

He, Yi; Wang, Bin; Chen, Wanping; Cox, Russell J; He, Jingren; Chen, Fusheng

High throughput genome sequencing has revealed a multitude of potential secondary metabolites biosynthetic pathways that remain cryptic. Pathway reconstruction coupled with genetic engineering via heterologous expression enables discovery of novel compounds, elucidation of biosynthetic pathways, and optimization of product yields. Apart from Escherichia coli and yeast, fungi, especially Aspergillus spp., are well known and efficient heterologous hosts. This review summarizes recent advances in heterologous expression of microbial secondary metabolite biosynthesis in Aspergillus spp. We also discuss the technological challenges and successes in regard to heterologous host selection and DNA assembly behind the reconstruction of microbial secondary metabolite biosynthesis. Copyright © 2018 Elsevier Inc. All rights reserved.

PPAR{gamma} activates ABCA1 gene transcription but reduces the level of ABCA1 protein in HepG2 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mogilenko, Denis A., E-mail: denis@iem.sp.ru; Department of Embryology, St. Petersburg State University, 199034 St. Petersburg; Shavva, Vladimir S.

Research highlights: {yields} PPAR{gamma} activates ABCA1 gene expression but decreases ABCA1 protein content in human hepatoma cell line HepG2. {yields} Treatment of HepG2 cells with PPAR{gamma} agonist GW1929 leads to dissociation of LXR{beta} from ABCA1-LXR{beta} complex. {yields} Inhibition of protein kinases MEK1/2 abolishes PPAR{gamma}-mediated dissociation of LXR{beta} from ABCA1/LXR{beta} complex. {yields} Activation of PPAR{gamma} leads to increasing of the level of LXR{beta} associated with LXRE within ABCA1 gene promoter. -- Abstract: Synthesis of ABCA1 protein in liver is necessary for high-density lipoproteins (HDL) formation in mammals. Nuclear receptor PPAR{gamma} is known as activator of ABCA1 expression, but details of PPAR{gamma}-mediatedmore » regulation of ABCA1 at both transcriptional and post-transcriptional levels in hepatocytes have not still been well elucidated. In this study we have shown, that PPAR{gamma} activates ABCA1 gene transcription in human hepatoma cells HepG2 through increasing of LXR{beta} binding with promoter region of ABCA1 gene. Treatment of HepG2 cells with PPAR{gamma} agonist GW1929 leads to dissociation of LXR{beta} from ABCA1/LXR{beta} complex and to nuclear translocation of this nuclear receptor resulting in reduction of ABCA1 protein level 24 h after treatment. Inhibition of protein kinases MEK1/2 abolishes PPAR{gamma}-mediated dissociation of LXR{beta} from ABCA1/LXR{beta} complex, but does not block PPAR{gamma}-dependent down-regulation of ABCA1 protein in HepG2 cells. These data suggest that PPAR{gamma} may be important for regulation of the level of hepatic ABCA1 protein and indicate the new interplays between PPAR{gamma}, LXR{beta} and MEK1/2 in regulation of ABCA1 mRNA and protein expression.« less

A novel Rieske-type protein derived from an apoptosis-inducing factor-like (AIFL) transcript with a retained intron 4 induces change in mitochondrial morphology and growth arrest

DOE Office of Scientific and Technical Information (OSTI.GOV)

Murata, Yasuhiko, E-mail: 97318@ib.k.u-tokyo.ac.jp; Furuyama, Isao; Oda, Shoji

2011-04-01

Highlights: {yields} A novel major transcript, AIFL-I4, is found. {yields} Nuclear localization of AIFL-I4 induces mitochondrial morphology change and suppression of cell proliferation. {yields} AIFL-I4 mutant with a lesion in [2Fe-2S] cluster binding site does not induce these phenotypes. {yields} [2Fe-2S] cluster binding site is essential for these phenotypes. -- Abstract: Apoptosis-inducing factor-like (AIFL) protein contains a Rieske domain and pyridine nucleotide-disulfide oxidoreductase (Pyr{sub r}edox) domain that shows 35% homology to that of apoptosis-inducing factor (AIF) protein. We identified a novel major transcript of the medaka (Oryzias latipes) AIFL gene that retained intron 4 (AIFL-I4) in embryos and tissues frommore » adult fish. The product of this transcript, AIFL-I4 protein, lacked the Pyr{sub r}edox domain because of a nonsense codon in intron 4. Both AIFL-I4 and full-length AIFL (fAIFL) transcripts were highly expressed in the brain and late embryos, and relative fAIFL and AIFL-I4 expression levels differed among tissues. Transient expression of AIFL-I4 and fAIFL tagged with GFP showed that AIFL-I4 localized in the nucleus, while fAIFL localized throughout the cytoplasm. We also found that overexpression of AIFL-I4 induced a change in mitochondrial morphology and suppression of cell proliferation. AIFL-I4 mutant with a lesion in [2Fe-2S] cluster binding site of the Rieske domain did not induce these phenotypes. This report is the first to demonstrate nuclear localization of a Rieske-type protein translated from the AIFL gene. Our data suggested that the [2Fe-2S] cluster binding site was essential for the nuclear localization and involved in mitochondrial morphology and suppression of cell proliferation.« less

Controlling the spectral shape of nonlinear Thomson scattering with proper laser chirping

DOE PAGES

Rykovanov, S. G.; Geddes, C. G. R.; Schroeder, C. B.; ...

2016-03-18

Effects of nonlinearity in Thomson scattering of a high intensity laser pulse from electrons are analyzed. Analytic expressions for laser pulse shaping in frequency (chirping) are obtained which control spectrum broadening for high laser pulse intensities. These analytic solutions allow prediction of the spectral form and required laser parameters to avoid broadening. Results of analytical and numerical calculations agree well. The control over the scattered radiation bandwidth allows narrow bandwidth sources to be produced using high scattering intensities, which in turn greatly improves scattering yield for future x- and gamma-ray sources.

Recombinants proteins for industrial uses: utilization of Pichia pastoris expression system

PubMed Central

Rabert, Claudia; Weinacker, Daniel; Pessoa, Adalberto; Farías, Jorge G.

2013-01-01

The innovation in industrial process with impact in the efficient production is the major challenge for actual industry. A high numerous of enzymes are utilized in at different level of process; the search for new alternatives with better characteristic has become a field of study of great interest, the recombinant protein achievement in a different host system is an alternative widely assessed for production of this. The microorganism Pichia pastoris has been used like a successful expression system in diverse areas, improved the yield and extraction-recovery of the product expressed. The reported of diverse authors in the production of enzymes with different application in industry is varied, in this review the different industry areas and the characteristic of the enzymes produced are detailed. PMID:24294221

Traits and selection strategies to improve root systems and water uptake in water-limited wheat crops.

PubMed

Wasson, A P; Richards, R A; Chatrath, R; Misra, S C; Prasad, S V Sai; Rebetzke, G J; Kirkegaard, J A; Christopher, J; Watt, M

2012-05-01

Wheat yields globally will depend increasingly on good management to conserve rainfall and new varieties that use water efficiently for grain production. Here we propose an approach for developing new varieties to make better use of deep stored water. We focus on water-limited wheat production in the summer-dominant rainfall regions of India and Australia, but the approach is generally applicable to other environments and root-based constraints. Use of stored deep water is valuable because it is more predictable than variable in-season rainfall and can be measured prior to sowing. Further, this moisture is converted into grain with twice the efficiently of in-season rainfall since it is taken up later in crop growth during the grain-filling period when the roots reach deeper layers. We propose that wheat varieties with a deeper root system, a redistribution of branch root density from the surface to depth, and with greater radial hydraulic conductivity at depth would have higher yields in rainfed systems where crops rely on deep water for grain fill. Developing selection systems for mature root system traits is challenging as there are limited high-throughput phenotyping methods for roots in the field, and there is a risk that traits selected in the lab on young plants will not translate into mature root system traits in the field. We give an example of a breeding programme that combines laboratory and field phenotyping with proof of concept evaluation of the trait at the beginning of the selection programme. This would greatly enhance confidence in a high-throughput laboratory or field screen, and avoid investment in screens without yield value. This approach requires careful selection of field sites and years that allow expression of deep roots and increased yield. It also requires careful selection and crossing of germplasm to allow comparison of root expression among genotypes that are similar for other traits, especially flowering time and disease and toxicity resistances. Such a programme with field and laboratory evaluation at the outset will speed up delivery of varieties with improved root systems for higher yield.

Milk yield differences between 1x and 4x milking are associated with changes in mammary mitochondrial number and milk protein gene expression, but not mammary cell apoptosis or "SOCS" gene expression

USDA-ARS?s Scientific Manuscript database

Milking frequency is known to affect milk production and lactation persistence in dairy cows. Despite this, the mechanisms underlying this effect are only partially understood. Previous work in dairy cows examining increases in milk yield due to increased milking frequency have identified changes in...

A simple approach for human recombinant apolipoprotein E4 expression and purification.

PubMed

Argyri, Letta; Skamnaki, Vassiliki; Stratikos, Efstratios; Chroni, Angeliki

2011-10-01

We report a simple expression and purification procedure for the production of recombinant apolipoprotein E4 (apoE4), an important protein for the lipid homeostasis in humans that plays critical roles in the pathogenesis of cardiovascular and neurodegenerative diseases. Our approach is based on the expression of a thioredoxin-apoE4 fusion construct in bacterial cells and subsequent removal of the fused thioredoxin using the highly specific 3C protease, avoiding costly and laborious lipidation-delipidation steps used before. Our approach results in rapid, high-yield production of structurally and functionally competent apoE4 as evidenced by secondary structure measurements, thermal and chemical melting profiles and the kinetic profile of solubilization of dimyristoyl-phosphatidylcholine (DMPC) vesicles. This protocol is appropriate for laboratories with little experience in apolipoprotein biochemistry and will facilitate future studies on the role of apoE4 in the pathogenesis of cardiovascular disease and neurodegenerative diseases, including Alzheimer's disease. Copyright © 2011 Elsevier Inc. All rights reserved.

Rapid synthesis of DNA-cysteine conjugates for expressed protein ligation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lovrinovic, Marina; Niemeyer, Christof M.

2005-09-30

We report a rapid method for the covalent modification of commercially available amino-modified DNA oligonucleotides with a cysteine moiety. The resulting DNA-cysteine conjugates are versatile reagents for the efficient preparation of covalent DNA-protein conjugates by means of expressed protein ligation (EPL). The EPL method allows for the site-specific coupling of cysteine-modified DNA oligomers with recombinant intein-fusion proteins, the latter of which contain a C-terminal thioester enabling the mild and highly specific reaction with N-terminal cysteine compounds. We prepared a cysteine-modifier reagent in a single-step reaction which allows for the rapid and near quantitative synthesis of cysteine-DNA conjugates. The latter weremore » ligated with the green fluorescent protein mutant EYFP, recombinantly expressed as an intein-fusion protein, allowing for the mild and selective formation of EYFP-DNA conjugates in high yields of about 60%. We anticipate many applications of our approach, ranging from protein microarrays to the arising field of nanobiotechnology.« less

Identification of Essential Genetic Baculoviral Elements for Recombinant Protein Expression by Transactivation in Sf21 Insect Cells.

PubMed

Bleckmann, Maren; Schürig, Margitta; Chen, Fang-Fang; Yen, Zen-Zen; Lindemann, Nils; Meyer, Steffen; Spehr, Johannes; van den Heuvel, Joop

2016-01-01

The Baculovirus Expression Vector System (BEVS) is widely used to produce high amounts of recombinant proteins. Nevertheless, generating recombinant baculovirus in high quality is rather time-consuming and labor-intensive. Alternatively, virus-free expression in insect cells did not achieve similar expression levels for most proteins so far. The transactivation method is a promising approach for protein expression in Sf21 cells. It combines advantages of BEVS and plasmid-based expression by activating strong virus-dependent promoters on a transfected plasmid by baculoviral coinfection. Here, we identified expression elements required for transactivation. Therefore, we designed several vectors comprising different viral promoters or promoter combinations and tested them for eGFP expression using the automated BioLector microcultivation system. Remarkably, only the combination of the very late promoter p10 together with the homologous region 5 (hr5) could boost expression during transactivation. Other elements, like p10 alone or the late viral promoter polH, did not respond to transactivation. A new combination of hr5 and p10 with the strongest immediate early OpMNPV viral promoter OpIE2 improved the yield of eGFP by ~25% in comparison to the previous applied hr5-IE1-p10 expression cassette. Furthermore, we observed a strong influence of the transcription termination sequence and vector backbone on the level of expression. Finally, the expression levels for transactivation, BEVS and solely plasmid-based expression were compared for the marker protein eGFP, underlining the potential of transactivation for fast recombinant protein expression in Sf21 cells. In conclusion, essential elements for transactivation could be identified. The optimal elements were applied to generate an improved vector applicable in virus-free plasmid-based expression, transactivation and BEVS.

The redox-sensing protein Rex modulates ethanol production in Thermoanaerobacterium saccharolyticum

PubMed Central

Lanahan, Anthony A.; Lynd, Lee R.

2018-01-01

Thermoanaerobacterium saccharolyticum is a thermophilic anaerobe that has been engineered to produce high amounts of ethanol, reaching ~90% theoretical yield at a titer of 70 g/L. Here we report the physiological changes that occur upon deleting the redox-sensing transcriptional regulator Rex in wild type T. saccharolyticum: a single deletion of rex resulted in a two-fold increase in ethanol yield (from 40% to 91% theoretical yield), but the resulting strains grew only about a third as fast as the wild type strain. Deletion of the rex gene also had the effect of increasing expression of alcohol dehydrogenase genes, adhE and adhA. After several serial transfers, the ethanol yield decreased from an average of 91% to 55%, and the growth rates had increased. We performed whole-genome resequencing to identify secondary mutations in the Δrex strains adapted for faster growth. In several cases, secondary mutations had appeared in the adhE gene. Furthermore, in these strains the NADH-linked alcohol dehydrogenase activity was greatly reduced. Complementation studies were done to reintroduce rex into the Δrex strains: reintroducing rex decreased ethanol yield to below wild type levels in the Δrex strain without adhE mutations, but did not change the ethanol yield in the Δrex strain where an adhE mutation occurred. PMID:29621294

Evaluation of plant biomass resources available for replacement of fossil oil

PubMed Central

Henry, Robert J

2010-01-01

The potential of plants to replace fossil oil was evaluated by considering the scale of production required, the area of land needed and the types of plants available. High yielding crops (50 tonnes/ha) that have a high conversion efficiency (75%) would require a global land footprint of around 100 million ha to replace current (2008) oil consumption. Lower yielding or less convertible plants would require a larger land footprint. Domestication of new species as dedicated energy crops may be necessary. A systematic analysis of higher plants and their current and potential uses is presented. Plant biotechnology provides tools to improve the prospects of replacing oil with plant-derived biomass by increasing the amount of biomass produced per unit area of land and improving the composition of the biomass to increase the efficiency of conversion to biofuel and biomaterials. Options for the production of high value coproducts and the expression of processing aids such as enzymes in the plant may add further value to plants as bioenergy resources. PMID:20070873

Piperidine alkaloids from Piperretrofractum Vahl. protect against high-fat diet-induced obesity by regulating lipid metabolism and activating AMP-activated protein kinase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Kyung Jin; Lee, Myoung-Su; Jo, Keunae

Highlights: {yields} Piperidine alkaloids from Piperretrofractum Vahl. (PRPAs), including piperine, pipernonaline, and dehydropipernonaline, are isolated as the anti-obesity constituents. {yields} PRPA administration significantly reduces body weight gain without altering food intake and fat pad mass. {yields} PRPA reduces high-fat diet-induced triglyceride accumulation in liver. {yields} PRPAs attenuate HFD-induced obesity by activating AMPK and PPAR{delta}, and regulate lipid metabolism, suggesting their potential anti-obesity effects. -- Abstract: The fruits of Piperretrofractum Vahl. have been used for their anti-flatulent, expectorant, antitussive, antifungal, and appetizing properties in traditional medicine, and they are reported to possess gastroprotective and cholesterol-lowering properties. However, their anti-obesity activity remainsmore » unexplored. The present study was conducted to isolate the anti-obesity constituents from P. retrofractum Vahl. and evaluate their effects in high-fat diet (HFD)-induced obese mice. Piperidine alkaloids from P. retrofractum Vahl. (PRPAs), including piperine, pipernonaline, and dehydropipernonaline, were isolated as the anti-obesity constituents through a peroxisome proliferator-activated receptor {delta} (PPAR{delta}) transactivation assay. The molecular mechanism was investigated in 3T3-L1 adipocytes and L6 myocytes. PRPA treatment activated AMP-activated protein kinase (AMPK) signaling and PPAR{delta} protein and also regulated the expression of lipid metabolism-related proteins. In the animal model, oral PRPA administration (50, 100, or 300 mg/kg/day for 8 weeks) significantly reduced HFD-induced body weight gain without altering the amount of food intake. Fat pad mass was reduced in the PRPA treatment groups, as evidenced by reduced adipocyte size. In addition, elevated serum levels of total cholesterol, low-density lipoprotein cholesterol, total lipid, leptin, and lipase were suppressed by PRPA treatment. PRPA also protected against the development of nonalcoholic fatty liver by decreasing hepatic triglyceride accumulation. Consistent with the in vitro results, PRPA activated AMPK signaling and altered the expression of lipid metabolism-related proteins in liver and skeletal muscle. Taken together, these findings demonstrate that PRPAs attenuate HFD-induced obesity by activating AMPK and PPAR{delta}, and regulate lipid metabolism, suggesting their potential anti-obesity effects.« less

Light-Weight Multispectral Uav Sensors and Their Capabilities for Predicting Grain Yield and Detecting Plant Diseases

NASA Astrophysics Data System (ADS)

Nebiker, S.; Lack, N.; Abächerli, M.; Läderach, S.

