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961.
Baculovirus has emerged as a promising vector for in vivo or ex vivo gene therapy. To date, the infectious titer and multiplicity of infection (MOI) based on the ability of baculovirus to infect insect cells are commonly adopted to indicate the virus dosage. However, the infectious titer and MOI do not reliably represent the baculovirus transducing ability, making the comparison of baculovirus-mediated gene transfer difficult. To determine the baculovirus transducing ability more rapidly and reliably, we developed a protocol to evaluate the transducing titers of baculovirus stocks. The virus was diluted twofold serially and used to transduce HeLa cells. The resultant transduction efficiencies were measured by flow cytometry for the calculation of transducing titers. Compared to the infectious titer, the determination of transducing titer is more reproducible as the standard deviations among measurements are smaller. Also, the transducing titers can be obtained in 24 h, which is significantly faster as opposed to 4-7 days to obtain the infectious titer. More importantly, we demonstrated that baculoviruses with higher transducing titers could transduce cells at higher efficiency and yield stronger and longer transgene expression, confirming that the transducing titer was representative of the baculovirus transducing ability. This finding is particularly significant for ex vivo gene delivery whereby unconcentrated viruses are used for transduction and long-term transgene expression is desired. In this regard, our titration protocol provides a simple, fast, and reliable measure to evaluate the quality of virus stocks during virus production and purification, and is helpful to predict the performance of vector supernatants and ensure reproducible gene delivery experiments. 相似文献
962.
High-level production of 1,3-propanediol from crude glycerol by <Emphasis Type="Italic">Clostridium butyricum</Emphasis> AKR102a 总被引:1,自引:0,他引:1
Wilkens E Ringel AK Hortig D Willke T Vorlop KD 《Applied microbiology and biotechnology》2012,93(3):1057-1063
The aim of this study was to optimize a biotechnological process for the production of 1,3-propanediol (1,3-PD) based on low-quality
crude glycerol derived from biodiesel production. Clostridium butyricum AKR102a was used in fed-batch fermentations in 1-L and 200-L scale. The newly discovered strain is characterized by rapid
growth, high product tolerance, and the ability to use crude glycerol at the lowest purity directly gained from a biodiesel
plant side stream. Using pure glycerol, the strain AKR102 reached 93.7 g/L 1,3-PD with an overall productivity of 3.3 g/(L*h).
With crude glycerol under the same conditions, 76.2 g/L 1,3-PD was produced with a productivity of 2.3 g/(L*h). These are
among the best results published so far for natural producers. The scale up to 200 L was possible. Due to the simpler process
design, only 61.5 g/L 1,3-PD could be reached with a productivity of 2.1 g/(L*h). 相似文献
963.
Jensen KK Previs SF Zhu L Herath K Wang SP Bhat G Hu G Miller PL McLaren DG Shin MK Vogt TF Wang L Wong KK Roddy TP Johns DG Hubbard BK 《American journal of physiology. Endocrinology and metabolism》2012,302(2):E209-E217
The liver is a crossroad for metabolism of lipid and carbohydrates, with acetyl-CoA serving as an important metabolic intermediate and a precursor for fatty acid and cholesterol biosynthesis pathways. A better understanding of the regulation of these pathways requires an experimental approach that provides both quantitative metabolic flux measurements and mechanistic insight. Under conditions of high carbohydrate availability, excess carbon is converted into free fatty acids and triglyceride for storage, but it is not clear how excessive carbohydrate availability affects cholesterol biosynthesis. To address this, C57BL/6J mice were fed either a low-fat, high-carbohydrate diet or a high-fat, carbohydrate-free diet. At the end of the dietary intervention, the two groups received (2)H(2)O to trace de novo fatty acid and cholesterol synthesis, and livers were collected for gene expression analysis. Expression of lipid and glucose metabolism genes was determined using a custom-designed pathway focused PCR-based gene expression array. The expression analysis showed downregulation of cholesterol biosynthesis genes and upregulation of fatty acid synthesis genes in mice receiving the high-carbohydrate diet compared with the carbohydrate-free diet. In support of these findings, (2)H(2)O tracer data showed that fatty acid synthesis was increased 10-fold and cholesterol synthesis was reduced by 1.6-fold in mice fed the respective diets. In conclusion, by applying gene expression analysis and tracer methodology, we show that fatty acid and cholesterol synthesis are differentially regulated when the carbohydrate intake in mice is altered. 相似文献
964.
Vanesa M. Fernández Andrés Arenas Walter M. Farina 《Journal of comparative physiology. A, Neuroethology, sensory, neural, and behavioral physiology》2009,195(8):759-768
Honeybees of different ages and reproductive castes cohabit in the hive where they are exposed to many odors that might affect
associative learning. Our aim was to analyze the role of odors pre-exposed as volatiles on appetitive learning in honeybees
of different ages and search for their long-term effect both under natural and laboratory conditions. By evaluating memory
acquisition and retention through a differential proboscis extension response conditioning, we found that hive-exposed odors
offered as a reinforced conditioned stimulus during training promoted a learning-reduced effect [latent inhibition (LI)].
