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1.
Optimization of 5-(and-6)-Carboxyfluorescein Diacetate Succinimidyl Ester for Labeling Human Intervertebral Disc Cells in Vitro 总被引:4,自引:0,他引:4
Helen E. Gruber Kelly P. Leslie Jane A. Ingram Edward N. Hanley jr. 《Biotechnic & histochemistry》2000,75(3):118-123
We have assessed the utility of an intracellular fluorochrome, 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE), as a tracking label for human intervertebral disc cells in vitro. Although 5 JJIM provides adequate intracellular labeling for whole cell fluorescent microscopic identification of labeled cells, 20 JJLM was preferable for immunocytochemical localization of paraffin embedded labeled cells. Electron dense vesicles are seen at the ultra-structural level in labeled cells. Discrete vesicular labeling can also be observed in whole cell mounts viewed with fluorescence microscopy. Whole cells retain good label for 6 weeks. CFSE labeling is relatively easy, nontoxic to cells and nonradiocactive. Initial optimization of dose with specific cells types is recommended when confirmation of positive immunocytochemistry is needed for tissue engineering studies. 相似文献
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《Biotechnic & histochemistry》2013,88(3):118-123
We have assessed the utility of an intracellular fluorochrome, 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE), as a tracking label for human intervertebral disc cells in vitro. Although 5 JJIM provides adequate intracellular labeling for whole cell fluorescent microscopic identification of labeled cells, 20 JJLM was preferable for immunocytochemical localization of paraffin embedded labeled cells. Electron dense vesicles are seen at the ultra-structural level in labeled cells. Discrete vesicular labeling can also be observed in whole cell mounts viewed with fluorescence microscopy. Whole cells retain good label for 6 weeks. CFSE labeling is relatively easy, nontoxic to cells and nonradiocactive. Initial optimization of dose with specific cells types is recommended when confirmation of positive immunocytochemistry is needed for tissue engineering studies. 相似文献
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Efremenko A. V. Dyakova E. D. Ostroverkhov P. V. Kirin N. S. Mironov A. F. Grin M. A. Feofanov A. V. 《Russian Journal of Bioorganic Chemistry》2021,47(4):845-853
Russian Journal of Bioorganic Chemistry - It has been found that the photosensitizer 131-[2-(guanidinyl)ethylamino] chlorin e6 dimethyl ester accumulates predominantly in lysosomes, partially in... 相似文献
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The 5- (and 6-)carboxyfluorescein succinimidyl ester (cFDASE) is a pH-dependent fluorescent probe which conjugates with the aliphatic amine functions of the cells. Because of this property, the fluorescence efflux rate is low, 10 to 40% after 30 min of fermentable carbon source incubation. Thus, this technique allows a continuous high-time measurement (>30 min) without correction of the signal, but also a dynamic study phenomenon of few seconds. Due to the advantages presented by this technique, the method was adapted for Escherichia coli. The internal pH measurement obtained under various conditions are in accuracy with those from radiolabelled technique and from other fluorescence probe, with the advantages mentioned previously. 相似文献
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Bromidge SM Griffith K Heightman TD Jennings A King FD Moss SF Newman H Riley G Routledge C Serafinowska HT Thomas DR 《Bioorganic & medicinal chemistry letters》2001,11(21):2843-2846
The discovery of (4-piperazin-1-ylquinolin-6-yl) arylsulfonamides and their binding affinities for a selection of 5-HT and dopamine subreceptors is described. Many compounds show high affinity (pK(i)>8) for the 5-HT(6) receptor and >100-fold selectivity against a range of other receptors. Structure-activity relationships of these compounds are discussed. 相似文献
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Intracellular pH shifts in cultured kidney (A6) cells: effects on apical Na+ transport 总被引:1,自引:0,他引:1
Zeiske Wolfgang; Smets Ilse; Ameloot Marcel; Steels Paul; van Driessche Willy 《American journal of physiology. Cell physiology》1999,277(3):C469