2016-06-01

In this paper we investigate the performance of new light-weight multispectral sensors for micro UAV and their application to selected tasks in agronomical research and agricultural practice. The investigations are based on a series of flight campaigns in 2014 and 2015 covering a number of agronomical test sites with experiments on rape, barley, onion, potato and other crops. In our sensor comparison we included a high-end multispectral multiSPEC 4C camera with bandpass colour filters and reference channel in zenith direction and a low-cost, consumer-grade Canon S110 NIR camera with Bayer pattern colour filters. Ground-based reference measurements were obtained using a terrestrial hyperspectral field spectrometer. The investigations show that measurements with the high-end system consistently match very well with ground-based field spectrometer measurements with a mean deviation of just 0.01-0.04 NDVI values. The low-cost system, while delivering better spatial resolutions, expressed significant biases. The sensors were subsequently used to address selected agronomical questions. These included crop yield estimation in rape and barley and plant disease detection in potato and onion cultivations. High levels of correlation between different vegetation indices and reference yield measurements were obtained for rape and barley. In case of barley, the NDRE index shows an average correlation of 87% with reference yield, when species are taken into account. With high geometric resolutions and respective GSDs of down to 2.5 cm the effects of a thrips infestation in onion could be analysed and potato blight was successfully detected at an early stage of infestation.

Cell Wall Remodeling in Abscission Zone Cells during Ethylene-Promoted Fruit Abscission in Citrus

PubMed Central

Merelo, Paz; Agustí, Javier; Arbona, Vicent; Costa, Mário L.; Estornell, Leandro H.; Gómez-Cadenas, Aurelio; Coimbra, Silvia; Gómez, María D.; Pérez-Amador, Miguel A.; Domingo, Concha; Talón, Manuel; Tadeo, Francisco R.

2017-01-01

Abscission is a cell separation process by which plants can shed organs such as fruits, leaves, or flowers. The process takes place in specific locations termed abscission zones. In fruit crops like citrus, fruit abscission represents a high percentage of annual yield losses. Thus, understanding the molecular regulation of abscission is of capital relevance to control production. To identify genes preferentially expressed within the citrus fruit abscission zone (AZ-C), we performed a comparative transcriptomics assay at the cell type resolution level between the AZ-C and adjacent fruit rind cells (non-abscising tissue) during ethylene-promoted abscission. Our strategy combined laser microdissection with microarray analysis. Cell wall modification-related gene families displayed prominent representation in the AZ-C. Phylogenetic analyses of such gene families revealed a link between phylogenetic proximity and expression pattern during abscission suggesting highly conserved roles for specific members of these families in abscission. Our transcriptomic data was validated with (and strongly supported by) a parallel approach consisting on anatomical, histochemical and biochemical analyses on the AZ-C during fruit abscission. Our work identifies genes potentially involved in organ abscission and provides relevant data for future biotechnology approaches aimed at controlling such crucial process for citrus yield. PMID:28228766

Mutant Transcriptome Sequencing Provides Insights into Pod Development in Peanut (Arachis hypogaea L.)

PubMed Central

Wan, Liyun; Li, Bei; Lei, Yong; Yan, Liying; Ren, Xiaoping; Chen, Yuning; Dai, Xiaofeng; Jiang, Huifang; Zhang, Juncheng; Guo, Wei; Chen, Ao; Liao, Boshou

2017-01-01

Pod size is the major yield component and a key target trait that is selected for in peanut breeding. However, although numerous quantitative trait loci (QTLs) for peanut pod size have been described, the molecular mechanisms underlying the development of this characteristic remain elusive. A peanut mutant with a narrower pod was developed in this study using ethyl methanesulfonate (EMS) mutagenesis and designated as the “pod width” mutant line (pw). The fresh pod weight of pw was only about 40% of that seen in the wild-type (WT) Zhonghua16, while the hull and seed filling of the mutant both also developed at earlier stages. Pods from both pw and WT lines were sampled 20, 40, and 60 days after flowering (DAF) and used for RNA-Seq analysis; the results revealed highly differentially expressed lignin metabolic pathway genes at all three stages, but especially at DAF 20 and DAF 40. At the same time, expression of genes related to auxin signal transduction was found to be significantly repressed during the pw early pod developmental stage. A genome-wide comparative analysis of expression profiles revealed 260 differentially expressed genes (DEGs) across all three stages, and two candidate genes, c26901_g1 (CAD) and c37339_g1 (ACS), responsible for pod width were identified by integrating expression patterns and function annotation of the common DEGs within the three stages. Taken together, the information provided in this study illuminates the processes underlying peanut pod development, and will facilitate further identification of causal genes and the development of improved peanut varieties with higher yields. PMID:29170673

Glycoprotein production for structure analysis with stable, glycosylation mutant CHO cell lines established by fluorescence-activated cell sorting.

PubMed

Wilke, Sonja; Krausze, Joern; Gossen, Manfred; Groebe, Lothar; Jäger, Volker; Gherardi, Ermanno; van den Heuvel, Joop; Büssow, Konrad

2010-06-01

Stable mammalian cell lines are excellent tools for the expression of secreted and membrane glycoproteins. However, structural analysis of these molecules is generally hampered by the complexity of N-linked carbohydrate side chains. Cell lines with mutations are available that result in shorter and more homogenous carbohydrate chains. Here, we use preparative fluorescence-activated cell sorting (FACS) and site-specific gene excision to establish high-yield glycoprotein expression for structural studies with stable clones derived from the well-established Lec3.2.8.1 glycosylation mutant of the Chinese hamster ovary (CHO) cell line. We exemplify the strategy by describing novel clones expressing single-chain hepatocyte growth factor/scatter factor (HGF/SF, a secreted glycoprotein) and a domain of lysosome-associated membrane protein 3 (LAMP3d). In both cases, stable GFP-expressing cell lines were established by transfection with a genetic construct including a GFP marker and two rounds of cell sorting after 1 and 2 weeks. The GFP marker was subsequently removed by heterologous expression of Flp recombinase. Production of HGF/SF and LAMP3d was stable over several months. 1.2 mg HGF/SF and 0.9 mg LAMP3d were purified per litre of culture, respectively. Homogenous glycoprotein preparations were amenable to enzymatic deglycosylation under native conditions. Purified and deglycosylated LAMP3d protein was readily crystallized. The combination of FACS and gene excision described here constitutes a robust and fast procedure for maximizing the yield of glycoproteins for structural analysis from glycosylation mutant cell lines.

An optimized expression vector for improving the yield of dengue virus-like particles from transfected insect cells.

PubMed

Charoensri, Nicha; Suphatrakul, Amporn; Sriburi, Rungtawan; Yasanga, Thippawan; Junjhon, Jiraphan; Keelapang, Poonsook; Utaipat, Utaiwan; Puttikhunt, Chunya; Kasinrerk, Watchara; Malasit, Prida; Sittisombut, Nopporn

2014-09-01

Recombinant virus-like particles (rVLPs) of flaviviruses are non-infectious particles released from cells expressing the envelope glycoproteins prM and E. Dengue virus rVLPs are recognized as a potential vaccine candidate, but large scale production of these particles is hindered by low yields and the occurrence of cytopathic effects. In an approach to improve the yield of rVLPs from transfected insect cells, several components of a dengue serotype 2 virus prM+E expression cassette were modified and the effect of these modifications was assessed during transient expression. Enhancement of extracellular rVLP levels by simultaneous substitutions of the prM signal peptide and the stem-anchor region of E with homologous cellular and viral counterparts, respectively, was further augmented by codon optimization. Extensive formation of multinucleated cells following transfection with the codon-optimized expression cassette was abrogated by introducing an E fusion loop mutation. This mutation also helped restore the extracellular E levels affected negatively by alteration of a charged residue at the pr-M junction, which was intended to promote maturation of rVLPs during export. Optimized expression cassettes generated in this multiple add-on modification approach should be useful in the generation of stably expressing clones and production of dengue virus rVLPs for immunogenicity studies. Copyright © 2014 Elsevier B.V. All rights reserved.

Freebies for Investors--Precise Incremental Yield Value

ERIC Educational Resources Information Center

Michelson, Irving

1977-01-01

Competition for savings dollars has led to free gift bonus offers as incentive for new deposits. A concise new formula presented here permits calculation of the total yield using an inexpensive minicomputer. Yield is expressed in terms of interest rate, effective discount value of gift bonus, and period of deposit. (Author/MA)

A rapid and cost-effective method of producing recombinant proBNP and NT-proBNP variants in Escherichia coli for immunoassay of heart failure.

PubMed

Soleh, Muhammad Tarmizi; Foo, Jared Yong Yang; Bailey, Ulla-Maja; Tan, Nikki Yi; Wan, Yunxia; Cooper-White, Justin; Schulz, Benjamin Luke; Punyadeera, Chamindie

2014-01-01

The measurements of plasma natriuretic peptides (NT-proBNP, proBNP and BNP) are used to diagnose heart failure but these are expensive to produce. We describe a rapid, cheap and facile production of proteins for immunoassays of heart failure. DNA encoding N-terminally His-tagged NT-proBNP and proBNP were cloned into the pJexpress404 vector. ProBNP and NT-proBNP peptides were expressed in Escherichia coli, purified and refolded in vitro. The analytical performance of these peptides were comparable with commercial analytes (NT-proBNP EC50 for the recombinant is 2.6 ng/ml and for the commercial material is 5.3 ng/ml) and the EC50 for recombinant and commercial proBNP, are 3.6 and 5.7 ng/ml respectively). Total yield of purified refolded NT-proBNP peptide was 1.75 mg/l and proBNP was 0.088 mg/l. This approach may also be useful in expressing other protein analytes for immunoassay applications. To develop a cost effective protein expression method in E. coli to obtain high yields of NT-proBNP (1.75 mg/l) and proBNP (0.088 mg/l) peptides for immunoassay use.

Roles of Soybean Plasma Membrane Intrinsic Protein GmPIP2;9 in Drought Tolerance and Seed Development.

PubMed

Lu, Linghong; Dong, Changhe; Liu, Ruifang; Zhou, Bin; Wang, Chuang; Shou, Huixia

2018-01-01

Aquaporins play an essential role in water uptake and transport in vascular plants. The soybean genome contains a total of 22 plasma membrane intrinsic protein (PIP) genes. To identify candidate PIPs important for soybean yield and stress tolerance, we studied the transcript levels of all 22 soybean PIPs. We found that a GmPIP2 subfamily member, GmPIP2;9, was predominately expressed in roots and developing seeds. Here, we show that GmPIP2;9 localized to the plasma membrane and had high water channel activity when expressed in Xenopus oocytes. Using transgenic soybean plants expressing a native GmPIP2;9 promoter driving a GUS-reporter gene, it was found high GUS expression in the roots, in particular, in the endoderm, pericycle, and vascular tissues of the roots of transgenic plants. In addition, GmPIP2;9 was also highly expressed in developing pods. GmPIP2;9 expression significantly increased in short term of polyethylene glycol (PEG)-mediated drought stress treatment. GmPIP2;9 overexpression increased tolerance to drought stress in both solution cultures and soil plots. Drought stress in combination with GmPIP2;9 overexpression increased net CO 2 assimilation of photosynthesis, stomata conductance, and transpiration rate, suggesting that GmPIP2;9- overexpressing transgenic plants were less stressed than wild-type (WT) plants. Furthermore, field experiments showed that GmPIP2;9 -overexpressing plants had significantly more pod numbers and larger seed sizes than WT plants. In summary, the study demonstrated that GmPIP2;9 has water transport activity. Its relative high expression levels in roots and developing pods are in agreement with the phenotypes of GmPIP2;9 -overexpressing plants in drought stress tolerance and seed development.

Roles of Soybean Plasma Membrane Intrinsic Protein GmPIP2;9 in Drought Tolerance and Seed Development

PubMed Central

Lu, Linghong; Dong, Changhe; Liu, Ruifang; Zhou, Bin; Wang, Chuang; Shou, Huixia

2018-01-01

Aquaporins play an essential role in water uptake and transport in vascular plants. The soybean genome contains a total of 22 plasma membrane intrinsic protein (PIP) genes. To identify candidate PIPs important for soybean yield and stress tolerance, we studied the transcript levels of all 22 soybean PIPs. We found that a GmPIP2 subfamily member, GmPIP2;9, was predominately expressed in roots and developing seeds. Here, we show that GmPIP2;9 localized to the plasma membrane and had high water channel activity when expressed in Xenopus oocytes. Using transgenic soybean plants expressing a native GmPIP2;9 promoter driving a GUS-reporter gene, it was found high GUS expression in the roots, in particular, in the endoderm, pericycle, and vascular tissues of the roots of transgenic plants. In addition, GmPIP2;9 was also highly expressed in developing pods. GmPIP2;9 expression significantly increased in short term of polyethylene glycol (PEG)-mediated drought stress treatment. GmPIP2;9 overexpression increased tolerance to drought stress in both solution cultures and soil plots. Drought stress in combination with GmPIP2;9 overexpression increased net CO2 assimilation of photosynthesis, stomata conductance, and transpiration rate, suggesting that GmPIP2;9-overexpressing transgenic plants were less stressed than wild-type (WT) plants. Furthermore, field experiments showed that GmPIP2;9-overexpressing plants had significantly more pod numbers and larger seed sizes than WT plants. In summary, the study demonstrated that GmPIP2;9 has water transport activity. Its relative high expression levels in roots and developing pods are in agreement with the phenotypes of GmPIP2;9-overexpressing plants in drought stress tolerance and seed development. PMID:29755491

Wheat differential gene expression induced by different races of Puccinia triticina.

PubMed

Neugebauer, Kerri A; Bruce, Myron; Todd, Tim; Trick, Harold N; Fellers, John P

2018-01-01

Puccinia triticina, the causal agent of wheat leaf rust, causes significant losses in wheat yield and quality each year worldwide. During leaf rust infection, the host plant recognizes numerous molecules, some of which trigger host defenses. Although P. triticina reproduces clonally, there is still variation within the population due to a high mutation frequency, host specificity, and environmental adaptation. This study explores how wheat responds on a gene expression level to different P. triticina races. Six P. triticina races were inoculated onto a susceptible wheat variety and samples were taken at six days post inoculation, just prior to pustule eruption. RNA sequence data identified 63 wheat genes differentially expressed between the six races. A time course, conducted over the first seven days post inoculation, was used to examine the expression pattern of 63 genes during infection. Forty-seven wheat genes were verified to have differential expression. Three common expression patterns were identified. In addition, two genes were associated with race specific gene expression. Differential expression of an ER molecular chaperone gene was associated with races from two different P. triticina lineages. Also, differential expression in an alanine glyoxylate aminotransferase gene was associated with races with virulence shifts for leaf rust resistance genes.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Konishi, Hiroyuki, E-mail: konishi@med.osaka-cu.ac.jp; The 21st Century COE Program 'Base to Overcome Fatigue', Osaka City University Graduate School of Medicine, Osaka; Department of Anatomy and Neuroscience, Nagoya University, Graduate School of Medicine, Nagoya

Research highlights: {yields} We focused on the rat pituitary intermediate lobe (IL) under continuous stress (CS). {yields} CS induced PAP-I and PAP-II expression in melanotrophs of the IL. {yields} This gene induction was triggered by CS-related dopamine dysregulation. {yields} PAP-I and PAP-II may sustain homeostasis of the IL under CS. -- Abstract: Under continuous stress (CS) in rats, melanotrophs, the predominant cell-type in the intermediate lobe (IL) of the pituitary, are hyperactivated to secrete {alpha}-melanocyte-stimulating hormone and thereafter degenerate. Although these phenomena are drastic, the molecular mechanisms underlying the cellular changes are mostly unknown. In this study, we focused onmore » the pancreatitis-associated protein (PAP) family members of the secretory lectins and characterized their expression in the IL of CS model rats because we had identified two members of this family as up-regulated genes in our previous microarray analysis. RT-PCR and histological studies demonstrated that prominent PAP-I and PAP-II expression was induced in melanotrophs in the early stages of CS, while another family member, PAP-III, was not expressed. We further examined the regulatory mechanisms of PAP-I and PAP-II expression and revealed that both were induced by the decreased dopamine levels in the IL under CS. Because the PAP family members are implicated in cell survival and proliferation, PAP-I and PAP-II secreted from melanotrophs may function to sustain homeostasis of the IL under CS conditions in an autocrine or a paracrine manner.« less

High-level expression, purification and antibacterial activity of bovine lactoferricin and lactoferrampin in Photorhabdus luminescens.

