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61.
Comparison of published methods for the quantification of adherent cell numbers by the measurement of absorbance of bound stain indicates a wide variation in their sensitivity. This study aimed at comparing the sensitivities of five different staining procedures (Coomassie brilliant blue G in perchloric acid, Coomassie brilliant blue G in phosphoric acid, methylene blue, crystal violet, and toluidine blue) applied to three separate types of cultured fibroblasts (3T3 cells, Vero cells, and human gingival fibroblasts) at concentrations from 0.125 x 10(4) to 10 x 10(4) per well in 96-well microplates. Absorbance values of Coomassie blue-stained cells were measured in situ. Those of the remaining cells were measured after solubilization of the dye with 1% sodium dodecyl sulfate. All absorbance values were measured using an Elisa reader at 620 or 570 nm for crystal violet. The relationship between cell number and absorbance over the entire cell concentration range was best fitted with quadratic regression analysis, in contrast with the linear relationship described elsewhere. The order of sensitivity of the staining procedures was the same for each cell type: Coomassie blue in perchloric acid less than Coomassie blue in phosphoric acid less than methylene blue less than crystal violet less than toluidine blue. With the latter two stains absorbance values began to plateau at approximately 8 x 10(4) cells per well. However, staining with Coomassie blue in perchloric acid and methylene blue resulted in an almost linear relationship between cell number and absorbance over the entire concentration range tested.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
62.
A novel, automated method is described for the determination of Nτ-methylhistidine in human urine. The method uses a modification (H. Nakamura and J. J. Pisano (1976) Arch. Biochem. Biophys.177, 334–335) of the reaction of fluorescamine with amines, which renders it specific for certain imidazoles. Interference due to histidine and histamine is selectively removed by prior reaction with aldehydes. The fluorescence yield for Nτ-methylhistamine is 280, 440, 50, and 1.7, respectively. The concentrations of Nτ-methylhistidine in human urine as determined by this technique correlate well (r = 0.99) with those determined by ion-exchange chromatography. Furthermore, the technique is rapid (6–7 samples/h net throughout), is reproducible (coefficient of variation 1.8%), requires no prior treatment of the sample, and is implemented with widely available equipment. 相似文献
63.
The mechanism of action of Cu2+ when applied to the external side of the frog skin preparation was investigated. Cu2+ acts most probably on the external barrier of this preparation, since it increases the transport pool of Na+ proportionally to the increase in the short circuit current (Isc). Cu2+ does not open new routes for the Na+ entry since the stimulated Isc is still completely abolished by amiloride. The Isc dependence of Na+ concentration in the external medium is modified by copper, since the Km value increases in addition to changes in V. It is suggested that copper acts at the external barrier Na channels in a way similar to that proposed by Zeiske and Lindemann ((1974) Biochim. Biophys. Acta 352, 323--326) for benzoylimidazole-2 guanidine and benzoylthiazole-2 guanidine and by Dick and Lindemann ((1975) Pflügers Arch. ges. Physiol. 355, R72) for para-chloromercuribenzenosulfonate and para-chloromercuribenzoate. 相似文献
64.
- 1. 1. Cu2+ at a concentration of 10−4 M, when applied to the external side of the frog skin produces an increase in the short-circuit current (Isc).
- 2. 2. This effect was studied in skins of Rana temporaria adapted to cold (5°C) and room temperature (20°C), skins of Rana pipiens adapted to cold, and the results compared with those obtained previously with Rana ribibunda.
- 3. 3. The observed effect is less dependent upon the adaptation to cold than upon the functional state of the skin: skins with low short circuit currents have a bigger response to Cu2+ than skins with high Isc.
- 4. 4. A species difference cannot be ruled out since skins of Rana ribibunda exhibiting high Isc give good responses to Cu2+.
- 5. 5. 5,5′-dithiobis(2-nitrobenzoic acid), a sulphydryl-oxidizing reagent, produces an effect similar to that of Cu2+, and dithiothreitol an SH-reducing agent, reverses the effect of this ion.
- 6. 6. Cu2+ also induces an increase in the unidirectional K+ fluxes and unmasks a net outward potassium flux.
- 7. 7. The outward K+ flux induced by Cu2+ is sensitive to ouabain.
- 8. 8. It is concluded that Cu2+ increases the permeability of the external barrier of the frog skin to Na+ and K+, probably by reacting with SH groups.
Abbreviations: DTNB; 5; 5′-dithiobis(2-nitrobenzoic acid) 相似文献
65.