We report, for the epithelialNa+ channel (ENaC) in A6 cells,the modulation by cell pH (pHc)of the transepithelial Na+ current(INa), thecurrent through the individual Na+channel (i), the openNa+ channel density(No), and thekinetic parameters of the relationship betweenINa and theapical Na+ concentration. Thei andNo were evaluatedfrom the Lorentzian INa noise inducedby the apical Na+ channel blocker6-chloro-3,5-diaminopyrazine-2-carboxamide.pHc shifts were induced, understrict and volume-controlled experimental conditions, byapical/basolateral NH4Cl pulses orbasolateral arrest of theNa+/H+exchanger (Na+ removal; block byethylisopropylamiloride) and were measured with the pH-sensitive probe2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Thechanges in pHc were positivelycorrelated to changes inINa and theapically dominated transepithelial conductance. The sole pHc-sensitive parameter underlyingINa wasNo. Only thesaturation value of theINa kinetics wassubject to changes in pHc.pHc-dependent changes inNo may be causedby influencingPo, the ENaC openprobability, or/and the total channel number,NT = No/Po. 相似文献
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Isabelle Rasolonjatovo Simon R. Sarfati 《Nucleosides, nucleotides & nucleic acids》2013,32(9-11):2021-2025
Abstract 6-Amino-4-oxo-hexanoic acid with a fluorescent probe attached to the amino function, derivative of the levulinic acid has been developed for protection of hydroxyl groups. It is introduced by reaction of its symetrical anhydride and rapidly removed under mild conditions using a hydrazine-pyridinium acetate buffer at near neutral pH and room temperature. It can be used within the scope of a new DNA sequencing method and as a sensitive detectable protecting group. 相似文献
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Determination of the Number of Respiring Thiobacillus ferrooxidans Cells in Water Samples by Using Combined Fluorescent Antibody-2-(p-Iodophenyl)-3-(p-Nitrophenyl)-5-Phenyltetrazolium Chloride Staining 总被引:9,自引:7,他引:2
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Fluorescent antibody staining was combined with 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride reduction in a procedure termed FAINT to allow for the direct microscopic determination of specific actively respiring populations of bacteria in a variety of aquatic habitats. The FAINT procedure is simple, precise, and appropriate for use in a wide variety of autecological studies. The distribution of Thiobacillus ferrooxidans was examined by FAINT enumerations in both acidic and nonacidic sites. Comparisons among the FAINT technique and fluorescent antibody staining alone or most-probable-number determinations in 9K broth showed that the use of most-probable-number determinations resulted in an underestimation of the number of viable cells by one to three orders of magnitude, whereas fluorescent antibody counts resulted in an overestimation of the number of viable T. ferrooxidans. The amount of difference was not consistent but varied, depending on the sample site. 相似文献
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M. Stępińska O. Olszewska-Sosińska M. Lau-Dworak B. Zielnik-Jurkiewicz E. A. Trafny 《Current microbiology》2014,68(1):21-29
Monocyte/macrophage cells from human nasopharyngeal lymphoid tissue can be a source of bacteria responsible for human chronic and recurrent upper respiratory tract infection. Detection and characterization of pathogens surviving intracellularly could be a key element in bacteriological diagnosis of the infections as well as in the study on interactions between bacteria and their host. The present study was undertaken to assess the possibility of isolation of viable bacteria from the cells expressing monocyte/macrophage marker CD14 in nasopharyngeal lymphoid tissue. Overall, 74 adenotonsillectomy specimens (adenoids and tonsils) from 37 children with adenoid hypertrophy and recurrent infections as well as 15 specimens from nine children with adenoid hypertrophy, which do not suffer from upper respiratory tract infections (the control group), were studied. The suitability of immunomagnetic separation for extraction of CD14+ cells from lymphoid tissue and for further isolation of the intracellular pathogens has been shown. The coexistence of living pathogens including Haemophilus influenzae, Staphylococcus aureus, and Streptococcus pyogenes with the bacteria representing normal nasopharyngeal microbiota inside CD14+ cells was demonstrated. Twenty-four strains of these pathogens from 32.4 % of the lysates of CD14+ cells were isolated. Concurrently, the fluorescent in situ hybridization (FISH) with a universal EUB388, and the species-specific probes demonstrated twice more often the persistence of these bacterial species in the lysates of CD14+ cells than conventional culture. Although the FISH technique appears to be more sensitive than traditional culture in the intracellular bacteria identification, the doubts on whether the bacteria are alive, and therefore, pathogenic would still exist without the strain cultivation. 相似文献