PubMed

Tang, Zhiru; Zhang, Youming; Stewart, Adrian Francis; Geng, Meimei; Tang, Xiangsha; Tu, Qiang; Yin, Yulong

2010-10-01

Bovine lactoferricin (LFC) and bovine lactoferrampin (LFA) are two active fragments located in the N(1)-domain of bovine lactoferrin. Recent studies suggested that LFC and LFA have broad-spectrum activity against Gram-positive and Gram-negative bacteria. To date, LFC and LFA have usually been produced from milk. We report here the high-level expression, purification and characterization of LFC and LFA using the Photorhabdus luminescens expression system. After the cipA and cipB genes were deleted by ET recombination, the expression host P. luminescens TZR(001) was constructed. A synthetic LFC-LFA gene containing LFC and LFA was fused with the cipB gene to form a cipB-LFC-LFA gene. To obtain the expression vector pBAD-cipB-LFC-LFA, the cipB-LFC-LFA gene was cloned on the L-arabinose-inducible expression vector pBAD24. pBAD-cipB-LFC-LFA was transformed into P. luminescens TZR(001). The cipB-LFC-LFA fusion protein was expressed under the induction of L-arabinose and its yield reached 12 mg L(-1) bacterial culture. Recombinant LFC-LFA was released from cipB by pepsin. The MIC of recombinant LFC-LFA toward E. coli 0149, 0141 and 020 was 6.25, 12.5 and 3.175 microg ml(-1), respectively. Copyright 2010 Elsevier Inc. All rights reserved.

Identification of two integration sites in favor of transgene expression in Trichoderma reesei.

PubMed

Qin, Lina; Jiang, Xianzhang; Dong, Zhiyang; Huang, Jianzhong; Chen, Xiuzhen

2018-01-01

The ascomycete fungus Trichoderma reesei was widely used as a biotechnological workhorse for production of cellulases and recombinant proteins due to its large capacity of protein secretion. Transgenesis by random integration of a gene of interest (GOI) into the genome of T. reesei can generate series of strains that express different levels of the indicated transgene. The insertion site of the GOI plays an important role in the ultimate production of the targeted proteins. However, so far no systematic studies have been made to identify transgene integration loci for optimal expression of the GOI in T. reesei . Currently, only the locus of exocellobiohydrolases I encoding gene ( cbh1) is widely used as a promising integration site to lead to high expression level of the GOI. No additional sites associated with efficient gene expression have been characterized. To search for gene integration sites that benefit for the secreted expression of GOI, the food-and-mouth disease virus 2A protein was applied for co-expression of an Aspergillus niger lipA gene and Discosoma sp. DsRed1 gene in T. reesei, by random integration of the expression cassette into the genome. We demonstrated that the fluorescent intensity of RFP (red fluorescent protein) inside of the cell was well correlated with the secreted lipase yields, based on which, we successfully developed a high-throughput screening method to screen strains with relatively higher secreted expression of the GOI (in this study, lipase). The copy number and the insertion sites of the transgene were investigated among the selected highly expressed strains. Eventually, in addition to cbh1 gene locus, two other genome insertion loci that efficiently facilitate gene expression in T. reesei were identified. We have successfully developed a high-throughput screening method to screen strains with optimal expression of the indicated secreted proteins in T. reesei . Moreover, we identified two optimal genome loci for transgene expression, which could provide new approach to modulate gene expression levels while retaining the indicated promoter and culture conditions.

Alternate Bearing in Citrus: Changes in the Expression of Flowering Control Genes and in Global Gene Expression in ON- versus OFF-Crop Trees

PubMed Central

Shalom, Liron; Samuels, Sivan; Zur, Naftali; Shlizerman, Lyudmila; Zemach, Hanita; Weissberg, Mira; Ophir, Ron; Blumwald, Eduardo; Sadka, Avi

2012-01-01

Alternate bearing (AB) is the process in fruit trees by which cycles of heavy yield (ON crop) one year are followed by a light yield (OFF crop) the next. Heavy yield usually reduces flowering intensity the following year. Despite its agricultural importance, how the developing crop influences the following year's return bloom and yield is not fully understood. It might be assumed that an ‘AB signal’ is generated in the fruit, or in another organ that senses fruit presence, and moves into the bud to determine its fate—flowering or vegetative growth. The bud then responds to fruit presence by altering regulatory and metabolic pathways. Determining these pathways, and when they are altered, might indicate the nature of this putative AB signal. We studied bud morphology, the expression of flowering control genes, and global gene expression in ON- and OFF-crop buds. In May, shortly after flowering and fruit set, OFF-crop buds were already significantly longer than ON-crop buds. The number of differentially expressed genes was higher in May than at the other tested time points. Processes differentially expressed between ON- and OFF-crop trees included key metabolic and regulatory pathways, such as photosynthesis and secondary metabolism. The expression of genes of trehalose metabolism and flavonoid metabolism was validated by nCounter technology, and the latter was confirmed by metabolomic analysis. Among genes induced in OFF-crop trees was one homologous to SQUAMOSA PROMOTER BINDING-LIKE (SPL), which controls juvenile-to-adult and annual phase transitions, regulated by miR156. The expression pattern of SPL-like, miR156 and other flowering control genes suggested that fruit load affects bud fate, and therefore development and metabolism, a relatively long time before the flowering induction period. Results shed light on some of the metabolic and regulatory processes that are altered in ON and OFF buds. PMID:23071667

WNT signaling controls expression of pro-apoptotic BOK and BAX in intestinal cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zeilstra, Jurrit; Joosten, Sander P.J.; Wensveen, Felix M.

Research highlights: {yields} Intestinal adenomas initiated by aberrant activation of the WNT pathway displayed an increased sensitivity to apoptosis. {yields} Expression profiling of apoptosis-related genes in Apc{sup Min/+} mice revealed the differential expression of pro-apoptotic Bok and Bax. {yields} APC-mutant adenomatous crypts in FAP patients showed strongly increased BAX immunoreactivity. {yields} Blocking of {beta}-catenin/TCF-4-mediated signaling in colon cancer cells reduced the expression of BOK and BAX. -- Abstract: In a majority of cases, colorectal cancer is initiated by aberrant activation of the WNT signaling pathway. Mutation of the genes encoding the WNT signaling components adenomatous polyposis coli or {beta}-catenin causesmore » constitutively active {beta}-catenin/TCF-mediated transcription, driving the transformation of intestinal crypts to cancer precursor lesions, called dysplastic aberrant crypt foci. Deregulated apoptosis is a hallmark of adenomatous colon tissue. However, the contribution of WNT signaling to this process is not fully understood. We addressed this role by analyzing the rate of epithelial apoptosis in aberrant crypts and adenomas of the Apc{sup Min/+} mouse model. In comparison with normal crypts and adenomas, aberrant crypts displayed a dramatically increased rate of apoptotic cell death. Expression profiling of apoptosis-related genes along the crypt-villus axis and in Apc mutant adenomas revealed increased expression of two pro-apoptotic Bcl-2 family members in intestinal adenomas, Bok and Bax. Analysis of the colon of familial adenomatous polyposis (FAP) patients along the crypt-to-surface axis, and of dysplastic crypts, corroborated this expression pattern. Disruption of {beta}-catenin/TCF-4-mediated signaling in the colorectal cancer cell line Ls174T significantly decreased BOK and BAX expression, confirming WNT-dependent regulation in intestinal epithelial cells. Our results suggest a feedback mechanism by which uncontrolled epithelial cell proliferation in the stem cell compartment can be counterbalanced by an increased propensity to undergo cell death.« less

Alternate bearing in citrus: changes in the expression of flowering control genes and in global gene expression in ON- versus OFF-crop trees.

PubMed

Shalom, Liron; Samuels, Sivan; Zur, Naftali; Shlizerman, Lyudmila; Zemach, Hanita; Weissberg, Mira; Ophir, Ron; Blumwald, Eduardo; Sadka, Avi

2012-01-01

Alternate bearing (AB) is the process in fruit trees by which cycles of heavy yield (ON crop) one year are followed by a light yield (OFF crop) the next. Heavy yield usually reduces flowering intensity the following year. Despite its agricultural importance, how the developing crop influences the following year's return bloom and yield is not fully understood. It might be assumed that an 'AB signal' is generated in the fruit, or in another organ that senses fruit presence, and moves into the bud to determine its fate-flowering or vegetative growth. The bud then responds to fruit presence by altering regulatory and metabolic pathways. Determining these pathways, and when they are altered, might indicate the nature of this putative AB signal. We studied bud morphology, the expression of flowering control genes, and global gene expression in ON- and OFF-crop buds. In May, shortly after flowering and fruit set, OFF-crop buds were already significantly longer than ON-crop buds. The number of differentially expressed genes was higher in May than at the other tested time points. Processes differentially expressed between ON- and OFF-crop trees included key metabolic and regulatory pathways, such as photosynthesis and secondary metabolism. The expression of genes of trehalose metabolism and flavonoid metabolism was validated by nCounter technology, and the latter was confirmed by metabolomic analysis. Among genes induced in OFF-crop trees was one homologous to SQUAMOSA PROMOTER BINDING-LIKE (SPL), which controls juvenile-to-adult and annual phase transitions, regulated by miR156. The expression pattern of SPL-like, miR156 and other flowering control genes suggested that fruit load affects bud fate, and therefore development and metabolism, a relatively long time before the flowering induction period. Results shed light on some of the metabolic and regulatory processes that are altered in ON and OFF buds.

Maximizing RNA yield from archival renal tumors and optimizing gene expression analysis.

PubMed

Glenn, Sean T; Head, Karen L; Teh, Bin T; Gross, Kenneth W; Kim, Hyung L

2010-01-01

Formalin-fixed, paraffin-embedded tissues are widely available for gene expression analysis using TaqMan PCR. Five methods, including 4 commercial kits, for recovering RNA from paraffin-embedded renal tumor tissue were compared. The MasterPure kit from Epicentre produced the highest RNA yield. However, the difference in RNA yield between the kit from Epicenter and Invitrogen's TRIzol method was not significant. Using the top 3 RNA isolation methods, the manufacturers' protocols were modified to include an overnight Proteinase K digestion. Overnight protein digestion resulted in a significant increase in RNA yield. To optimize the reverse transcription reaction, conventional reverse transcription with random oligonucleotide primers was compared to reverse transcription using primers specific for genes of interest. Reverse transcription using gene-specific primers significantly increased the quantity of cDNA detectable by TaqMan PCR. Therefore, expression profiling of formalin-fixed, paraffin-embedded tissue using TaqMan qPCR can be optimized by using the MasterPure RNA isolation kit modified to include an overnight Proteinase K digestion and gene-specific primers during the reverse transcription.

Generating high temperature tolerant transgenic plants: Achievements and challenges.

PubMed

Grover, Anil; Mittal, Dheeraj; Negi, Manisha; Lavania, Dhruv

2013-05-01

Production of plants tolerant to high temperature stress is of immense significance in the light of global warming and climate change. Plant cells respond to high temperature stress by re-programming their genetic machinery for survival and reproduction. High temperature tolerance in transgenic plants has largely been achieved either by over-expressing heat shock protein genes or by altering levels of heat shock factors that regulate expression of heat shock and non-heat shock genes. Apart from heat shock factors, over-expression of other trans-acting factors like DREB2A, bZIP28 and WRKY proteins has proven useful in imparting high temperature tolerance. Besides these, elevating the genetic levels of proteins involved in osmotic adjustment, reactive oxygen species removal, saturation of membrane-associated lipids, photosynthetic reactions, production of polyamines and protein biosynthesis process have yielded positive results in equipping transgenic plants with high temperature tolerance. Cyclic nucleotide gated calcium channel proteins that regulate calcium influxes across the cell membrane have recently been shown to be the key players in induction of high temperature tolerance. The involvement of calmodulins and kinases in activation of heat shock factors has been implicated as an important event in governing high temperature tolerance. Unfilled gaps limiting the production of high temperature tolerant transgenic plants for field level cultivation are discussed. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

High Throughput Engineering to Revitalize a Vestigial Electron Transfer Pathway in Bacterial Photosynthetic Reaction Centers*

PubMed Central

Faries, Kaitlyn M.; Kressel, Lucas L.; Wander, Marc J.; Holten, Dewey; Laible, Philip D.; Kirmaier, Christine; Hanson, Deborah K.

2012-01-01

Photosynthetic reaction centers convert light energy into chemical energy in a series of transmembrane electron transfer reactions, each with near 100% yield. The structures of reaction centers reveal two symmetry-related branches of cofactors (denoted A and B) that are functionally asymmetric; purple bacterial reaction centers use the A pathway exclusively. Previously, site-specific mutagenesis has yielded reaction centers capable of transmembrane charge separation solely via the B branch cofactors, but the best overall electron transfer yields are still low. In an attempt to better realize the architectural and energetic factors that underlie the directionality and yields of electron transfer, sites within the protein-cofactor complex were targeted in a directed molecular evolution strategy that implements streamlined mutagenesis and high throughput spectroscopic screening. The polycistronic approach enables efficient construction and expression of a large number of variants of a heteroligomeric complex that has two intimately regulated subunits with high sequence similarity, common features of many prokaryotic and eukaryotic transmembrane protein assemblies. The strategy has succeeded in the discovery of several mutant reaction centers with increased efficiency of the B pathway; they carry multiple substitutions that have not been explored or linked using traditional approaches. This work expands our understanding of the structure-function relationships that dictate the efficiency of biological energy-conversion reactions, concepts that will aid the design of bio-inspired assemblies capable of both efficient charge separation and charge stabilization. PMID:22247556

Intracellular high cholesterol content disorders the clock genes, apoptosis-related genes and fibrinolytic-related genes rhythmic expressions in human plaque-derived vascular smooth muscle cells.

PubMed

Lin, Changpo; Tang, Xiao; Xu, Lirong; Qian, Ruizhe; Shi, Zhenyu; Wang, Lixin; Cai, Tingting; Yan, Dong; Fu, Weiguo; Guo, Daqiao

2017-07-10

The clock genes are involved in regulating cardiovascular functions, and their expression disorders would lead to circadian rhythm disruptions of clock-controlled genes (CCGs), resulting in atherosclerotic plaque formation and rupture. Our previous study revealed the rhythmic expression of clock genes were attenuated in human plaque-derived vascular smooth muscle cells (PVSMCs), but failed to detect the downstream CCGs expressions and the underlying molecular mechanism. In this study, we examined the difference of CCGs rhythmic expression between human normal carotid VSMCs (NVSMCs) and PVSMCs. Furthermore, we compared the cholesterol and triglycerides levels between two groups and the link to clock genes and CCGs expressions. Seven health donors' normal carotids and 19 carotid plaques yielded viable cultured NVSMCs and PVSMCs. The expression levels of target genes were measured by quantitative real-time PCR and Western-blot. The intracellular cholesterol and triglycerides levels were measured by kits. The circadian expressions of apoptosis-related genes and fibrinolytic-related genes were disordered. Besides, the cholesterol levels were significant higher in PVSMCs. After treated with cholesterol or oxidized low density lipoprotein (ox-LDL), the expressions of clock genes were inhibited; and the rhythmic expressions of clock genes, apoptosis-related genes and fibrinolytic-related genes were disturbed in NVSMCs, which were similar to PVSMCs. The results suggested that intracellular high cholesterol content of PVSMCs would lead to the disorders of clock genes and CCGs rhythmic expressions. And further studies should be conducted to demonstrate the specific molecular mechanisms involved.