An indirect antiglobulin immunoperoxidase test was developed for the serological diagnosis of American Trypanosomiasis. Purified rabbit antihuman IgG was labeled with the enzyme and the conjugate so obtained was characterized according to its immune and enzymatic activities, with the help of such parameters as the authors have recently described. For a maximal sensitivity in the tests, high antibody levels and a high-labeling ratio were chosen, as well as dilutions of conjugate ensuring maximal reactivity. Positive tests were found in all 90 serum samples from patients with Chagas' disease and titers did not differ significantly from those observed in immunofluorescence tests done in parallel. The specificity of both tests was also similar, as indicated by the results found for serum samples from 60 patients with other diseases, parasitic or not, showing high levels of antibodies against other infective agents or autoantibodies, and in 15 normals. 相似文献
66.
M Goossens M C Garel J Auvinet O Basset P Ferreira Gomes J Rosa N Arous 《FEBS letters》1975,58(1):149-154
67.
Jayr de Paiva Campello Sebastião Ferreira Fonseca Ching-Jer Chang Ernest Wenkert 《Phytochemistry》1975,14(1):243-248
Sitosterol and the following terpenic compounds have been isolated from the bark of Podocarpus lambertius: 3β-hydroxytotarol, 4β-carboxynortotarol, and macrophyllic and lambertic acids. The leaves yielded sitosterol, stigmastan-3β,5α-diol-6-one, isopimaric acid, phyllocladene, isophyllocladene, 8,9-abieten-15-ol and 17-isophyllocladenol. 相似文献
68.
Tomas Urbaitis Giedrius Gasiunas Joshua K Young Zhenglin Hou Sushmitha Paulraj Egle Godliauskaite Mantvyda M Juskeviciene Migle Stitilyte Monika Jasnauskaite Megumu Mabuchi G Brett Robb Virginijus Siksnys 《EMBO reports》2022,23(12)
Most CRISPR‐type V nucleases are stimulated to cleave double‐stranded (ds) DNA targets by a T‐rich PAM, which restricts their targeting range. Here, we identify and characterize a new family of type V RNA‐guided nuclease, Cas12l, that exclusively recognizes a C‐rich (5''‐CCY‐3′) PAM. The organization of genes within its CRISPR locus is similar to type II‐B CRISPR‐Cas9 systems, but both sequence analysis and functional studies establish it as a new family of type V effector. Biochemical experiments show that Cas12l nucleases function optimally between 37 and 52°C, depending on the ortholog, and preferentially cut supercoiled DNA. Like other type V nucleases, it exhibits collateral nonspecific ssDNA and ssRNA cleavage activity that is triggered by ssDNA or dsDNA target recognition. Finally, we show that one family member, Asp2Cas12l, functions in a heterologous cellular environment, altogether, suggesting that this new group of CRISPR‐associated nucleases may be harnessed as genome editing reagents. 相似文献
69.
70.
Phosphorus Limitation in Boreal Forests: Effects of Aluminum and Iron Accumulation in the Humus Layer 总被引:1,自引:1,他引:1
Plant growth in boreal forests is generally considered to be predominantly nitrogen (N) limited, but forested groundwater
discharge areas may be exceptions. In this study, we conducted tests to determine whether highly productive forested groundwater
discharge areas generally differ from adjacent groundwater recharge areas in terms of humus chemistry and the availability
of phosphorus (P) and N to plants. We investigated six forested sites, divided into groundwater discharge and adjacent groundwater
recharge areas, in northern Sweden. The humus layers of the forested groundwater discharge areas were clearly distinguished
from the adjacent groundwater recharge areas by having higher acid-digestible calcium (Ca) and/or aluminium (Al) and iron
(Fe) content and higher organic P and N content. Soil solution inorganic N (NH4
+ and NO3
−) and pH were higher in the groundwater discharge areas than in the groundwater recharge areas. The organic P content showed
a positive linear relationship to the Al and Fe content in the humus layer, indicating that organic P is associated with Al
and Fe compounds in the humus. A plant bioassay using humus substrate from one groundwater discharge area and the adjacent
groundwater recharge area found that plants grown in groundwater discharge area humus (with a high P-fixation capacity) increased
their biomass upon P fertilization, whereas no growth response was found for N additions. By contrast, plants grown in humus
from the groundwater recharge area did not respond to added P unless N was added too. This study suggests that groundwater
discharge can affect the nutrient availability of N and P both directly, via increased P fixation due to the redistribution
of Al and Fe, and indirectly, via the inflow of groundwater high in Ca and alkalinity, maintaining a high pH in the humus
layer that favors in situ N turnover processes.
Received 2 March 2001; Accepted 9 November 2001. 相似文献