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寡核苷酸探针(CAC)5/(GTG) 5指纹的非放射性标记和检测 总被引:1,自引:0,他引:1
蓝 翎张小为 霍振义段秉章 史秀芬LAN Ling ZHANG Xiao-Wei HOU Zhen-Yi DUAN Bing-Zhang SHI Xiu-Fen 《遗传》1994,16(3):33-34
在DNA指纹分析中,通常采用的是同位素32P标记DNA分子探针,可检测到微微克(pg)靶序列。但32P半衰期很短(仅有14天),使用和运输均不方便,32P还放出硬射线,对实验者身体造成损害,需要一定的防护措施,并且容易污染环境。因此,科学家们研制出了... 相似文献
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Hasina Yasmin Mohammed Shafikur Rahman Takayuki Shibata Tsutomu Kabashima Masaaki Kai 《International journal of peptide research and therapeutics》2014,20(4):441-446
Fluorescence (FL) derivatization reactions have often been used for the selective determination of bioactive peptides. Herein, a sensitive and selective fluorometric method has been developed for Pro-Gly and Pro-Gly-Pro using a derivatizing reagent 3,4-dihydroxybenzoic acid (3,4-DHBA). In the presence of borate buffer (pH 8.0) and sodium periodate, peptides were reacted with 3,4-DHBA at 37 °C for 30 min. The resulting FL intensity was measured by spectrofluorometer with the excitation wavelength of 450 nm and the emission wavelength of 535 nm. Different reaction conditions such as concentrations of the reagents, reaction time and pH were optimized to develop the method. Under the optimized conditions, a linear relationship was obtained between FL intensity and peptide concentration from 5–30 µM with a lower detection limit of 5 µM. We found that 3,4-DHBA showed strong preference for Pro-Gly and Pro-Gly-Pro amongst all the peptides tested and no other biogenic substances such as amino acids or proteins produced any FL. The reaction is selective, sensitive and simple which can be applied for the determination of peptides as biomarkers in biological samples or for the assay of various protease activities. 相似文献
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Ichiro Tomida Shin’ichi Senda Takahisa Kuwabara Kaoru Katayama 《Bioscience, biotechnology, and biochemistry》2013,77(10):2033-2036
It was observed that the dl-dipeptide derivative was formed predominantly over the ll-compound, only when l-Pro-OR was allowed to react as amino-component to the pseudooxazolone-(5), in contrast to the other l-amino acid esters. From the observation of the influence of the solvent, the added base and H-Gly-OMe, some of the mechanism in this reaction was discussed. The preparative isolation of the dl-compound from the reaction product, the synthesis of Tfp-l-Ileu-OH and the corresponding pseudooxazolone-(5) compound were also described. 相似文献
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The 5-position of tetrahydrofolate was found to be unusually reactive with low concentrations of formic acid in the presence of a water-soluble carbodiimide. The product of this reaction has neutral and acid ultraviolet spectra and chromatographic behavior consistent with its identity as 5-formyltetrahydrofolate (leucovorin). When enzymatically synthesized (6S)-tetrahydrofolate was used as starting material, the product supported the growth of folatedepleted L1210 cells at one-half the concentration required for authentic (6R,S)-leucovorin. This reaction has been used to produce high specific activity (44 Ci/mmol) [3H](6S)-5-formyltetrahydrofolate in high yield. Experiments with [14C]formic acid indicate that 1 mol of formate reacted per mol of tetrahydrofolate but that no reaction occurred with a variety of other folate compounds. (6S)-5-Formyltetrahydrofolate, labeled in the formyl group with 14C, has also been synthesized using this reaction. These easily produced, labeled folates should allow close examination of the transport and utillization of leucovorin and of the mechanism of reversal of methotrexate toxicity by reduced folate cofactors. 相似文献
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Matthew R. Sapio Monique Vessaz Pierre Thomas Pierre Genton Lloyd D. Fricker Annick Salzmann 《PloS one》2015,10(4)