Genomic resources for songbird research and their use in characterizing gene expression during brain development

PubMed Central

Li, XiaoChing; Wang, Xiu-Jie; Tannenhauser, Jonathan; Podell, Sheila; Mukherjee, Piali; Hertel, Moritz; Biane, Jeremy; Masuda, Shoko; Nottebohm, Fernando; Gaasterland, Terry

2007-01-01

Vocal learning and neuronal replacement have been studied extensively in songbirds, but until recently, few molecular and genomic tools for songbird research existed. Here we describe new molecular/genomic resources developed in our laboratory. We made cDNA libraries from zebra finch (Taeniopygia guttata) brains at different developmental stages. A total of 11,000 cDNA clones from these libraries, representing 5,866 unique gene transcripts, were randomly picked and sequenced from the 3′ ends. A web-based database was established for clone tracking, sequence analysis, and functional annotations. Our cDNA libraries were not normalized. Sequencing ESTs without normalization produced many developmental stage-specific sequences, yielding insights into patterns of gene expression at different stages of brain development. In particular, the cDNA library made from brains at posthatching day 30–50, corresponding to the period of rapid song system development and song learning, has the most diverse and richest set of genes expressed. We also identified five microRNAs whose sequences are highly conserved between zebra finch and other species. We printed cDNA microarrays and profiled gene expression in the high vocal center of both adult male zebra finches and canaries (Serinus canaria). Genes differentially expressed in the high vocal center were identified from the microarray hybridization results. Selected genes were validated by in situ hybridization. Networks among the regulated genes were also identified. These resources provide songbird biologists with tools for genome annotation, comparative genomics, and microarray gene expression analysis. PMID:17426146

Evaluation of reference genes for quantitative real-time PCR in oil palm elite planting materials propagated by tissue culture.

PubMed

Chan, Pek-Lan; Rose, Ray J; Abdul Murad, Abdul Munir; Zainal, Zamri; Low, Eng-Ti Leslie; Ooi, Leslie Cheng-Li; Ooi, Siew-Eng; Yahya, Suzaini; Singh, Rajinder

2014-01-01

The somatic embryogenesis tissue culture process has been utilized to propagate high yielding oil palm. Due to the low callogenesis and embryogenesis rates, molecular studies were initiated to identify genes regulating the process, and their expression levels are usually quantified using reverse transcription quantitative real-time PCR (RT-qPCR). With the recent release of oil palm genome sequences, it is crucial to establish a proper strategy for gene analysis using RT-qPCR. Selection of the most suitable reference genes should be performed for accurate quantification of gene expression levels. In this study, eight candidate reference genes selected from cDNA microarray study and literature review were evaluated comprehensively across 26 tissue culture samples using RT-qPCR. These samples were collected from two tissue culture lines and media treatments, which consisted of leaf explants cultures, callus and embryoids from consecutive developmental stages. Three statistical algorithms (geNorm, NormFinder and BestKeeper) confirmed that the expression stability of novel reference genes (pOP-EA01332, PD00380 and PD00569) outperformed classical housekeeping genes (GAPDH, NAD5, TUBULIN, UBIQUITIN and ACTIN). PD00380 and PD00569 were identified as the most stably expressed genes in total samples, MA2 and MA8 tissue culture lines. Their applicability to validate the expression profiles of a putative ethylene-responsive transcription factor 3-like gene demonstrated the importance of using the geometric mean of two genes for normalization. Systematic selection of the most stably expressed reference genes for RT-qPCR was established in oil palm tissue culture samples. PD00380 and PD00569 were selected for accurate and reliable normalization of gene expression data from RT-qPCR. These data will be valuable to the research associated with the tissue culture process. Also, the method described here will facilitate the selection of appropriate reference genes in other oil palm tissues and in the expression profiling of genes relating to yield, biotic and abiotic stresses.

In silico analysis and expression profiling of miRNAs targeting genes of steviol glycosides biosynthetic pathway and their relationship with steviol glycosides content in different tissues of Stevia rebaudiana.

PubMed

Saifi, Monica; Nasrullah, Nazima; Ahmad, Malik Mobeen; Ali, Athar; Khan, Jawaid A; Abdin, M Z

2015-09-01

miRNAs are emerging as potential regulators of the gene expression. Their proven promising role in regulating biosynthetic pathways related gene networks may hold the key to understand the genetic regulation of these pathways which may assist in selection and manipulation to get high performing plant genotypes with better secondary metabolites yields and increased biomass. miRNAs associated with genes of steviol glycosides biosynthetic pathway, however, have not been identified so far. In this study miRNAs targeting genes of steviol glycosides biosynthetic pathway were identified for the first time whose precursors were potentially generated from ESTs and nucleotide sequences of Stevia rebaudiana. Thereafter, stem-loop coupled real time PCR based expressions of these miRNAs in different tissues of Stevia rebaudiana were investigated and their relationship pattern was analysed with the expression levels of their target mRNAs as well as steviol glycoside contents. All the miRNAs investigated showed differential expressions in all the three tissues studied, viz. leaves, flowers and stems. Out of the eleven miRNAs validated, the expression levels of nine miRNAs (miR319a, miR319b, miR319c, miR319d, miR319e, miR319f, miR319h, miRstv_7, miRstv_9) were found to be inversely related, while expression levels of the two, i.e. miR319g and miRstv_11 on the contrary, showed direct relation with the expression levels of their target mRNAs and steviol glycoside contents in the leaves, flowers and stems. This study provides a platform for better understanding of the steviol glycosides biosynthetic pathway and these miRNAs can further be employed to manipulate the biosynthesis of these metabolites to enhance their contents and yield in S. rebaudiana. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhong, Yiming; Program in Molecular, Cellular, and Developmental Biology, The Ohio State University, Columbus, OH; Sullenbarger, Brent

Research highlights: {yields} HoxB4 overexpression in human TF1 cells increased the expression of CD61 and CD41a. {yields} HoxB4 fusion protein enhanced megakaryocytic development of CD34{sup +} cord blood cells. {yields} Ectopic HoxB4 increased Tpo receptor expression and decreased c-Myb expression. {yields} HoxB4 RNA silencing increased c-Myb expression and decreased Fli-1 expression. -- Abstract: In order to produce clinically useful quantities of platelets ex vivo we may need to firstly enhance early self-renewal of hematopoietic stem cells (HSCs) and/or megakaryocyte (Mk) progenitors. The homeodomain transcription factor HoxB4 has been shown to be an important regulator of stem cell renewal and hematopoiesis;more » however, its effect on megakaryopoiesis is unclear. In this study, we investigated the effect of HoxB4 overexpression or RNA silencing on megakaryocytic development in the human TF1 progenitor cell line; we then used recombinant tPTD-HoxB4 fusion protein to study the effect of exogenous HoxB4 on megakaryocytic development of human CD34 positively-selected cord blood cells. We found that ectopic HoxB4 in TF1 cells increased the antigen expression of CD61and CD41a, increased the gene expression of thrombopoietin receptor (TpoR), Scl-1, Cyclin D1, Fog-1 and Fli-1 while it decreased c-Myb expression. HoxB4 RNA silencing in TF1 cells decreased the expression of CD61 and CD41a and decreased Fli-1 expression while it increased the expression of c-Myb. Recombinant tPTD-HoxB4 fusion protein increased the percentages and absolute numbers of CD41a and CD61 positive cells during megakaryocytic differentiation of CD34 positively-selected cord blood cells and increased the numbers of colony-forming unit-megakaryocyte (CFU-Mk). Adding tPTD-HoxB4 fusion protein increased the gene expression of TpoR, Cyclin D1, Fog-1 and Fli-1 while it inhibited c-Myb expression. Our data suggest that increased HoxB4 enhanced early megakaryocytic development in human TF1 cells and CD34 positively-selected cord blood cells primarily by upregulating TpoR and Fli-1 expression and downregulating c-Myb expression. Increasing HoxB4 expression or adding recombinant HoxB4 protein might be a way to expand Mks for the production of platelets for use in transfusion medicine.« less

Experimental Assessment of Recycled Diesel Spill-Contaminated Domestic Wastewater Treated by Reed Beds for Irrigation of Sweet Peppers

PubMed Central

Almuktar, Suhad A.A.A.N.; Scholz, Miklas

2016-01-01

The aim of this experimental study is to assess if urban wastewater treated by ten different greenhouse-based sustainable wetland systems can be recycled to irrigate Capsicum annuum L. (Sweet Pepper; California Wonder) commercially grown either in compost or sand within a laboratory environment. The design variables were aggregate diameter, contact time, resting time and chemical oxygen demand. The key objectives were to assess: (i) the suitability of different treated (recycled) wastewaters for irrigation; (ii) response of peppers in terms of growth when using recycled wastewater subject to different growth media and hydrocarbon contamination; and (iii) the economic viability of different experimental set-ups in terms of marketable yield. Ortho-phosphate-phosphorus, ammonia-nitrogen, potassium and manganese concentrations in the irrigation water considerably exceeded the corresponding water quality thresholds. A high yield in terms of economic return (marketable yield expressed in monetary value) was linked to raw wastewater and an organic growth medium, while the plants grown in organic medium and wetlands of large aggregate size, high contact and resting times, diesel-spill contamination and low inflow loading rate produced the best fruits in terms of their dimensions and fresh weights, indicating the role of diesel in reducing too high nitrogen concentrations. PMID:26861370

Heavy ion mutagenesis combined with triclosan screening provides a new strategy for improving the arachidonic acid yield in Mortierella alpina.

PubMed

Zhang, Huidan; Lu, Dong; Li, Xin; Feng, Yingang; Cui, Qiu; Song, Xiaojin

2018-05-02

Arachidonic acid (ARA), which is a ω-6 polyunsaturated fatty acid, has a wide range of biological activities and is an essential component of cellular membranes in some human tissues. Mortierella alpina is the best strain for industrial production of ARA. To increase its yield of arachidonic acid, heavy ion beam irradiation mutagenesis of Mortierella alpina was carried out in combination with triclosan and octyl gallate treatment. The obtained mutant strain F-23 ultimately achieved an ARA yield of 5.26 g L - 1 , which is 3.24 times higher than that of the wild-type strain. In addition, quantitative real-time PCR confirmed that the expression levels of fatty acid synthase (FAS), Δ5-desaturase, Δ6-desaturase, and Δ9-desaturase were all significantly up-regulated in the mutant F-23 strain, especially Δ6- and Δ9-desaturase, which were up-regulated 3- and 2-fold, respectively. This study confirmed a feasible mutagenesis breeding strategy for improving ARA production and provided a mutant of Mortierella alpina with high ARA yield.

Tobacco BY-2 cell-free lysate: an alternative and highly-productive plant-based in vitro translation system.

PubMed

Buntru, Matthias; Vogel, Simon; Spiegel, Holger; Schillberg, Stefan

2014-05-03

Cell-free protein synthesis is a rapid and efficient method for the production of recombinant proteins. Usage of prokaryotic cell-free extracts often leads to non-functional proteins. Eukaryotic counterparts such as wheat germ extract (WGE) and rabbit reticulocyte lysate (RLL) may improve solubility and promote the correct folding of eukaryotic multi-domain proteins that are difficult to express in bacteria. However, the preparation of WGEs is complex and time-consuming, whereas RLLs suffer from low yields. Here we report the development of a novel cell-free system based on tobacco Bright Yellow 2 (BY-2) cells harvested in the exponential growth phase. The highly-productive BY-2 lysate (BYL) can be prepared quickly within 4-5 h, compared to 4-5 d for WGE. The efficiency of the BYL was tested using three model proteins: enhanced yellow fluorescent protein (eYFP) and two versions of luciferase. The added mRNA was optimized by testing different 5' and 3' untranslated regions (UTRs). The protein yield in batch and dialysis reactions using BYL was much higher than that of a commercial Promega WGE preparation, achieving a maximum yield of 80 μg/mL of eYFP and 100 μg/mL of luciferase, compared to only 45 μg/mL of eYFP and 35 μg/mL of luciferase in WGEs. In dialysis reactions, the BYL yielded about 400 μg/mL eYFP, representing up to 50% more of the target protein than the Promega WGE, and equivalent to the amount using 5Prime WGE system. Due to the high yield and the short preparation time the BYL represents a remarkable improvement over current eukaryotic cell-free systems.

Expression of the Aspergillus terreus itaconic acid biosynthesis cluster in Aspergillus niger

PubMed Central

2014-01-01

Background Aspergillus terreus is a natural producer of itaconic acid and is currently used to produce itaconic acid on an industrial scale. The metabolic process for itaconic acid biosynthesis is very similar to the production of citric acid in Aspergillus niger. However, a key enzyme in A. niger, cis-aconitate decarboxylase, is missing. The introduction of the A. terreus cadA gene in A. niger exploits the high level of citric acid production (over 200 g per liter) and theoretically can lead to production levels of over 135 g per liter of itaconic acid in A. niger. Given the potential for higher production levels in A. niger, production of itaconic acid in this host was investigated. Results Expression of Aspergillus terreus cis-aconitate decarboxylase in Aspergillus niger resulted in the production of a low concentration (0.05 g/L) of itaconic acid. Overexpression of codon-optimized genes for cis-aconitate decarboxylase, a mitochondrial transporter and a plasma membrane transporter in an oxaloacetate hydrolase and glucose oxidase deficient A. niger strain led to highly increased yields and itaconic acid production titers. At these higher production titers, the effect of the mitochondrial and plasma membrane transporters was much more pronounced, with levels being 5–8 times higher than previously described. Conclusions Itaconic acid can be produced in A. niger by the introduction of the A. terreus cis-aconitate decarboxylase encoding cadA gene. This results in a low itaconic acid production level, which can be increased by codon-optimization of the cadA gene for A. niger. A second crucial requirement for efficient production of itaconic acid is the expression of the A. terreus mttA gene, encoding a putative mitochondrial transporter. Expression of this transporter results in a twenty-fold increase in the secretion of itaconic acid. Expression of the A. terreus itaconic acid cluster consisting of the cadA gene, the mttA gene and the mfsA gene results in A. niger strains that produce over twenty five-fold higher levels of itaconic acid and show a twenty-fold increase in yield compared to a strain expressing only CadA. PMID:24438100

Expression of the Aspergillus terreus itaconic acid biosynthesis cluster in Aspergillus niger.

PubMed

van der Straat, Laura; Vernooij, Marloes; Lammers, Marieke; van den Berg, Willy; Schonewille, Tom; Cordewener, Jan; van der Meer, Ingrid; Koops, Andries; de Graaff, Leo H

2014-01-17

Aspergillus terreus is a natural producer of itaconic acid and is currently used to produce itaconic acid on an industrial scale. The metabolic process for itaconic acid biosynthesis is very similar to the production of citric acid in Aspergillus niger. However, a key enzyme in A. niger, cis-aconitate decarboxylase, is missing. The introduction of the A. terreus cadA gene in A. niger exploits the high level of citric acid production (over 200 g per liter) and theoretically can lead to production levels of over 135 g per liter of itaconic acid in A. niger. Given the potential for higher production levels in A. niger, production of itaconic acid in this host was investigated. Expression of Aspergillus terreus cis-aconitate decarboxylase in Aspergillus niger resulted in the production of a low concentration (0.05 g/L) of itaconic acid. Overexpression of codon-optimized genes for cis-aconitate decarboxylase, a mitochondrial transporter and a plasma membrane transporter in an oxaloacetate hydrolase and glucose oxidase deficient A. niger strain led to highly increased yields and itaconic acid production titers. At these higher production titers, the effect of the mitochondrial and plasma membrane transporters was much more pronounced, with levels being 5-8 times higher than previously described. Itaconic acid can be produced in A. niger by the introduction of the A. terreus cis-aconitate decarboxylase encoding cadA gene. This results in a low itaconic acid production level, which can be increased by codon-optimization of the cadA gene for A. niger. A second crucial requirement for efficient production of itaconic acid is the expression of the A. terreus mttA gene, encoding a putative mitochondrial transporter. Expression of this transporter results in a twenty-fold increase in the secretion of itaconic acid. Expression of the A. terreus itaconic acid cluster consisting of the cadA gene, the mttA gene and the mfsA gene results in A. niger strains that produce over twenty five-fold higher levels of itaconic acid and show a twenty-fold increase in yield compared to a strain expressing only CadA.

Molecular Testing for miRNA, mRNA, and DNA on Fine-Needle Aspiration Improves the Preoperative Diagnosis of Thyroid Nodules With Indeterminate Cytology.

