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51.
Background
Nucleic acids based therapeutic approaches have gained significant interest in recent years towards the development of therapeutics against many diseases. Recently, research on aptamers led to the marketing of Macugen®, an inhibitor of vascular endothelial growth factor (VEGF) for the treatment of age related macular degeneration (AMD). Aptamer technology may prove useful as a therapeutic alternative against an array of human maladies. Considering the increased interest in aptamer technology globally that rival antibody mediated therapeutic approaches, a simplified selection, possibly in one-step, technique is required for developing aptamers in limited time period.Principal Findings
Herein, we present a simple one-step selection of DNA aptamers against α-bungarotoxin. A toxin immobilized glass coverslip was subjected to nucleic acid pool binding and extensive washing followed by PCR enrichment of the selected aptamers. One round of selection successfully identified a DNA aptamer sequence with a binding affinity of 7.58 µM.Conclusion
We have demonstrated a one-step method for rapid production of nucleic acid aptamers. Although the reported binding affinity is in the low micromolar range, we believe that this could be further improved by using larger targets, increasing the stringency of selection and also by combining a capillary electrophoresis separation prior to the one-step selection. Furthermore, the method presented here is a user-friendly, cheap and an easy way of deriving an aptamer unlike the time consuming conventional SELEX-based approach. The most important application of this method is that chemically-modified nucleic acid libraries can also be used for aptamer selection as it requires only one enzymatic step. This method could equally be suitable for developing RNA aptamers. 相似文献52.
53.
Alicia Lundby Lasse Steen Ravn Jesper Hastrup Svendsen Stig Hauns S?ren-Peter Olesen Nicole Schmitt 《Cellular physiology and biochemistry》2008,21(1-3):47-54
BACKGROUND: Atrial fibrillation (AF) is the most common cardiac rhythm disorder with a lifetime risk for development of 25% for people aged 40 or older. In this study we aim for the functional assessment of a mutation in KCNE3 identified in a proband with early-onset lone AF. METHODS: Screening of genomic DNA from the proband led to identification of a KCNE3 V17M missense mutation. We heterologously expressed the accessory channel subunit in Xenopus laevis oocytes together with its known interacting potassium channel alpha-subunits. Further, we applied RT-PCR on human total RNA from left and right atria and ventricle. RESULTS: Electrophysiological recordings revealed an increased activity of Kv4.3/KCNE3 and Kv11.1/KCNE3 generated currents by the mutation, thereby conferring susceptibility of mutation carriers to faster cardiac action potential repolarization and thus vulnerability to re-entrant wavelets in the atria and thereby AF. CONCLUSION: Here we report a novel mutation in KCNE3 identified in a proband with early-onset lone AF possibly leading to gain-of-function of several cardiac currents. We suggest abnormalities in the KCNE3 gene as a potential genetic risk factor for initiation and/or maintenance of AF. 相似文献
54.
The number of biotransformation processes is increasing rapidly. Part of this success is based on the inherent properties of enzymes as chemo-, regio-, and enantioselective catalysts. Supercritical fluids (scF) are superior solvents inheriting adjustable and partly unique physical properties. These can be advantageously combined with biotransformations, as solvent power responds to pressure and temperature changes according to the reaction requirements. Among the scF, supercritical carbon dioxide has undoubtedly gained the highest attention. However, other scF are also recognized to enlarge the possibilities. Among these CH(4), C(2)H(6), C(2)H(4), C(3)H(8), CHF(3), and SF(6) are used as scF for biocatalysis. This review focuses on the use of non-CO(2) based scF for biotransformations. Wherever possible, special emphasis is given on the industrial viability of different biocatalytic processes. 相似文献
55.
Co-cultures of neurons and astrocytes were prepared from dissociated embryonic mouse cerebral cortex and cultured for 7 days.
To investigate if these cultures may serve as a functional model system to study neuron-glia interaction with regard to GABA
biosynthesis, the cells were incubated either in media containing [U-13C]glutamine (0.1, 0.3 and 0.5 mM) or 1 mM acetate plus 2.5 mM glucose plus 1 mM lactate. In the latter case one of the 3 substrates
was uniformly 13C labeled. Cellular contents and 13C labeling of glutamate, GABA, aspartate and glutamine were determined in the cells after an incubation period of 2.5 h. The
GABA biosynthetic machinery exhibited the expected complexity with regard to metabolic compartmentation and involvement of
TCA cycle activity as seen in other culture systems containing GABAergic neurons. Metabolism of acetate clearly demonstrated
glial synthesis of glutamine and its transfer to the neuronal compartment. It is concluded that this co-culture system serves
as a reliable model in which functional and pharmacological aspects of GABA biosynthesis can be investigated.
Special issue article in honor of Dr. Anna Maria Giuffrida-Stella.
An erratum to this article can be found at 相似文献
56.
