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31.
32.
Constance Vind Niels Grunnet 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1982,720(3):295-302
This paper describes the transfer of tritium from [2-3H]xylitol or (1R)-[1-3H]ethanol into lactate in cells from fed rats either untreated or triiodothyronine-treated. The labelling pattern of lactate during the metabolism of [2-3H]xylitol or (1R)-[1-3H]ethanol follows the equation (μmol tritium/μmol lactate). The yield in lactate together with the minimum value of the total flux of reducing equivalents are used to estimate the specific radioactivity of NADH. We have calculated the lactate dehydrogenase-catalysed oxidation rate of NADH from the experimental values of lactate labelling and the specific radioactivity of NADH. We found the calculated flux of reducing equivalents from NADH to pyruvate to be of the same order of magnitude whether labelled ethanol or labelled xylitol was metabolized. We found the flux to be only a few percent of the maximal activity of lactate dehydrogenase. The results obtained suggest that the cytoplasm can be regarded as one compartment, containing a single pool of NAD(H). 相似文献
33.
Steen Pedersen Solveig V. Reeh Jack Parker Robert J. Watson James D. Friesen Niels P. Fiil 《Molecular & general genetics : MGG》1976,144(3):339-343
Summary The presence of EF-Tu, RNA polymerase subunit , and EF-G on the dfus-3 genome and EF-Tu, ribosomal proteins L7/L12, and RNA polymerase subunit on the drif
d
18 genome has been confirmed using a two-dimensional gel electrophoresis technique sensitive to changes in isoelectric point and molecular weight. In this system two EF-Tu gene products could not be resolved. Following infection of ultraviolet light-irradiated Escherichia coli with either dfus-3 or drif
d
18, the EF-Tu gene, tufA, near 65 minutes on the genetic map is expressed as 3–4 copies per EF-G molecule. The EF-Tu gene, tufB, near 79 minutes on the genetic map, is expressed at about one-third of this rate. is expressed as 1 copy per EF-G molecule, as 0.14 per EF-G molecule and L7/L12 as 2.5 per EF-G. These figures compare well with the relative amounts found in exponentially-growing cells, in which the ratio of EF-Tu to EF-G is approximately 5. Almost 90% of the total number of proteins (calculated on a molecular weight basis) which theoretically can be encoded on the drif
d
18 have been identified on the two-dimensional gel. 相似文献
34.
CYTODYNAMICS IN THE THYMUS OF YOUNG ADULT MICE: 总被引:1,自引:0,他引:1
Cell proliferation and cell loss in the thymic blast cell population were studied in young adult mice by (1) stathmokinetic methods combined with an analysis of the PLMe-curve after a pulse 3H-TdR, and (2) nigrosin-dye exclusion combined with 3H-TdR-autoradiography. It was calculated that about 17% of the blast cells do not progress into mitosis within the period of an average cell cycle. The dye exclusion studies indicated a rate of blast cell death of about 2–5 %/hr. The two methods of assessing blast cell loss (death) support each other very well. In spite of these findings scintillation countings on thymuses removed from 1 to 17 hr after 3H-TdR injection showed fairly constant levels of thymic radioactivity. This suggests a very extensive reutilization of 3H-labelled break-down products from dying blast cells. The very sparse labelling of pyknotic thymocytes strongly suggests that thymic blast cells do not become pyknotic. The rate of small thymocyte production and disappearance was studied by pulse and repeated 3H-TdR labelling techniques combined with dye exclusion studies and pyknotic counts. The data from the repeated labelling experiment were analysed by use of a model based on the assumption of first order kinetics of small viable, dead, and pyknotic thymocytes. The rate of cell production was estimated to 1–6 %/hr whereas the rates of cell loss due to disintegration, i.e. supravital stainability and nuclear pyknosis, were calculated to 0–02 %/hr and 0–0006 %/hr respectively. Cell loss due to disintegration was less than 2 % of the total loss of small thymocytes. It was concluded that pyknotic counts are a useless method of assessing the cell death in the population of thymic blast cells and small thymocytes. On the basis of a model for thymocyte proliferation, production and loss it is suggested that about 45 % of the small viable thymocytes re-enter the generative cell pool, whereas about 55% disappear by emigration. 相似文献
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CRISPR/Cas12a is a single effector nuclease that, like CRISPR/Cas9, has been harnessed for genome editing based on its ability to generate targeted DNA double strand breaks (DSBs). Unlike the blunt-ended DSB generated by Cas9, Cas12a generates sticky-ended DSB that could potentially aid precise genome editing, but this unique feature has thus far been underutilized. In the current study, we found that a short double-stranded DNA (dsDNA) repair template containing a sticky end that matched one of the Cas12a-generated DSB ends and a homologous arm sharing homology with the genomic region adjacent to the other end of the DSB enabled precise repair of the DSB and introduced a desired nucleotide substitution. We termed this strategy ‘Ligation-Assisted Homologous Recombination’ (LAHR). Compared to the single-stranded oligo deoxyribonucleotide (ssODN)-mediated homology directed repair (HDR), LAHR yields relatively high editing efficiency as demonstrated for both a reporter gene and endogenous genes. We found that both HDR and microhomology-mediated end joining (MMEJ) mechanisms are involved in the LAHR process. Our LAHR genome editing strategy, extends the repertoire of genome editing technologies and provides a broader understanding of the type and role of DNA repair mechanisms involved in genome editing. 相似文献
38.