PubMed

Labourier, Emmanuel; Shifrin, Alexander; Busseniers, Anne E; Lupo, Mark A; Manganelli, Monique L; Andruss, Bernard; Wylie, Dennis; Beaudenon-Huibregtse, Sylvie

2015-07-01

Molecular testing for oncogenic mutations or gene expression in fine-needle aspirations (FNAs) from thyroid nodules with indeterminate cytology identifies a subset of benign or malignant lesions with high predictive value. This study aimed to evaluate a novel diagnostic algorithm combining mutation detection and miRNA expression to improve the diagnostic yield of molecular cytology. Surgical specimens and preoperative FNAs (n = 638) were tested for 17 validated gene alterations using the miRInform Thyroid test and with a 10-miRNA gene expression classifier generating positive (malignant) or negative (benign) results. Cross-sectional sampling of thyroid nodules with atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS) or follicular neoplasm/suspicious for a follicular neoplasm (FN/SFN) cytology (n = 109) was conducted at 12 endocrinology centers across the United States. Qualitative molecular results were compared with surgical histopathology to determine diagnostic performance and model clinical effect. Mutations were detected in 69% of nodules with malignant outcome. Among mutation-negative specimens, miRNA testing correctly identified 64% of malignant cases and 98% of benign cases. The diagnostic sensitivity and specificity of the combined algorithm was 89% (95% confidence interval [CI], 73-97%) and 85% (95% CI, 75-92%), respectively. At 32% cancer prevalence, 61% of the molecular results were benign with a negative predictive value of 94% (95% CI, 85-98%). Independently of variations in cancer prevalence, the test increased the yield of true benign results by 65% relative to mRNA-based gene expression classification and decreased the rate of avoidable diagnostic surgeries by 69%. Multiplatform testing for DNA, mRNA, and miRNA can accurately classify benign and malignant thyroid nodules, increase the diagnostic yield of molecular cytology, and further improve the preoperative risk-based management of benign nodules with AUS/FLUS or FN/SFN cytology.

The Expression and Characterization of Functionally Active Soluble CD83 by Pichia pastoris Using High-Density Fermentation

PubMed Central

Song, Xiaoping; Zhong, Yongjun; Wang, Chenguang; Jia, Hao; Wu, Lidan; Wang, Dong; Fang, Fang; Ma, Jiajia; Kang, Wenyao; Sun, Jie; Tian, Zhigang; Xiao, Weihua

2014-01-01

CD83 is a highly glycosylated type I transmembrane glycoprotein that belongs to the immunoglobulin superfamily. CD83 is upregulated during dendritic cell (DC) maturation, which is critical for the initiation of adaptive immune responses. The soluble isoform of CD83 (sCD83) is encoded by alternative splicing from full-length CD83 mRNA and inhibits DC maturation, which suggests that sCD83 acts as a potential immune suppressor. In this study, we developed a sound strategy to express functional sCD83 from Pichia pastoris in extremely high-density fermentation. Purified sCD83 was expressed as a monomer at a yield of more than 200 mg/L and contained N-linked glycosylation sites that were characterized by PNGase F digestion. In vitro tests indicated that recombinant sCD83 bound to its putative counterpart on monocytes and specifically blocked the binding of anti-CD83 antibodies to cell surface CD83 on DCs. Moreover, sCD83 from yeast significantly suppressed ConA-stimulated PBMC proliferation. Therefore, sCD83 that was expressed from the P. pastoris was functionally active and may be used for in vivo and in vitro studies as well as future clinical applications. PMID:24586642

Phenotypic Characteristics Associated with Virulence of Clinical Isolates from the Sporothrix Complex

PubMed Central

Almeida-Paes, Rodrigo; de Oliveira, Luã Cardoso; Oliveira, Manoel Marques Evangelista; Gutierrez-Galhardo, Maria Clara; Nosanchuk, Joshua Daniel; Zancopé-Oliveira, Rosely Maria

2015-01-01

The Sporothrix complex members cause sporotrichosis, a subcutaneous mycosis with a wide spectrum of clinical manifestations. Several specific phenotypic characteristics are associated with virulence in many fungi, but studies in this field involving the Sporothrix complex species are scarce. Melanization, thermotolerance, and production of proteases, catalase, and urease were investigated in 61 S. brasiliensis, one S. globosa, and 10 S. schenckii strains. The S. brasiliensis strains showed a higher expression of melanin and urease compared with S. schenckii. These two species, however, presented similar thermotolerances. Our S. globosa strain had low expression of all studied virulence factors. The relationship between these phenotypes and clinical aspects of sporotrichosis was also evaluated. Strains isolated from patients with spontaneous regression of infection were heavily melanized and produced high urease levels. Melanin was also related to dissemination of internal organs and protease production was associated with HIV-coinfection. A murine sporotrichosis model showed that a S. brasiliensis strain with high expression of virulence factors was able to disseminate and yield a high fungal burden in comparison with a control S. schenckii strain. Our results show that virulence-related phenotypes are variably expressed within the Sporothrix complex species and might be involved in clinical aspects of sporotrichosis. PMID:25961005

Suppression of HPV E6 and E7 expression by BAF53 depletion in cervical cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Kiwon; Lee, Ah-Young; Kwon, Yunhee Kim

Highlights: {yields} Integration of HPV into host genome critical for activation of E6 and E7 oncogenes. {yields} BAF53 is essential for higher-order chromatin structure. {yields} BAF53 knockdown suppresses E6 and E7 from HPV integrants, but not from episomal HPVs. {yields} BAF53 knockdown decreases H3K9Ac and H4K12Ac on P105 promoter of integrated HPV 18. {yields} BAF53 knockdown restores the p53-dependent signaling pathway in HeLa and SiHa cells. -- Abstract: Deregulation of the expression of human papillomavirus (HPV) oncogenes E6 and E7 plays a pivotal role in cervical carcinogenesis because the E6 and E7 proteins neutralize p53 and Rb tumor suppressor pathways,more » respectively. In approximately 90% of all cervical carcinomas, HPVs are found to be integrated into the host genome. Following integration, the core-enhancer element and P105 promoter that control expression of E6 and E7 adopt a chromatin structure that is different from that of episomal HPV, and this has been proposed to contribute to activation of E6 and E7 expression. However, the molecular basis underlying this chromatin structural change remains unknown. Previously, BAF53 has been shown to be essential for the integrity of higher-order chromatin structure and interchromosomal interactions. Here, we examined whether BAF53 is required for activated expression of E6 and E7 genes. We found that BAF53 knockdown led to suppression of expression of E6 and E7 genes from HPV integrants in cervical carcinoma cell lines HeLa and SiHa. Conversely, expression of transiently transfected HPV18-LCR-Luciferase was not suppressed by BAF53 knockdown. The level of the active histone marks H3K9Ac and H4K12Ac on the P105 promoter of integrated HPV 18 was decreased in BAF53 knockdown cells. BAF53 knockdown restored the p53-dependent signaling pathway in HeLa and SiHa cells. These results suggest that activated expression of the E6 and E7 genes of integrated HPV is dependent on BAF53-dependent higher-order chromatin structure or nuclear motor activity.« less

The activation of p38 MAPK primarily contributes to UV-induced RhoB expression by recruiting the c-Jun and p300 to the distal CCAAT box of the RhoB promoter

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ahn, Jiwon; Department of Microbiology, Chungnam National University, Daejeon 305-764; Choi, Jeong-Hae

2011-06-03

Highlights: {yields} Regulation of transcriptional activation of RhoB is still unclear. {yields} We examine the effect of p38 MAPK inhibition, and c-Jun and RhoB depletion on UV-induced RhoB expression and apoptosis. {yields} We identify the regions of RhoB promoter necessary to confer UV responsiveness using pRhoB-luciferase reporter assays. {yields} c-Jun, ATF2 and p300 are dominantly associated with NF-Y on the distal CCAAT box. {yields} The activation of p38 MAPK primarily contribute to UV-induced RhoB expression by recruiting the c-Jun and p300 proteins on distal CCAAT box of RhoB promoter. -- Abstract: The Ras-related small GTP-binding protein RhoB is rapidly inducedmore » in response to genotoxic stresses caused by ionizing radiation. It is known that UV-induced RhoB expression results from the binding of activating transcription factor 2 (ATF2) via NF-Y to the inverted CCAAT box (-23) of the RhoB promoter. Here, we show that the association of c-Jun with the distal CCAAT box (-72) is primarily involved in UV-induced RhoB expression and p38 MAPK regulated RhoB induction through the distal CCAAT box. UV-induced RhoB expression and apoptosis were markedly attenuated by pretreatment with the p38 MAPK inhibitor. siRNA knockdown of RhoB, ATF2 and c-Jun resulted in decreased RhoB expression and eventually restored the growth of UV-irradiated Jurkat cells. In the reporter assay using luciferase under the RhoB promoter, inhibition of RhoB promoter activity by the p38 inhibitor and knockdown of c-Jun using siRNA occurred through the distal CCAAT box. Immunoprecipitation and DNA affinity protein binding assays revealed the association of c-Jun and p300 via NF-YA and the dissociation of histone deacetylase 1 (HDAC1) via c-Jun recruitment to the CCAAT boxes of the RhoB promoter. These results suggest that the activation of p38 MAPK primarily contributes to UV-induced RhoB expression by recruiting the c-Jun and p300 proteins to the distal CCAAT box of the RhoB promoter in Jurkat cells.« less

Notch Signaling Augments BMP9-Induced Bone Formation by Promoting the Osteogenesis-Angiogenesis Coupling Process in Mesenchymal Stem Cells (MSCs).

PubMed

Liao, Junyi; Wei, Qiang; Zou, Yulong; Fan, Jiaming; Song, Dongzhe; Cui, Jing; Zhang, Wenwen; Zhu, Yunxiao; Ma, Chao; Hu, Xue; Qu, Xiangyang; Chen, Liqun; Yu, Xinyi; Zhang, Zhicai; Wang, Claire; Zhao, Chen; Zeng, Zongyue; Zhang, Ruyi; Yan, Shujuan; Wu, Tingting; Wu, Xingye; Shu, Yi; Lei, Jiayan; Li, Yasha; Luu, Hue H; Lee, Michael J; Reid, Russell R; Ameer, Guillermo A; Wolf, Jennifer Moriatis; He, Tong-Chuan; Huang, Wei

2017-01-01

Mesenchymal stem cells (MSCs) are multipotent progenitors that can differentiate into several lineages including bone. Successful bone formation requires osteogenesis and angiogenesis coupling of MSCs. Here, we investigate if simultaneous activation of BMP9 and Notch signaling yields effective osteogenesis-angiogenesis coupling in MSCs. Recently-characterized immortalized mouse adipose-derived progenitors (iMADs) were used as MSC source. Transgenes BMP9, NICD and dnNotch1 were expressed by adenoviral vectors. Gene expression was determined by qPCR and immunohistochem¡stry. Osteogenic activity was assessed by in vitro assays and in vivo ectopic bone formation model. BMP9 upregulated expression of Notch receptors and ligands in iMADs. Constitutively-active form of Notch1 NICD1 enhanced BMP9-induced osteogenic differentiation both in vitro and in vivo, which was effectively inhibited by dominant-negative form of Notch1 dnNotch1. BMP9- and NICD1-transduced MSCs implanted with a biocompatible scaffold yielded highly mature bone with extensive vascularization. NICD1 enhanced BMP9-induced expression of key angiogenic regulators in iMADs and Vegfa in ectopic bone, which was blunted by dnNotch1. Notch signaling may play an important role in BMP9-induced osteogenesis and angiogenesis. It's conceivable that simultaneous activation of the BMP9 and Notch pathways should efficiently couple osteogenesis and angiogenesis of MSCs for successful bone tissue engineering. © 2017 The Author(s)Published by S. Karger AG, Basel.

Differential effect of soil and environment on metabolic expression of turmeric (Curcuma longa cv. Roma).

PubMed

Sandeep, I S; Sanghamitra, Nayak; Sujata, Mohanty

2015-06-01

Curcuma longa (Zingiberaceae) is known for its uses in medicine, cosmetics, food flavouring and textile industries. The secondary metabolites of turmeric like essential oil, oleoresin and curcumin are important for its multipurpose uses. These traits of turmeric vary from place to place due to the influence of environment, soil and agro-climatic conditions. Here, we analyzed turmeric from different agroclimatic regions for influence of various factors on its growth and yield of important phytochemicals. A high curcumin yielding cultivar i.e., Roma was collected from high altitude research station, Koraput (HARS) and planted in nine agroclimatic regions of Odisha. Analysis of soil texture, pH, organic carbon, micro and macro nutrients were done from all the studied zones up to 2nd generation. Plants grown in their released station i.e., Eastern Ghat High Land showed 5% of curcumin and were taken as control. Plants grown in different agroclimatic zones showed a range of 1.4-5% of curcumin and 0.3-0.7% of rhizome essential oil and 0.3-1% of leaf essential oil content. Gas chromatography and mass spectra analysis showed tumerone and alpha phellandrene as the major compounds in all the zones with 10-20% variation. The present study will be immensely helpful for standardization and management of environmental and ecological factors for high phytochemical yield in turmeric plant.

RNA isolation from loquat and other recalcitrant woody plants with high quality and yield.

PubMed

Morante-Carriel, Jaime; Sellés-Marchart, Susana; Martínez-Márquez, Ascensión; Martínez-Esteso, María José; Luque, Ignacio; Bru-Martínez, Roque

2014-05-01

RNA isolation is difficult in plants that contain large amounts of polysaccharides and polyphenol compounds. To date, no commercial kit has been developed for the isolation of high-quality RNA from tissues with these characteristics, especially for fruit. The common protocols for RNA isolation are tedious and usually result in poor yields when applied to recalcitrant plant tissues. Here an efficient RNA isolation protocol based on cetyltrimethylammonium bromide (CTAB) and two successive precipitations with 10 M lithium chloride (LiCl) was developed specifically for loquat fruits, but it was proved to work efficiently in other tissues of loquat and woody plants. The RNA isolated by this improved protocol was not only of high purity and integrity (A260/A280 ratios ranged from 1.90 to 2.04 and A260/A230 ratios were>2.0) but also of high yield (up to 720 μg on average [coefficient of variation=21%] total RNA per gram fresh tissue). The protocol was tested on loquat fruit (different stages of development, postharvest, ripening, and bruising), leaf, root, flower, stem, and bud; quince fruit and root; grapevine cells in liquid culture; and rose petals. The RNA obtained with this method is amenable to enzymatic treatments and can be efficiently applied for research on gene characterization, expression, and function. Copyright © 2014 Elsevier Inc. All rights reserved.

A Comparative Analysis of Industrial Escherichia coli K–12 and B Strains in High-Glucose Batch Cultivations on Process-, Transcriptome- and Proteome Level

PubMed Central

Marisch, Karoline; Bayer, Karl; Scharl, Theresa; Mairhofer, Juergen; Krempl, Peter M.; Hummel, Karin; Razzazi-Fazeli, Ebrahim; Striedner, Gerald

2013-01-01

Escherichia coli K–12 and B strains are among the most frequently used bacterial hosts for production of recombinant proteins on an industrial scale. To improve existing processes and to accelerate bioprocess development, we performed a detailed host analysis. We investigated the different behaviors of the E. coli production strains BL21, RV308, and HMS174 in response to high-glucose concentrations. Tightly controlled cultivations were conducted under defined environmental conditions for the in-depth analysis of physiological behavior. In addition to acquisition of standard process parameters, we also used DNA microarray analysis and differential gel electrophoresis (EttanTM DIGE). Batch cultivations showed different yields of the distinct strains for cell dry mass and growth rate, which were highest for BL21. In addition, production of acetate, triggered by excess glucose supply, was much higher for the K–12 strains compared to the B strain. Analysis of transcriptome data showed significant alteration in 347 of 3882 genes common among all three hosts. These differentially expressed genes included, for example, those involved in transport, iron acquisition, and motility. The investigation of proteome patterns additionally revealed a high number of differentially expressed proteins among the investigated hosts. The subsequently selected 38 spots included proteins involved in transport and motility. The results of this comprehensive analysis delivered a full genomic picture of the three investigated strains. Differentially expressed groups for targeted host modification were identified like glucose transport or iron acquisition, enabling potential optimization of strains to improve yield and process quality. Dissimilar growth profiles of the strains confirm different genotypes. Furthermore, distinct transcriptome patterns support differential regulation at the genome level. The identified proteins showed high agreement with the transcriptome data and suggest similar regulation within a host at both levels for the identified groups. Such host attributes need to be considered in future process design and operation. PMID:23950949

A comparative analysis of industrial Escherichia coli K-12 and B strains in high-glucose batch cultivations on process-, transcriptome- and proteome level.