On the other hand, no effect was found when the non-reinforced conditioned stimulus was pre-exposed. The LI effect varied
with the odor identity. However, only slight differences were found with the age of the bees. Exposure-conditioning intervals
longer than 24 h did not show an LI effect unless the odor concentration was increased or exposure was prolonged. Our results
show that pre-exposed volatiles could either reduce learning performance, if this odor is later associated with food, or be
irrelevant in the case that alternative scented resources circulate within the colony. The differential effects found according
to the olfactory exposure characteristics could strongly influence the propagation of chemosensory information within the
hive. 相似文献
965.
966.
Natalia A. Kolganova Vadim A. Vasiliskov Viktoriya E. Kuznetsova Valeriy E. Shershov Sergey A. Lapa Timur O. Guseinov Maksim A. Spitsyn Edward N. Timofeev Alexander V. Chudinov 《Molecular biotechnology》2018,60(12):879-886
The transferase activity of non-proofreading DNA polymerases is a well-known phenomenon that has been utilized in cloning and sequencing applications. The non-templated addition of modified nucleotides at DNA blunt ends is a potentially useful feature of DNA polymerases that can be used for selective transformation of DNA 3′ ends. In this paper, we characterized the tailing reaction at perfectly matched and mismatched duplex ends with Cy3- and Cy5-modified pyrimidine nucleotides. It was shown that the best DNA tailing substrate does not have a perfect Watson–Crick base pair at the end. Mismatched duplexes with a 3′ dC were the most efficient in the Taq DNA polymerase-catalysed tailing reaction with a Cy5-modified dUTP. We further demonstrated that the arrangement of the dye residue relative to the nucleobase notably affects the outcome of the tailing reaction. A comparative study of labelled deoxycytidine and deoxyuridine nucleotides showed higher efficiency for dUTP derivatives. The non-templated addition of modified nucleotides by Taq polymerase at a duplex blunt end was generally complicated by the pyrophosphorolysis and 5′ exonuclease activity of the enzyme. 相似文献
967.
The acrosome reaction is a complex, calcium-dependent reaction that results in an exocytotic event required for successful fertilization of the egg. It has long been thought that the acrosome reaction occurs upon sperm binding to the zona pellucida, a viscoelastic layer surrounding the oocyte. Recent studies have suggested that the reaction may even occur before the sperm encounters the zona, perhaps mediated by progesterone or some other agonist. It has been particularly difficult to understand differences between progesterone-induced and zona-induced reactions experimentally and whether one substance is the more biologically relevant trigger. Until this present work, there has been little effort to mathematically model the acrosome reaction in sperm as a whole. Instead, attention has been paid to modeling portions of the pathways involved in other cell types. Here we present a base model for the acrosome reaction which characterizes the known biochemical reactions and behaviors of the system. Our model allows us to analyze several pathways that may act as a stabilizing mechanism for avoiding sustained oscillatory calcium responses often observed in other cell types. Such an oscillatory regime might otherwise prevent acrosomal exocytosis and therefore inhibit fertilization. Results indicate that the acrosome reaction may rely upon multiple redundant mechanisms to avoid entering an oscillatory state and instead maintain a high resting level of calcium, known to be required for successful acrosomal exocytosis and, ultimately, fertilization of the oocyte. 相似文献
968.
969.
Eun Jung Thak Jungho Kim Dong-Jik Lee Jeong Yoon Kim Hyun Ah Kang 《Journal of microbiology (Seoul, Korea)》2018,56(1):11-23
Protein glycosylation, the most universal and diverse post-translational modification, can affect protein secretion, stability, and immunogenicity. The structures of glycans attached to proteins are quite diverse among different organisms and even within yeast species. In yeast, protein glycosylation plays key roles in the quality control of secretory proteins, and particularly in maintaining cell wall integrity. Moreover, in pathogenic yeasts, glycans assembled on cell-surface glycoproteins can mediate their interactions with host cells. Thus, a comprehensive understanding of protein glycosylation in various yeast species and defining glycan structure characteristics can provide useful information for their biotechnological and clinical implications. Yeast-specific glycans are a target for glyco-engineering; implementing human-type glycosylation pathways in yeast can aid the production of recombinant glycoproteins with therapeutic potential. The virulenceassociated glycans of pathogenic yeasts could be exploited as novel targets for antifungal agents. Nowadays, several glycomics techniques facilitate the generation of species-and strain-specific glycome profiles and the delineation of modified glycan structures in mutant and engineered yeast cells. Here, we present the protocols employed in our laboratory to investigate the N-and O-glycan chains released from purified glycoproteins or cell wall mannoproteins in several yeast species. 相似文献
970.
Qingbin Song Ye Wu Jinhui Li Zhishi Wang Danfeng Yu Huabo Duan 《The International Journal of Life Cycle Assessment》2018,23(10):1916-1927