Emil D Jensen Marcos Laloux Beata J Lehka Lasse E Pedersen Tadas Jako
inas Michael
K Jensen Jay
D Keasling 《Nucleic acids research》2021,49(15):e88
Laboratory evolution is a powerful approach to search for genetic adaptations to new or improved phenotypes, yet either relies on labour-intensive human-guided iterative rounds of mutagenesis and selection, or prolonged adaptation regimes based on naturally evolving cell populations. Here we present CRISPR- and RNA-assisted in vivo directed evolution (CRAIDE) of genomic loci using evolving chimeric donor gRNAs continuously delivered from an error-prone T7 RNA polymerase, and directly introduced as RNA repair donors into genomic targets under either Cas9 or dCas9 guidance. We validate CRAIDE by evolving novel functional variants of an auxotrophic marker gene, and by conferring resistance to a toxic amino acid analogue in baker''s yeast Saccharomyces cerevisiae with a mutation rate >3,000-fold higher compared to spontaneous native rate, thus enabling the first demonstrations of in vivo delivery and information transfer from long evolving RNA donor templates into genomic context without the use of in vitro supplied and pre-programmed repair donors. 相似文献
57.
Mertz Christoffer Krarup Sara Jensen Cecilie D. Lindholm Sandy E. H. Kjær Christina Pinborg Lars H. Bak Lasse K. 《Neurochemical research》2020,45(6):1247-1255
Neurochemical Research - Epilepsy is one of the most common chronic neurological conditions. Today, close to 30 different medications to prevent epileptic seizures are in use; yet, far from all... 相似文献
58.
Ruth-Anne Sandaa Laura Gómez-Consarnau Jarone Pinhassi Lasse Riemann rea Malits Markus G. Weinbauer Josep M. Gasol T. Frede Thingstad 《Environmental microbiology》2009,11(10):2585-2597
We demonstrate here results showing that bottom-up and top-down control mechanisms can operate simultaneously and in concert in marine microbial food webs, controlling prokaryote diversity by a combination of viral lysis and substrate limitation. Models in microbial ecology predict that a shift in the type of bacterial growth rate limitation is expected to have a major effect on species composition within the community of bacterial hosts, with a subsequent shift in the composition of the viral community. Only moderate effects would, however, be expected in the absolute number of coexisting virus–host pairs. We investigated these relationships in nutrient-manipulated systems, under simulated in situ conditions. There was a strong correlation in the clustering of the viral and bacterial community data supporting the existence of an important link between the bacterial and viral communities. As predicted, the total number of viral populations was the same in all treatments, while the composition of the viral community varied. Our results support the theoretical prediction that there is one control mechanism for the number of niches for coexisting virus–host pairs (top-down control), and another mechanism that controls which virus–host pairs occupy these niches (bottom-up control). 相似文献
59.
Temperature responses of photosynthetic capacity parameters were not affected by foliar nitrogen content in mature Pinus sylvestris
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Lasse Tarvainen Martina Lutz Mats Räntfors Torgny Näsholm Göran Wallin 《Physiologia plantarum》2018,162(3):370-378
A key weakness in current Earth System Models is the representation of thermal acclimation of photosynthesis in response to changes in growth temperatures. Previous studies in boreal and temperate ecosystems have shown leaf‐scale photosynthetic capacity parameters, the maximum rates of carboxylation (Vcmax) and electron transport (Jmax), to be positively correlated with foliar nitrogen (N) content at a given reference temperature. It is also known that Vcmax and Jmax exhibit temperature optima that are affected by various environmental factors and, further, that N partitioning among the foliar photosynthetic pools is affected by N availability. However, despite the strong recent anthropogenic influence on atmospheric temperatures and N deposition to forests, little is known about the role of foliar N contents in controlling the photosynthetic temperature responses. In this study, we investigated the temperature dependencies of Vcmax and Jmax in 1‐year‐old needles of mature boreal Pinus sylvestris (Scots pine) trees growing under low and high N availabilities in northern Sweden. We found that needle N status did not significantly affect the temperature responses of Vcmax or Jmax when the responses were fitted to a peaked function. If such N insensitivity is a common tree trait it will simplify the interpretation of the results from gradient and multi‐species studies, which commonly use sites with differing N availabilities, on temperature acclimation of photosynthetic capacity. Moreover, it will simplify modeling efforts aimed at understanding future carbon uptake by precluding the need to adjust the shape of the temperature response curves to variation in N availability. 相似文献
60.
Bacillus thuringiensis in Fecal Samples from Greenhouse Workers after Exposure to B. thuringiensis-Based Pesticides
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Gert B. Jensen Preben Larsen Bodil L. Jacobsen Bodil Madsen Lasse Smidt Lars Andrup 《Applied microbiology》2002,68(10):4900-4905
In a study of occupational exposure to Bacillus thuringiensis, 20 exposed greenhouse workers were examined for Bacillus cereus-like bacteria in fecal samples and on biomonitoring filters. Bacteria with the following characteristics were isolated from eight individuals: intracellular crystalline inclusions characteristic of B. thuringiensis, genes for and production of B. cereus enterotoxins, and positivity for cry11 as determined by PCR. DNA fingerprints of the fecal isolates were identical to those of strains isolated from the commercial products used. Work processes (i.e., spraying) correlated with the presence of B. thuringiensis in the fecal samples (102 to 103 CFU/g of feces). However, no gastrointestinal symptoms correlated with the presence of B. thuringiensis in the fecal samples. 相似文献