PubMed

Marisch, Karoline; Bayer, Karl; Scharl, Theresa; Mairhofer, Juergen; Krempl, Peter M; Hummel, Karin; Razzazi-Fazeli, Ebrahim; Striedner, Gerald

2013-01-01

Escherichia coli K-12 and B strains are among the most frequently used bacterial hosts for production of recombinant proteins on an industrial scale. To improve existing processes and to accelerate bioprocess development, we performed a detailed host analysis. We investigated the different behaviors of the E. coli production strains BL21, RV308, and HMS174 in response to high-glucose concentrations. Tightly controlled cultivations were conducted under defined environmental conditions for the in-depth analysis of physiological behavior. In addition to acquisition of standard process parameters, we also used DNA microarray analysis and differential gel electrophoresis (Ettan(TM) DIGE). Batch cultivations showed different yields of the distinct strains for cell dry mass and growth rate, which were highest for BL21. In addition, production of acetate, triggered by excess glucose supply, was much higher for the K-12 strains compared to the B strain. Analysis of transcriptome data showed significant alteration in 347 of 3882 genes common among all three hosts. These differentially expressed genes included, for example, those involved in transport, iron acquisition, and motility. The investigation of proteome patterns additionally revealed a high number of differentially expressed proteins among the investigated hosts. The subsequently selected 38 spots included proteins involved in transport and motility. The results of this comprehensive analysis delivered a full genomic picture of the three investigated strains. Differentially expressed groups for targeted host modification were identified like glucose transport or iron acquisition, enabling potential optimization of strains to improve yield and process quality. Dissimilar growth profiles of the strains confirm different genotypes. Furthermore, distinct transcriptome patterns support differential regulation at the genome level. The identified proteins showed high agreement with the transcriptome data and suggest similar regulation within a host at both levels for the identified groups. Such host attributes need to be considered in future process design and operation.

Simple and accurate sum rules for highly relativistic systems

NASA Astrophysics Data System (ADS)

Cohen, Scott M.

2005-03-01

In this paper, I consider the Bethe and Thomas-Reiche-Kuhn sum rules, which together form the foundation of Bethe's theory of energy loss from fast charged particles to matter. For nonrelativistic target systems, the use of closure leads directly to simple expressions for these quantities. In the case of relativistic systems, on the other hand, the calculation of sum rules is fraught with difficulties. Various perturbative approaches have been used over the years to obtain relativistic corrections, but these methods fail badly when the system in question is very strongly bound. Here, I present an approach that leads to relatively simple expressions yielding accurate sums, even for highly relativistic many-electron systems. I also offer an explanation for the difference between relativistic and nonrelativistic sum rules in terms of the Zitterbewegung of the electrons.

Glucosylation of Smoke-Derived Volatiles in Grapevine (Vitis vinifera) is Catalyzed by a Promiscuous Resveratrol/Guaiacol Glucosyltransferase.

PubMed

Härtl, Katja; Huang, Fong-Chin; Giri, Ashok P; Franz-Oberdorf, Katrin; Frotscher, Johanna; Shao, Yang; Hoffmann, Thomas; Schwab, Wilfried

2017-07-19

Vinification of grapes (Vitis vinifera) exposed to forest fire smoke can yield unpalatable wine due to the presence of taint compounds from smoke and the release of smoke derived volatiles from their respective glycosides during the fermentation process or in-mouth during consumption. To identify glycosyltransferases (GTs) involved in the formation of glycosidically bound smoke-derived volatiles we performed gene expression analysis of candidate GTs in different grapevine tissues. Second, substrates derived from bushfire smoke or naturally occurring in grapes were screened with the candidate recombinant GTs. A resveratrol GT (UGT72B27) gene, highly expressed in grapevine leaves and berries was identified to be responsible for the production of the phenolic glucosides. UGT72B27 converted the stilbene trans-resveratrol mainly to the 3-O-glucoside. Kinetic analyses yielded specificity constants (k cat /K M ) of 114, 17, 9, 8, and 2 mM -1 s -1 for guaiacol, trans-resveratrol, syringol, methylsyringol, and methylguaiacol, respectively. This knowledge will help to design strategies for managing the risk of producing smoke-affected wines.

Environmentally safe production of 7-aminodeacetoxycephalosporanic acid (7-ADCA) using recombinant strains of Acremonium chrysogenum.

PubMed

Velasco, J; Luis Adrio, J; Angel Moreno, M; Díez, B; Soler, G; Barredo, J L

2000-08-01

Medically useful semisynthetic cephalosporins are made from 7-aminodeacetoxycephalosporanic acid (7-ADCA) or 7-aminocephalosporanic acid (7-ACA). Here we describe a new industrially amenable bioprocess for the production of the important intermediate 7-ADCA that can replace the expensive and environmentally unfriendly chemical method classically used. The method is based on the disruption and one-step replacement of the cefEF gene, encoding the bifunctional expandase/hydroxylase activity, of an actual industrial cephalosporin C production strain of Acremonium chrysogenum. Subsequent cloning and expression of the cefE gene from Streptomyces clavuligerus in A. chrysogenum yield recombinant strains producing high titers of deacetoxycephalosporin C (DAOC). Production level of DAOC is nearly equivalent (75-80%) to the total beta-lactams biosynthesized by the parental overproducing strain. DAOC deacylation is carried out by two final enzymatic bioconversions catalyzed by D-amino acid oxidase (DAO) and glutaryl acylase (GLA) yielding 7-ADCA. In contrast to the data reported for recombinant strains of Penicillium chrysogenum expressing ring expansion activity, no detectable contamination with other cephalosporin intermediates occurred.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Calva-Tellez, E.

The angular distributions of the process e/sup +/e/sup -/ yields e/sup +/e/sup / measured by the Bologna-CERN-Frascati group at the electron-positron colliding-beam facility Adonc are analyzed in terms of standard quantum 3/ including soft-and hard-photon emission. An analytical expression is given for the cross section of the process e/sup +/e/sup -/ yields e/sup +/e gamma for hard photons. Detailed numerical r esults for that specific experimental setup are obtained by a Monte Carlo progrm. It is found that this bremsstrahlung process is responsible for the noncollinear and noncoplanar events observed at Frasscati. Therefore, these data, together with the present calculation,more » provide a test of QED for this particular physical situation involving high energies and large momentum transfers. (auth)« less

Transgenic plants with enhanced growth characteristics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Unkefer, Pat J.; Anderson, Penelope S.; Knight, Thomas J.

The invention relates to transgenic plants exhibiting dramatically enhanced growth rates, greater seed and fruit/pod yields, earlier and more productive flowering, more efficient nitrogen utilization, increased tolerance to high salt conditions, and increased biomass yields. In one embodiment, transgenic plants engineered to over-express both glutamine phenylpyruvate transaminase (GPT) and glutamine synthetase (GS) are provided. The GPT+GS double-transgenic plants of the invention consistently exhibit enhanced growth characteristics, with T0 generation lines showing an increase in biomass over wild type counterparts of between 50% and 300%. Generations that result from sexual crosses and/or selfing typically perform even better, with some of themore » double-transgenic plants achieving an astounding four-fold biomass increase over wild type plants.« less

Unified analytic representation of physical sputtering yield

NASA Astrophysics Data System (ADS)

Janev, R. K.; Ralchenko, Yu. V.; Kenmotsu, T.; Hosaka, K.

2001-03-01

Generalized energy parameter η= η( ɛ, δ) and normalized sputtering yield Ỹ(η) , where ɛ= E/ ETF and δ= Eth/ ETF, are introduced to achieve a unified representation of all available experimental and sputtering data at normal ion incidence. The sputtering data in the new Ỹ(η) representation retain their original uncertainties. The Ỹ(η) data can be fitted to a simple three-parameter analytic expression with an rms deviation of 32%, well within the uncertainties of original data. Both η and Ỹ(η) have correct physical behavior in the threshold and high-energy regions. The available theoretical data produced by the TRIM.SP code can also be represented by the same single analytic function Ỹ(η) with a similar accuracy.

STRAP regulates c-Jun ubiquitin-mediated proteolysis and cellular proliferation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Reiner, Jennifer; Ye, Fei; Kashikar, Nilesh D.

2011-04-08

Highlights: {yields} STRAP is specifically correlated with c-Jun expression and activation in fibroblasts. {yields} STRAP inhibits c-Jun ubiquitylation in vivo and prolongs the half-life of c-Jun. {yields} STRAP expression increases expression of the AP-1 target gene, cyclin D1, and promotes cell autonomous growth. -- Abstract: STRAP is a ubiquitous WD40 protein that has been implicated in tumorigenesis. Previous studies suggest that STRAP imparts oncogenic characteristics to cells by promoting ERK and pRb phosphorylation. While these findings suggest that STRAP can activate mitogenic signaling pathways, the effects of STRAP on other MAPK pathways have not been investigated. Herein, we report thatmore » STRAP regulates the expression of the c-Jun proto-oncogene in mouse embryonic fibroblasts. Loss of STRAP expression results in reduced phospho-c-Jun and total c-Jun but does not significantly reduce the level of two other early response genes, c-Myc and c-Fos. STRAP knockout also decreases expression of the AP-1 target gene, cyclin D1, which is accompanied by a reduction in cell growth. No significant differences in JNK activity or basal c-Jun mRNA levels were observed between wild type and STRAP null fibroblasts. However, proteasomal inhibition markedly increases c-Jun expression in STRAP knockout MEFs and STRAP over-expression decreases the ubiquitylation of c-Jun in 293T cells. Loss of STRAP accelerates c-Jun turnover in fibroblasts and ectopic over-expression of STRAP in STRAP null fibroblasts increases c-Jun expression. Collectively, our findings indicate that STRAP regulates c-Jun stability by decreasing the ubiquitylation and proteosomal degradation of c-Jun.« less

Cellular Response to the high protein digestibility/high-Lysine (hdhl) sorghum mutation.

PubMed

Benmoussa, Mustapha; Chandrashekar, Arun; Ejeta, Gebisa; Hamaker, Bruce R

2015-12-01

A high protein digestibility/high-lysine mutant P721Q (hdhl) with a multi-folded protein body morphology has been developed, with a 22kDa α-kafirin single point mutation having also been recently identified. Relatively little is known regarding the resulting cellular response in hdhl endosperm. The aim is to elucidate these biochemical changes. Two-dimentional gel electrophoresis showed an apparent increase of non-kafirin and a decrease in kafirins content in hdhl endosperm. Mass spectrometry data yielded the identity of differentially expressed non-kafirin proteins in hdhl, wild-type lines such as cytoskeleton and chaperones proteins, and also others involved in amino acids and carbohydrates biochemical synthesis pathways. Western blot analysis showed that chaperone proteins were more highly expressed in the hdhl than the wild-type sorghum and confirmed the non-kafirin proteins proteomic results. Two-dimentional gel electrophoresis showed that the γ-kafirin subunits content had decreased, and the 22kDa α-kafirin subunit was increased in hdhl without any apparent molecular mass change. The observed differential expression most likely led to proteins interactions between γ- and α-kafirin subunits in particular, which resulted in a kafirins packing differently to form the protein body's multi-folded morphology, while also improving its digestibility. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

The smallest natural high-active luciferase: cloning and characterization of novel 16.5-kDa luciferase from copepod Metridia longa.

PubMed

Markova, Svetlana V; Larionova, Marina D; Burakova, Ludmila P; Vysotski, Eugene S

2015-01-30

Coelenterazine-dependent copepod luciferases containing natural signal peptide for secretion are a very convenient analytical tool as they enable monitoring of intracellular events with high sensitivity, without destroying cells or tissues. This property is well suited for application in biomedical research and development of cell-based assays for high throughput screening. We report the cloning of cDNA gene encoding a novel secreted non-allelic 16.5-kDa isoform (MLuc7) of Metridia longa luciferase, which, in fact, is the smallest natural luciferase of known for today. Despite the small size, isoform contains 10 conservative Cys residues suggesting the presence of up to 5 SS bonds. This hampers the efficient production of functionally active recombinant luciferase in bacterial expression systems. With the use of the baculovirus expression system, we produced substantial amounts of the proper folded MLuc7 luciferase with a yield of ∼3 mg/L of a high purity protein. We demonstrate that MLuc7 produced in insect cells is highly active and extremely thermostable, and is well suited as a secreted reporter when expressed in mammalian cells ensuring higher sensitivity of detection as compared to another Metridia luciferase isoform (MLuc164) which is widely employed in real-time imaging. Copyright © 2014 Elsevier Inc. All rights reserved.

A comparison of torque expression between stainless steel, titanium molybdenum alloy, and copper nickel titanium wires in metallic self-ligating brackets.

PubMed

Archambault, Amy; Major, Thomas W; Carey, Jason P; Heo, Giseon; Badawi, Hisham; Major, Paul W

2010-09-01

The force moment providing rotation of the tooth around the x-axis (buccal-lingual) is referred to as torque expression in orthodontic literature. Many factors affect torque expression, including the wire material characteristics. This investigation aims to provide an experimental study into and comparison of the torque expression between wire types. With a worm-gear-driven torquing apparatus, wire was torqued while a bracket mounted on a six-axis load cell was engaged. Three 0.019 x 0.0195 inch wire (stainless steel, titanium molybdenum alloy [TMA], copper nickel titanium [CuNiTi]), and three 0.022 inch slot bracket combinations (Damon 3MX, In-Ovation-R, SPEED) were compared. At low twist angles (<12 degrees), the differences in torque expression between wires were not statistically significant. At twist angles over 24 degrees, stainless steel wire yielded 1.5 to 2 times the torque expression of TMA and 2.5 to 3 times that of nickel titanium (NiTi). At high angles of torsion (over 40 degrees) with a stiff wire material, loss of linear torque expression sometimes occurred. Stainless steel has the largest torque expression, followed by TMA and then NiTi.

Content of intrinsic disorder influences the outcome of cell-free protein synthesis.

PubMed

Tokmakov, Alexander A; Kurotani, Atsushi; Ikeda, Mariko; Terazawa, Yumiko; Shirouzu, Mikako; Stefanov, Vasily; Sakurai, Tetsuya; Yokoyama, Shigeyuki

2015-09-11

Cell-free protein synthesis is used to produce proteins with various structural traits. Recent bioinformatics analyses indicate that more than half of eukaryotic proteins possess long intrinsically disordered regions. However, no systematic study concerning the connection between intrinsic disorder and expression success of cell-free protein synthesis has been presented until now. To address this issue, we examined correlations of the experimentally observed cell-free protein expression yields with the contents of intrinsic disorder bioinformatically predicted in the expressed sequences. This analysis revealed strong relationships between intrinsic disorder and protein amenability to heterologous cell-free expression. On the one hand, elevated disorder content was associated with the increased ratio of soluble expression. On the other hand, overall propensity for detectable protein expression decreased with disorder content. We further demonstrated that these tendencies are rooted in some distinct features of intrinsically disordered regions, such as low hydrophobicity, elevated surface accessibility and high abundance of sequence motifs for proteolytic degradation, including sites of ubiquitination and PEST sequences. Our findings suggest that identification of intrinsically disordered regions in the expressed amino acid sequences can be of practical use for predicting expression success and optimizing cell-free protein synthesis.

A BioDesign Approach to Obtain High Yields of Biosimilars by Anti-apoptotic Cell Engineering: a Case Study to Increase the Production Yield of Anti-TNF Alpha Producing Recombinant CHO Cells.

PubMed

Gulce Iz, Sultan; Inevi, Muge Anil; Metiner, Pelin Saglam; Tamis, Duygu Ayyildiz; Kisbet, Nazli

2018-01-01

Recent developments in medical biotechnology have facilitated to enhance the production of monoclonal antibodies (mAbs) and recombinant proteins in mammalian cells. Human mAbs for clinical applications have focused on three areas, particularly cancer, immunological disorders, and infectious diseases. Tumor necrosis factor alpha (TNF-α), which has both proinflammatory and immunoregulatory functions, is an important target in biopharmaceutical industry. In this study, a humanized anti-TNF-α mAb producing stable CHO cell line which produces a biosimilar of Humira (adalimumab) was used. Adalimumab is a fully human anti-TNF mAb among the top-selling mAb products in recent years as a biosimilar. Products from mammalian cell bioprocesses are a derivative of cell viability and metabolism, which is mainly disrupted by cell death in bioreactors. Thus, different strategies are used to increase the product yield. Suppression of apoptosis, also called anti-apoptotic cell engineering, is the most remarkable strategy to enhance lifetime of cells for a longer production period. In fact, using anti-apoptotic cell engineering as a BioDesign approach was inspired by nature; nature gives prolonged life span to some cells like stem cells, tumor cells, and memory B and T cells, and researchers have been using this strategy for different purposes. In this study, as a biomimicry approach, anti-apoptotic cell engineering was used to increase the anti-TNF-α mAb production from the humanized anti-TNF-α mAb producing stable CHO cell line by Bcl-xL anti-apoptotic protein. It was shown that transient transfection of CHO cells by the Bcl-xL anti-apoptotic protein expressing plasmid prolonged the cell survival rate and protected cells from apoptosis. The transient expression of Bcl-xL using CHO cells enhanced the anti-TNF-α production. The production of anti-TNF-α in CHO cells was increased up to 215 mg/L with an increase of 160% after cells were transfected with Bcl-xL expressing plasmid with polyethylenimine (PEI) reagent at the ratio of 1:6 (DNA:PEI). In conclusion, the anti-apoptotic efficacy of the Bcl-xL expressing plasmid in humanized anti-TNF-α MAb producing stable CHO cells is compatible with curative effect for high efficiency recombinant protein production. Thus, this model can be used for large-scale production of biosimilars through transient Bcl-xL gene expression as a cost-effective method.