Use of Tetrahymena thermophila To Study the Role of Protozoa in Inactivation of Viruses in Water 总被引:1,自引:0,他引:1 下载免费PDF全文
Marcel D. O. Pinheiro Mary E. Power Barbara J. Butler Vivian R. Dayeh Robin Slawson Lucy E. J. Lee Denis H. Lynn Niels C. Bols 《Applied microbiology》2007,73(2):643-649
The ability of a ciliate to inactivate bacteriophage was studied because these viruses are known to influence the size and diversity of bacterial populations, which affect nutrient cycling in natural waters and effluent quality in sewage treatment, and because ciliates are ubiquitous in aquatic environments, including sewage treatment plants. Tetrahymena thermophila was used as a representative ciliate; T4 was used as a model bacteriophage. The T4 titer was monitored on Escherichia coli B in a double-agar overlay assay. T4 and the ciliate were incubated together under different conditions and for various times, after which the mixture was centrifuged through a step gradient, producing a top layer free of ciliates. The T4 titer in this layer decreased as coincubation time increased, but no decrease was seen if phage were incubated with formalin-fixed Tetrahymena. The T4 titer associated with the pellet of living ciliates was very low, suggesting that removal of the phage by Tetrahymena inactivated T4. When Tetrahymena cells were incubated with SYBR gold-labeled phage, fluorescence was localized in structures that had the shape and position of food vacuoles. Incubation of the phage and ciliate with cytochalasin B or at 4°C impaired T4 inactivation. These results suggest the active removal of T4 bacteriophage from fluid by macropinocytosis, followed by digestion in food vacuoles. Such ciliate virophagy may be a mechanism occurring in natural waters and sewage treatment, and the methods described here could be used to study the factors influencing inactivation and possibly water quality. 相似文献
39.
Cremer C Kaufmann R Gunkel M Pres S Weiland Y Müller P Ruckelshausen T Lemmer P Geiger F Degenhard S Wege C Lemmermann NA Holtappels R Strickfaden H Hausmann M 《Biotechnology journal》2011,6(9):1037-1051
For the improved understanding of biological systems on the nanoscale, it is necessary to enhance the resolution of light microscopy in the visible wavelength range beyond the limits of conventional epifluorescence microscopy (optical resolution of about 200 nm laterally, 600 nm axially). Recently, various far-field methods have been developed allowing a substantial increase of resolution ("superresolution microscopy", or "lightoptical nanoscopy"). This opens an avenue to 'nano-image' intact and even living cells, as well as other biostructures like viruses, down to the molecular detail. Thus, it is possible to combine light optical spatial nanoscale information with ultrastructure analyses and the molecular interaction information provided by molecular cell biology. In this review, we describe the principles of spectrally assigned localization microscopy (SALM) of biological nanostructures, focusing on a special SALM approach, spectral precision distance/position determination microscopy (SPDM) with physically modified fluorochromes (SPDM(Phymod) . Generally, this SPDM method is based on high-precision localization of fluorescent molecules, which can be discriminated using reversibly bleached states of the fluorophores for their optical isolation. A variety of application examples is presented, ranging from superresolution microscopy of membrane and cytoplasmic protein distribution to dual-color SPDM of nuclear proteins. At present, we can achieve an optical resolution of cellular structures down to the 20-nm range, with best values around 5 nm (~1/100 of the exciting wavelength). 相似文献
40.
The antimicrobial peptide LL-37 activates innate immunity at the airway epithelial surface by transactivation of the epidermal growth factor receptor 总被引:14,自引:0,他引:14
Tjabringa GS Aarbiou J Ninaber DK Drijfhout JW Sørensen OE Borregaard N Rabe KF Hiemstra PS 《Journal of immunology (Baltimore, Md. : 1950)》2003,171(12):6690-6696
Antimicrobial peptides produced by epithelial cells and neutrophils represent essential elements of innate immunity, and include the defensin and cathelicidin family of antimicrobial polypeptides. The human cathelicidin cationic antimicrobial protein-18 is an antimicrobial peptide precursor predominantly expressed in neutrophils, and its active peptide LL-37 is released from the precursor through the action of neutrophil serine proteinases. LL-37 has been shown to display antimicrobial activity against a broad spectrum of microorganisms, to neutralize LPS bioactivity, and to chemoattract neutrophils, monocytes, mast cells, and T cells. In this study we show that LL-37 activates airway epithelial cells as demonstrated by activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) and increased release of IL-8. Epithelial cell activation was inhibited by the MAPK/ERK kinase (MEK) inhibitors PD98059 and U0126, by the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor AG1478, by blocking anti-EGFR and anti-EGFR-ligand Abs, and by the metalloproteinase inhibitor GM6001. These data suggest that LL-37 transactivates the EGFR via metalloproteinase-mediated cleavage of membrane-anchored EGFR-ligands. LL-37 may thus constitute one of the mediators by which neutrophils regulate epithelial cell activity in the lung. 相似文献