Carboxypeptidase A6 (CPA6) is a peptidase that removes C-terminal hydrophobic amino acids from peptides and proteins. The CPA6 gene is expressed in the brains of humans and animals, with high levels of expression during development. It is translated with a prodomain (as proCPA6), which is removed before secretion. The active form of CPA6 binds tightly to the extracellular matrix (ECM) where it is thought to function in the processing of peptides and proteins. Mutations in the CPA6 gene have been identified in patients with temporal lobe epilepsy and febrile seizures. In the present study, we screened for CPA6 mutations in patients with juvenile myoclonic epilepsy and identified two novel missense mutations: Arg36His and Asn271Ser. Patients harboring these mutations also presented with generalized epilepsy. Neither of the novel mutations was found in a control population. Asn271 is highly conserved in CPA6 and other related metallocarboxypeptidases. Arg36 is present in the prodomain and is not highly conserved. To assess structural consequences of the amino acid substitutions, both mutants were modeled within the predicted structure of the enzyme. To examine the effects of these mutations on enzyme expression and activity, we expressed the mutated enzymes in human embryonic kidney 293T cells. These analyses revealed that Asn271Ser abolished enzymatic activity, while Arg36His led to a ~50% reduction in CPA6 levels in the ECM. Pulse-chase using radio-labeled amino acids was performed to follow secretion. Newly-synthesized CPA6 appeared in the ECM with peak levels between 2-8 hours. There was no major difference in time course between wild-type and mutant forms, although the amount of radiolabeled CPA6 in the ECM was lower for the mutants. Our experiments demonstrate that these mutations in CPA6 are deleterious and provide further evidence for the involvement of CPA6 mutations in the predisposition for several types of epilepsy. 相似文献
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Thimma R. Reddy Emma J. Kelsall Léna M.S. Fevat Sarah E. Munson Shaun M. Cowley 《Journal of visualized experiments : JoVE》2015,(95)
Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic mouse models using this method necessitates the construction of a ‘targeting’ vector, which contains homologous DNA sequences to the target gene, and has for many years been a limiting step in the process. Vector construction can be performed in vivo in Escherichia coli cells using homologous recombination mediated by phage recombinases using a technique termed recombineering. Recombineering is the preferred technique to subclone the long homology sequences (>4kb) and various targeting elements including selection markers that are required to mediate efficient allelic exchange between a targeting vector and its cognate genomic locus. Typical recombineering protocols follow an iterative scheme of step-wise integration of the targeting elements and require intermediate purification and transformation steps. Here, we present a novel recombineering methodology of vector assembly using a multiplex approach. Plasmid gap repair is performed by the simultaneous capture of genomic sequence from mouse Bacterial Artificial Chromosome libraries and the insertion of dual bacterial and mammalian selection markers. This subcloning plus insertion method is highly efficient and yields a majority of correct recombinants. We present data for the construction of different types of conditional gene knockout, or knock-in, vectors and BAC reporter vectors that have been constructed using this method. SPI vector construction greatly extends the repertoire of the recombineering toolbox and provides a simple, rapid and cost-effective method of constructing these highly complex vectors. 相似文献
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We report on a new method for the determination of lipid oxidation in lipoproteins and plasma. The biological lipid system is preloaded with a fluorescent analog of phosphatidylcholine containing diphenylhexatriene (DPH) propionic acid covalently linked to the sn-2 position. When externally added, the respective phospholipid label (DPHPC) localizes to the surface monolayer of a lipoprotein. Under oxidative conditions (e.g. in the presence of Cu2+ ions) the fluorophore undergoes decomposition, resulting in a continuous decrease of fluorescence intensity which reflects the oxidation of a chemically defined phospholipid molecule with well defined localization. When incorporated into LDL particles, the kinetics of the decrease in DPHPC fluorescence intensity upon exposure to Cu2+ is very similar to that of conjugated diene accumulation. Furthermore, our assay can be applied to follow the oxidation of lipids in diluted serum and may also be developed into a suitable test system for clinical studies of susceptibility of plasma lipids to oxidation. 相似文献
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