Enhanced recombinant factor VII expression in Chinese hamster ovary cells by optimizing signal peptides and fed-batch medium.

PubMed

Peng, Lin; Yu, Xiao; Li, Chengyuan; Cai, Yanfei; Chen, Yun; He, Yang; Yang, Jianfeng; Jin, Jian; Li, Huazhong

2016-04-01

Signal peptides play an important role in directing and efficiently transporting secretory proteins to their proper locations in the endoplasmic reticulum of mammalian cells. The aim of this study was to enhance the expression of recombinant coagulation factor VII (rFVII) in CHO cells by optimizing the signal peptides and type of fed-batch culture medium used. Five sub-clones (O2, I3, H3, G2 and M3) with different signal peptide were selected by western blot (WB) analysis and used for suspension culture. We compared rFVII expression levels of 5 sub-clones and found that the highest rFVII expression level was obtained with the IgK signal peptide instead of Ori, the native signal peptide of rFVII. The high protein expression of rFVII with signal peptide IgK was mirrored by a high transcription level during suspension culture. After analyzing culture and feed media, the combination of M4 and F4 media yielded the highest rFVII expression of 20 mg/L during a 10-day suspension culture. After analyzing cell density and cell cycle, CHO cells feeding by F4 had a similar percentage of cells in G0/G1 and a higher cell density compared to F2 and F3. This may be the reason for high rFVII expression in M4+F4. In summary, rFVII expression was successfully enhanced by optimizing the signal peptide and fed-batch medium used in CHO suspension culture. Our data may be used to improve the production of other therapeutic proteins in fed-batch culture.

Nuclear translocation of glutathione S-transferase {pi} is mediated by a non-classical localization signal

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kawakatsu, Miho; Goto, Shinji, E-mail: sgoto@nagasaki-u.ac.jp; Yoshida, Takako

2011-08-12

Highlights: {yields} Nuclear translocation of GST{pi} is abrogated by the deletion of the last 16 amino acid residues in the carboxy-terminal region, indicating that residues 195-208 of GST{pi} are required for nuclear translocation. {yields} The lack of a contiguous stretch of positively charged amino acid residues within the carboxy-terminal region of GST{pi}, suggests that the nuclear translocation of GST{pi} is mediated by a non-classical nuclear localization signal. {yields} An in vitro transport assay shows that the nuclear translocation of GST{pi} is dependent on cytosolic factors and ATP. -- Abstract: Glutathione S-transferase {pi} (GST{pi}), a member of the GST family ofmore » multifunctional enzymes, is highly expressed in human placenta and involved in the protection of cellular components against electrophilic compounds or oxidative stress. We have recently found that GST{pi} is expressed in the cytoplasm, mitochondria, and nucleus in some cancer cells, and that the nuclear expression of GST{pi} appears to correlate with resistance to anti-cancer drugs. Although the mitochondrial targeting signal of GST{pi} was previously identified in the amino-terminal region, the mechanism of nuclear translocation remains completely unknown. In this study, we find that the region of GST{pi}195-208 is critical for nuclear translocation, which is mediated by a novel and non-classical nuclear localization signal. In addition, using an in vitro transport assay, we demonstrate that the nuclear translocation of GST{pi} depends on the cytosolic extract and ATP. Although further experiments are needed to understand in depth the precise mechanism of nuclear translocation of GST{pi}, our results may help to establish more efficient anti-cancer therapy, especially with respect to resistance to anti-cancer drugs.« less

Low-energy N-ion beam biotechnology application in the induction of Thai jasmine rice mutant with improved seed storability

NASA Astrophysics Data System (ADS)

Semsang, Nuananong; Techarang, Jiranat; Yu, Liangdeng; Phanchaisri, Boonrak

2018-06-01

Low-energy heavy-ion beam is a novel biotechnology used for mutation induction in plants. We used a low-energy N-ion beam to induce mutations in Thai jasmine rice (Oryza sativa L. cv. KDML 105) to improve the yield and seed quality. Seeds of BKOS6, a Thai jasmine rice mutant previously induced by ion beams, were re-bombarded with 60-kV-accelerated N-ions (N++N2+) to fluences of 1-2 × 1016 ions/cm2. The resulting mutant, named HyKOS21, exhibited photoperiod insensitivity, semi-dwarfness, and high yield potential. Seed storability of the mutant was studied in natural and accelerated ageing conditions and compared to that of KDML 105 and six other Thai rice varieties. In both testing conditions, HyKOS21 mutant had the highest seed storability among the tested varieties. After storage in the natural condition for 18 months, HyKOS21 had a seed germination percentage nearly two times as that of the original KDML 105. Biochemical analysis showed that the lipid peroxidation level of the mutant seeds was the lowest among those of the tested varieties. Furthermore, an expression analysis of genes encoding lipoxygenase isoenzyme (lox1, lox2, and lox3) revealed that the mutant lacked expression of lox1 and lox2 and expressed only lox3 in seeds. These results may explain the improved seed longevity of the mutant after storage. This work provides further evidence of the modification of biological materials using a low-energy ion beam to produce rice mutants with improved yield and seed storability. The benefits of this technology, to create new varieties with improved values, could serve for local economic development.

Physiological and transcriptomic responses in the seed coat of field-grown soybean (Glycine max L. Merr.) to abiotic stress.

PubMed

Leisner, Courtney P; Yendrek, Craig R; Ainsworth, Elizabeth A

2017-12-12

Understanding how intensification of abiotic stress due to global climate change affects crop yields is important for continued agricultural productivity. Coupling genomic technologies with physiological crop responses in a dynamic field environment is an effective approach to dissect the mechanisms underpinning crop responses to abiotic stress. Soybean (Glycine max L. Merr. cv. Pioneer 93B15) was grown in natural production environments with projected changes to environmental conditions predicted for the end of the century, including decreased precipitation, increased tropospheric ozone concentrations ([O 3 ]), or increased temperature. All three environmental stresses significantly decreased leaf-level photosynthesis and stomatal conductance, leading to significant losses in seed yield. This was driven by a significant decrease in the number of pods per node for all abiotic stress treatments. To understand the underlying transcriptomic response involved in the yield response to environmental stress, RNA-Sequencing analysis was performed on the soybean seed coat, a tissue that plays an essential role in regulating carbon and nitrogen transport to developing seeds. Gene expression analysis revealed 49, 148 and 1,576 differentially expressed genes in the soybean seed coat in response to drought, elevated [O 3 ] and elevated temperature, respectively. Elevated [O 3 ] and drought did not elicit substantive transcriptional changes in the soybean seed coat. However, this may be due to the timing of sampling and does not preclude impacts of those stresses on different tissues or different stages in seed coat development. Expression of genes involved in DNA replication and metabolic processes were enriched in the seed coat under high temperate stress, suggesting that the timing of events that are important for cell division and proper seed development were altered in a stressful growth environment.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Leem, Yea-Hyun, E-mail: leemyy@empas.com; Lee, Young-Ik, E-mail: lee0ik@hanmail.net; Son, Hee-Jeong, E-mail: son1106@paran.com

Research highlights: {yields} The progress of neurodegeration are directly linked to the neuroinflammatory response. {yields} We investigate whether exercise improves the neuroinflammation using T{sub g}-NSE/htau23 mice. {yields} This provides insights that exercise may beneficial effects on the neuroinflammatory disorders. -- Abstract: The objective of the present study was to investigate whether chronic endurance exercise attenuates the neuroinflammation in the brain of mice with NSE/htau23. In this study, the tau-transgenic (Tg) mouse, Tg-NSE/htau23, which over expresses human Tau23 in its brain, was subjected to chronic exercise for 3 months, from 16 months of age. The brains of Tg mice exhibited increasedmore » immunoreactivity and active morphological changes in GFAP (astrocyte marker) and MAC-1 (microglia marker) expression in an age-dependent manner. To identify the effects of chronic exercise on gliosis, the exercised Tg mice groups were treadmill run at a speed of 12 m/min (intermediate exercise group) or 19 m/min (high exercise group) for 1 h/day and 5 days/week during the 3 month period. The neuroinflammatory response characterized by activated astroglia and microglia was significantly repressed in the exercised Tg mice in an exercise intensity-dependent manner. In parallel, chronic exercise in Tg mice reduced the increased expression of TNF-{alpha}, IL-6, IL-1{beta}, COX-2, and iNOS. Consistently with these changes, the levels of phospho-p38 and phospho-ERK were markedly downregulated in the brain of Tg mice after exercise. In addition, nuclear NF-{kappa}B activity was profoundly reduced after chronic exercise in an exercise intensity-dependent manner. These findings suggest that chronic endurance exercise may alleviate neuroinflammation in the Tau pathology of Alzheimer's disease.« less

Systematic characterization of maturation time of fluorescent proteins in living cells

PubMed Central

Balleza, Enrique; Kim, J. Mark; Cluzel, Philippe

2017-01-01

Slow maturation time of fluorescent proteins limits accurate measurement of rapid gene expression dynamics and effectively reduces fluorescence signal in growing cells. We used high-precision time-lapse microscopy to characterize, at two different temperatures in E. coli, the maturation kinetics of 50 FPs that span the visible spectrum. We identified fast-maturing FPs that yield the highest signal-to-noise ratio and temporal resolution in individual growing cells. PMID:29320486

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hamrick, Mark W., E-mail: mhamrick@mail.mcg.edu; Department of Orthopaedic Surgery, Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA; Herberg, Samuel

Research highlights: {yields} Aging is associated with muscle atrophy and loss of muscle mass, known as the sarcopenia of aging. {yields} We demonstrate that age-related muscle atrophy is associated with marked changes in miRNA expression in muscle. {yields} Treating aged mice with the adipokine leptin significantly increased muscle mass and the expression of miRNAs involved in muscle repair. {yields} Recombinant leptin therapy may therefore be a novel approach for treating age-related muscle atrophy. -- Abstract: Age-associated loss of muscle mass, or sarcopenia, contributes directly to frailty and an increased risk of falls and fractures among the elderly. Aged mice andmore » elderly adults both show decreased muscle mass as well as relatively low levels of the fat-derived hormone leptin. Here we demonstrate that loss of muscle mass and myofiber size with aging in mice is associated with significant changes in the expression of specific miRNAs. Aging altered the expression of 57 miRNAs in mouse skeletal muscle, and many of these miRNAs are now reported to be associated specifically with age-related muscle atrophy. These include miR-221, previously identified in studies of myogenesis and muscle development as playing a role in the proliferation and terminal differentiation of myogenic precursors. We also treated aged mice with recombinant leptin, to determine whether leptin therapy could improve muscle mass and alter the miRNA expression profile of aging skeletal muscle. Leptin treatment significantly increased hindlimb muscle mass and extensor digitorum longus fiber size in aged mice. Furthermore, the expression of 37 miRNAs was altered in muscles of leptin-treated mice. In particular, leptin treatment increased the expression of miR-31 and miR-223, miRNAs known to be elevated during muscle regeneration and repair. These findings suggest that aging in skeletal muscle is associated with marked changes in the expression of specific miRNAs, and that nutrient-related hormones such as leptin may be able to reverse muscle atrophy and alter the expression of atrophy-related miRNAs in aging skeletal muscle.« less

Methane Emission and Milk Production of Dairy Cows Grazing Pastures Rich in Legumes or Rich in Grasses in Uruguay.

PubMed

Dini, Yoana; Gere, José; Briano, Carolina; Manetti, Martin; Juliarena, Paula; Picasso, Valentin; Gratton, Roberto; Astigarraga, Laura

2012-06-08

Understanding the impact of changing pasture composition on reducing emissions of GHGs in dairy grazing systems is an important issue to mitigate climate change. The aim of this study was to estimate daily CH₄ emissions of dairy cows grazing two mixed pastures with contrasting composition of grasses and legumes: L pasture with 60% legumes on Dry Matter (DM) basis and G pasture with 75% grasses on DM basis. Milk production and CH₄ emissions were compared over two periods of two weeks during spring using eight lactating Holstein cows in a 2 × 2 Latin square design. Herbage organic matter intake (HOMI) was estimated by chromic oxide dilution and herbage organic matter digestibility (OMD) was estimated by faecal index. Methane emission was estimated by using the sulfur hexafluoride (SF6) tracer technique adapted to collect breath samples over 5-day periods. OMD (0.71) and HOMI (15.7 kg OM) were not affected by pasture composition. Milk production (20.3 kg/d), milk fat yield (742 g/d) and milk protein yield (667 g/d) were similar for both pastures. This may be explained by the high herbage allowance (30 kg DM above 5 cm/cow) which allowed the cows to graze selectively, in particular in grass sward. Similarly, methane emission expressed as absolute value (368 g/d or 516 L/d) or expressed as methane yield (6.6% of Gross Energy Intake (GEI)) was not affected by treatments. In conclusion, at high herbage allowance, the quality of the diet selected by grazing cows did not differ between pastures rich in legumes or rich in grasses, and therefore there was no effect on milk or methane production.

Perfluorodecalins and Hexenol as Inducers of Secondary Metabolism in Taxus media and Vitis vinifera Cell Cultures

PubMed Central

Vidal-Limon, Heriberto R.; Almagro, Lorena; Moyano, Elisabeth; Palazon, Javier; Pedreño, Maria A.; Cusido, Rosa M.

2018-01-01

Plant cell cultures constitute a potentially efficient and sustainable tool for the production of high added-value bioactive compounds. However, due to the inherent restrictions in the expression of secondary metabolism, to date the yields obtained have generally been low. Plant cell culture elicitation can boost production, sometimes leading to dramatic improvements in yield, as well as providing insight into the target biosynthetic pathways and the regulation of the genes involved. Among the secondary compounds successfully being produced in biotechnological platforms are taxanes and trans-resveratrol (t-R). In the current study, perfluorodecalins (PFDs) and hexenol (Hex) were tested for the first time with Taxus media and Vitis vinifera cell cultures to explore their effect on plant cell growth and secondary metabolite production, either alone or combined with other elicitors already established as highly effective, such as methyl jasmonate (MeJa), coronatine (Coro) or randomly methylated β-cyclodextrins (β-CDs). The total taxane content at the peak of production in T. media cell cultures treated with PFDs together with Coro plus β-CDs was 3.3-fold higher than in the control, whereas the t-R production in MeJa and β-CD-treated V. vinifera cell cultures increased 552.6-fold compared to the extremely low-yielding control. Hex was ineffective as an elicitor in V. vinifera cell cultures, and in T. media cell suspensions it blocked the taxol production but induced a clear enhancement of baccatin III. Regarding biosynthetic gene expression, a strong positive relationship was observed between the transcript level of targeted genes and taxol production in the T. media cell cultures, but not with t-R production in the elicited V. vinifera cell cultures. PMID:29616056

Perfluorodecalins and Hexenol as Inducers of Secondary Metabolism in Taxus media and Vitis vinifera Cell Cultures.

PubMed

Vidal-Limon, Heriberto R; Almagro, Lorena; Moyano, Elisabeth; Palazon, Javier; Pedreño, Maria A; Cusido, Rosa M

2018-01-01

Plant cell cultures constitute a potentially efficient and sustainable tool for the production of high added-value bioactive compounds. However, due to the inherent restrictions in the expression of secondary metabolism, to date the yields obtained have generally been low. Plant cell culture elicitation can boost production, sometimes leading to dramatic improvements in yield, as well as providing insight into the target biosynthetic pathways and the regulation of the genes involved. Among the secondary compounds successfully being produced in biotechnological platforms are taxanes and trans -resveratrol ( t -R). In the current study, perfluorodecalins (PFDs) and hexenol (Hex) were tested for the first time with Taxus media and Vitis vinifera cell cultures to explore their effect on plant cell growth and secondary metabolite production, either alone or combined with other elicitors already established as highly effective, such as methyl jasmonate (MeJa), coronatine (Coro) or randomly methylated β-cyclodextrins (β-CDs). The total taxane content at the peak of production in T. media cell cultures treated with PFDs together with Coro plus β-CDs was 3.3-fold higher than in the control, whereas the t -R production in MeJa and β-CD-treated V. vinifera cell cultures increased 552.6-fold compared to the extremely low-yielding control. Hex was ineffective as an elicitor in V. vinifera cell cultures, and in T. media cell suspensions it blocked the taxol production but induced a clear enhancement of baccatin III. Regarding biosynthetic gene expression, a strong positive relationship was observed between the transcript level of targeted genes and taxol production in the T. media cell cultures, but not with t -R production in the elicited V. vinifera cell cultures.

Overcoming the response plateau in multiple myeloma: a novel bortezomib-based strategy for secondary induction and high-yield CD34+ stem cell mobilization.

PubMed

Niesvizky, Ruben; Mark, Tomer M; Ward, Maureen; Jayabalan, David S; Pearse, Roger N; Manco, Megan; Stern, Jessica; Christos, Paul J; Mathews, Lena; Shore, Tsiporah B; Zafar, Faiza; Pekle, Karen; Xiang, Zhaoying; Ely, Scott; Skerret, Donna; Chen-Kiang, Selina; Coleman, Morton; Lane, Maureen E

2013-03-15

This phase II study evaluated bortezomib-based secondary induction and stem cell mobilization in 38 transplant-eligible patients with myeloma who had an incomplete and stalled response to, or had relapsed after, previous immunomodulatory drug-based induction. Patients received up to six 21-day cycles of bortezomib plus dexamethasone, with added liposomal doxorubicin for patients not achieving partial response or better by cycle 2 or very good partial response or better (≥VGPR) by cycle 4 (DoVeD), followed by bortezomib, high-dose cyclophosphamide, and filgrastim mobilization. Gene expression/signaling pathway analyses were conducted in purified CD34+ cells after bortezomib-based mobilization and compared against patients who received only filgrastim ± cyclophosphamide. Plasma samples were similarly analyzed for quantification of associated protein markers. The response rate to DoVeD relative to the pre-DoVeD baseline was 61%, including 39% ≥ VGPR. Deeper responses were achieved in 10 of 27 patients who received bortezomib-based mobilization; postmobilization response rate was 96%, including 48% ≥ VGPR, relative to the pre-DoVeD baseline. Median CD34+ cell yield was 23.2 × 10(6) cells/kg (median of 1 apheresis session). After a median follow-up of 46.6 months, median progression-free survival was 47.1 months from DoVeD initiation; 5-year overall survival rate was 76.4%. Grade ≥ 3 adverse events included thrombocytopenia (13%), hand-foot syndrome (11%), peripheral neuropathy (8%), and neutropenia (5%). Bortezomib-based mobilization was associated with modulated expression of genes involved in stem cell migration. Bortezomib-based secondary induction and mobilization could represent an alternative strategy for elimination of tumor burden in immunomodulatory drug-resistant patients that does not impact stem cell yield.

A highly functional synthetic phage display library containing over 40 billion human antibody clones.

PubMed

Weber, Marcel; Bujak, Emil; Putelli, Alessia; Villa, Alessandra; Matasci, Mattia; Gualandi, Laura; Hemmerle, Teresa; Wulhfard, Sarah; Neri, Dario

2014-01-01

Several synthetic antibody phage display libraries have been created and used for the isolation of human monoclonal antibodies. The performance of antibody libraries, which is usually measured in terms of their ability to yield high-affinity binding specificities against target proteins of interest, depends both on technical aspects (such as library size and quality of cloning) and on design features (which influence the percentage of functional clones in the library and their ability to be used for practical applications). Here, we describe the design, construction and characterization of a combinatorial phage display library, comprising over 40 billion human antibody clones in single-chain fragment variable (scFv) format. The library was designed with the aim to obtain highly stable antibody clones, which can be affinity-purified on protein A supports, even when used in scFv format. The library was found to be highly functional, as >90% of randomly selected clones expressed the corresponding antibody. When selected against more than 15 antigens from various sources, the library always yielded specific and potent binders, at a higher frequency compared to previous antibody libraries. To demonstrate library performance in practical biomedical research projects, we isolated the human antibody G5, which reacts both against human and murine forms of the alternatively spliced BCD segment of tenascin-C, an extracellular matrix component frequently over-expressed in cancer and in chronic inflammation. The new library represents a useful source of binding specificities, both for academic research and for the development of antibody-based therapeutics.

A Highly Functional Synthetic Phage Display Library Containing over 40 Billion Human Antibody Clones

PubMed Central

Weber, Marcel; Bujak, Emil; Putelli, Alessia; Villa, Alessandra; Matasci, Mattia; Gualandi, Laura; Hemmerle, Teresa; Wulhfard, Sarah; Neri, Dario

2014-01-01

Several synthetic antibody phage display libraries have been created and used for the isolation of human monoclonal antibodies. The performance of antibody libraries, which is usually measured in terms of their ability to yield high-affinity binding specificities against target proteins of interest, depends both on technical aspects (such as library size and quality of cloning) and on design features (which influence the percentage of functional clones in the library and their ability to be used for practical applications). Here, we describe the design, construction and characterization of a combinatorial phage display library, comprising over 40 billion human antibody clones in single-chain fragment variable (scFv) format. The library was designed with the aim to obtain highly stable antibody clones, which can be affinity-purified on protein A supports, even when used in scFv format. The library was found to be highly functional, as >90% of randomly selected clones expressed the corresponding antibody. When selected against more than 15 antigens from various sources, the library always yielded specific and potent binders, at a higher frequency compared to previous antibody libraries. To demonstrate library performance in practical biomedical research projects, we isolated the human antibody G5, which reacts both against human and murine forms of the alternatively spliced BCD segment of tenascin-C, an extracellular matrix component frequently over-expressed in cancer and in chronic inflammation. The new library represents a useful source of binding specificities, both for academic research and for the development of antibody-based therapeutics. PMID:24950200

Activation tagging in indica rice identifies ribosomal proteins as potential targets for manipulation of water-use efficiency and abiotic stress tolerance in plants.

PubMed

Moin, Mazahar; Bakshi, Achala; Saha, Anusree; Udaya Kumar, M; Reddy, Attipalli R; Rao, K V; Siddiq, E A; Kirti, P B

2016-11-01

We have generated 3900 enhancer-based activation-tagged plants, in addition to 1030 stable Dissociator-enhancer plants in a widely cultivated indica rice variety, BPT-5204. Of them, 3000 were screened for water-use efficiency (WUE) by analysing photosynthetic quantum efficiency and yield-related attributes under water-limiting conditions that identified 200 activation-tagged mutants, which were analysed for flanking sequences at the site of enhancer integration in the genome. We have further selected five plants with low Δ 13 C, high quantum efficiency and increased plant yield compared with wild type for a detailed investigation. Expression studies of 18 genes in these mutants revealed that in four plants one of the three to four tagged genes became activated, while two genes were concurrently up-regulated in the fifth plant. Two genes coding for proteins involved in 60S ribosomal assembly, RPL6 and RPL23A, were among those that became activated by enhancers. Quantitative expression analysis of these two genes also corroborated the results on activating-tagging. The high up-regulation of RPL6 and RPL23A in various stress treatments and the presence of significant cis-regulatory elements in their promoter regions along with the high up-regulation of several of RPL genes in various stress treatments indicate that they are potential targets for manipulating WUE/abiotic stress tolerance. © 2016 John Wiley & Sons Ltd.

Expression, purification, and functional analysis of the C-terminal domain of Herbaspirillum seropedicae NifA protein.

PubMed

Monteiro, Rose A; Souza, Emanuel M; Geoffrey Yates, M; Steffens, M Berenice R; Pedrosa, Fábio O; Chubatsu, Leda S

2003-02-01

The Herbaspirillum seropedicae NifA protein is responsible for nif gene expression. The C-terminal domain of the H. seropedicae NifA protein, fused to a His-Tag sequence (His-Tag-C-terminal), was over-expressed and purified by metal-affinity chromatography to yield a highly purified and active protein. Band-shift assays showed that the NifA His-Tag-C-terminal bound specifically to the H. seropedicae nifB promoter region in vitro. In vivo analysis showed that this protein inhibited the Central + C-terminal domains of NifA protein from activating the nifH promoter of K. pneumoniae in Escherichia coli, indicating that the protein must be bound to the NifA-binding site (UAS site) at the nifH promoter region to activate transcription. Copyright 2002 Elsevier Science (USA)

Breeding approaches and genomics technologies to increase crop yield under low-temperature stress.

PubMed

Jha, Uday Chand; Bohra, Abhishek; Jha, Rintu

2017-01-01

Improved knowledge about plant cold stress tolerance offered by modern omics technologies will greatly inform future crop improvement strategies that aim to breed cultivars yielding substantially high under low-temperature conditions. Alarmingly rising temperature extremities present a substantial impediment to the projected target of 70% more food production by 2050. Low-temperature (LT) stress severely constrains crop production worldwide, thereby demanding an urgent yet sustainable solution. Considerable research progress has been achieved on this front. Here, we review the crucial cellular and metabolic alterations in plants that follow LT stress along with the signal transduction and the regulatory network describing the plant cold tolerance. The significance of plant genetic resources to expand the genetic base of breeding programmes with regard to cold tolerance is highlighted. Also, the genetic architecture of cold tolerance trait as elucidated by conventional QTL mapping and genome-wide association mapping is described. Further, global expression profiling techniques including RNA-Seq along with diverse omics platforms are briefly discussed to better understand the underlying mechanism and prioritize the candidate gene (s) for downstream applications. These latest additions to breeders' toolbox hold immense potential to support plant breeding schemes that seek development of LT-tolerant cultivars. High-yielding cultivars endowed with greater cold tolerance are urgently required to sustain the crop yield under conditions severely challenged by low-temperature.

Improving the performance of industrial ethanol-producing yeast by expressing the aspartyl protease on the cell surface.

PubMed

Guo, Zhong-peng; Zhang, Liang; Ding, Zhong-yang; Wang, Zheng-Xiang; Shi, Gui-Yang

2010-12-01

The yeasts used in fuel ethanol manufacture are unable to metabolize soluble proteins. The PEP4 gene, encoding a vacuolar aspartyl protease in Saccharomyces cerevisiae, was either secretively or cell-surface anchored expressed in industrial ethanol-producing S. cerevisiae. The obtained recombinant strains APA (expressing the protease secretively) and APB (expressing the protease on the cell wall) were studied under ethanol fermentation conditions in feed barley cultures. The effects of expression of the protease on product formation, growth and cell protein content were measured. The biomass yield of the wild-type was clearly lower than that of the recombinant strains (0.578 ± 0.12 g biomass/g glucose for APA and 0.582 ± 0.08 g biomass/g glucose for APB). In addition, nearly 98-99% of the theoretical maximum level of ethanol yield was achieved (relative to the amount of substrate consumed) for the recombinant strains, while limiting the nitrogen source resulted in dissatisfactory fermentation for the wild-type and more than 30 g/l residual sugar was detected at the end of fermentation. In addition, higher growth rate, viability and lower yields of byproducts such as glycerol and pyruvic acid for recombinant strains were observed. Expressing acid protease can be expected to lead to a significant increase in ethanol productivity. Copyright © 2010 John Wiley & Sons, Ltd.

CPEB1 modulates lipopolysaccharide-mediated iNOS induction in rat primary astrocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Ki Chan; Hyun Joo, So; Shin, Chan Young, E-mail: chanyshin@kku.ac.kr

2011-06-17

Highlights: {yields} Expression and phosphorylation of CPEB1 is increased by LPS stimulation in rat primary astrocytes. {yields} JNK regulates expression and phosphorylation of CPEB1 in reactive astrocytes. {yields} Down-regulation of CPEB1 using siRNA inhibits oxidative stress and iNOS induction by LPS stimulation. {yields} CPEB1 may play an important role in regulating inflammatory responses in reactive astrocytes induced by LPS. -- Abstract: Upon CNS damage, astrocytes undergo a series of biological changes including increased proliferation, production of inflammatory mediators and morphological changes, in a response collectively called reactive gliosis. This process is an essential part of the brains response to injury,more » yet much is unknown about the molecular mechanism(s) that induce these changes. In this study, we investigated the role of cytoplasmic polyadenylation element binding protein 1 (CPEB1) in the regulation of inflammatory responses in a model of reactive gliosis, lipopolysaccharide-stimulated astrocytes. CPEB1 is an mRNA-binding protein recently shown to be expressed in astrocytes that may play a role in astrocytes migration. After LPS stimulation, the expression and phosphorylation of CPEB1 was increased in rat primary astrocytes in a JNK-dependent process. siRNA-induced knockdown of CPEB1 expression inhibited the LPS-induced up-regulation of iNOS as well as NO and ROS production, a hallmark of immunological activation of astrocytes. The results from the study suggest that CPEB1 is actively involved in the regulation of inflammatory responses in astrocytes, which might provide new insights into the regulatory mechanism after brain injury.« less

Associative electron detachment - O(-) + H yields OH + e(-)

NASA Astrophysics Data System (ADS)

Acharya, P. K.; Kendall, R. A.; Simons, J.

1985-10-01

Diatomic associative electron detachment (AED) involves the ejection of an electron when a atomic anion and another atom collisionally associate to produce a neutral diatomic molecule in a vibration-rotation state labeled V-prime, J-prime. Electron ejection rate calculations are discussed, taking into account aspects of rate expressions, calculations of ingredients in rate expression, initial-condition weighting factors, and the vibration and rotation dependence of ejection rates. The results of ab initio theoretical simulations indicate that AED in O(-) + H is so slow (approximately 10,000 per s) that it is likely to be inaccessible to present experimental observation. Propensity for producing OH in high vibrational levels does occur but the propensity is not sharp.

Expression and Purification of Rat Glucose Transporter 1 in Pichia pastoris.

PubMed

Venskutonytė, Raminta; Elbing, Karin; Lindkvist-Petersson, Karin

2018-01-01

Large amounts of pure and homogenous protein are a prerequisite for several biochemical and biophysical analyses, and in particular if aiming at resolving the three-dimensional protein structure. Here we describe the production of the rat glucose transporter 1 (GLUT1), a membrane protein facilitating the transport of glucose in cells. The protein is recombinantly expressed in the yeast Pichia pastoris. It is easily maintained and large-scale protein production in shaker flasks, as commonly performed in academic research laboratories, results in relatively high yields of membrane protein. The purification protocol describes all steps needed to obtain a pure and homogenous GLUT1 protein solution, including cell growth, membrane isolation, and chromatographic purification methods.

Steroid synthesis by primary human keratinocytes; implications for skin disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hannen, Rosalind F., E-mail: r.f.hannen@qmul.ac.uk; Michael, Anthony E.; Jaulim, Adil

2011-01-07

Research highlights: {yields} Primary keratinocytes express the steroid enzymes required for cortisol synthesis. {yields} Normal primary human keratinocytes can synthesise cortisol. {yields} Steroidogenic regulators, StAR and MLN64, are expressed in normal epidermis. {yields} StAR expression is down regulated in eczema and psoriatic epidermis. -- Abstract: Cortisol-based therapy is one of the most potent anti-inflammatory treatments available for skin conditions including psoriasis and atopic dermatitis. Previous studies have investigated the steroidogenic capabilities of keratinocytes, though none have demonstrated that these skin cells, which form up to 90% of the epidermis are able to synthesise cortisol. Here we demonstrate that primary humanmore » keratinocytes (PHK) express all the elements required for cortisol steroidogenesis and metabolise pregnenolone through each intermediate steroid to cortisol. We show that normal epidermis and cultured PHK express each of the enzymes (CYP11A1, CYP17A1, 3{beta}HSD1, CYP21 and CYP11B1) that are required for cortisol synthesis. These enzymes were shown to be metabolically active for cortisol synthesis since radiometric conversion assays traced the metabolism of [7-{sup 3}H]-pregnenolone through each steroid intermediate to [7-{sup 3}H]-cortisol in cultured PHK. Trilostane (a 3{beta}HSD1 inhibitor) and ketoconazole (a CYP17A1 inhibitor) blocked the metabolism of both pregnenolone and progesterone. Finally, we show that normal skin expresses two cholesterol transporters, steroidogenic acute regulatory protein (StAR), regarded as the rate-determining protein for steroid synthesis, and metastatic lymph node 64 (MLN64) whose function has been linked to cholesterol transport in steroidogenesis. The expression of StAR and MLN64 was aberrant in two skin disorders, psoriasis and atopic dermatitis, that are commonly treated with cortisol, suggesting dysregulation of epidermal steroid synthesis in these patients. Collectively these data show that PHK are capable of extra-adrenal cortisol synthesis, which could be a fundamental pathway in skin biology with implications in psoriasis and atopic dermatitis.« less