Novel dye for detection of callus embryo by confocal laser scanning fluorescence microscopy

In the present study a new luminescent dye 3‐N‐(2‐pyrrolidinylacetamido)benzanthrone (AZR) was synthesized. Spectroscopic measurements of the novel benzanthrone 3‐aminoderivative were performed in seven organic solvents showing strong fluorescence. The capability of the prepared dye for visualization has been tested on flax, red clover and alfalfa to determinate the embryo in plant callus tissue cultures. Callus cells were stained with AZR and further analysed utilizing confocal laser scanning fluorescence microscopy. Performed experiments show high visualization effectiveness of newly synthesized fluorescent dye AZR that is efficient in fast and relatively inexpensive diagnostics of callus embryos that are problematic due to in vitro culture specificity.     

活性X-3B红染料在水-土壤-作物连续体中的迁移模型研究

进入环境的自学成才性X-3B红染料是一典型的持久性有机污染物（POPs)。它从水分室向土壤分室再到作物分室的迁移,是一连续的生态过程,基于此,从理论上对活性X-3B红染料在水-土壤两相,土壤-作物两相以及水-土壤-作物整个连续体中的迁移进行了定量描述与探讨,并采用棕壤-大豆、褐土-小麦,红壤-萝卜和水稻土-水稻等系统给予了实验论证,表明活性X-3B红染料通过吸附机制从水分室到土壤分室的迁移符合Langmuir模型。通过作物根系吸收机制从土壤分室到作物分室的迁移可以用对数化的作物-土壤积累因子（CSAF）模型予以定量描述。     

Differential perturbation of erythrocyte membrane-associated transport and enzyme activities by structurally related lipophilic compounds

The alteration of two erythrocyte plasma membrane functions, acetylcholine hydrolysis and glucose exchange, by a series of structurally related small lipophilic compounds which exhibit similar antihemolytic behavior was studied. 2-Methyldimethylaminoazobenzene is a more potent inhibitor of acetylcholinesterase than the 3′-methyl analogue, while the unsubstituted compound fails to inhibit. Esterase inhibition by the 2-methyl compound is noncompetitive and dependent on the anion composition of the assay buffer. The temperature dependence of acetylcholinesterase activity in the presence of the 2-methyl compound suggests that interaction with inhibitor is influenced by the state of lipids tightly bound to the enzyme. Glucose exchange is inhibited to the same extent by both methyl derivatives but not by the unsubstituted dye, and the temperature dependence in the presence of inhibitor is not grossly altered. The lack of correlation between inhibition of membrane function and stabilization of erythrocytes against osmotic hemolysis is discussed.     

Cell Kinetic Evidence Suggests Elevated Oxidative Stress in Cultured Cells of Bloom's Syndrome

Bromodeoxyuridine/Hoechst flow cytometry was used to analyse disturbed cell proliferation of fibroblasts and lymphoblastoid cells from Bloom's syndrome (BS). Fibroblasts show poor activation, arrest in the G2 phase of the cell cycle along with a prolongation of the Gl phase. This pattern of perturbed cells proliferation is akin to that elicited in normal fibroblasts by 4-hydroxy-nonenal, a breakdown product of lipid peroxides. Treatment with vitamin E improved growth of BS fibroblasts more strongly than growth of normal fibroblasts. Lymphoblastoid cells from BS, to the contrary, experience only a minor arrest in the G2 phase after one round of bromodeoxyuridine incorporation, but are strongly inhibited during and after the second S phase. Thus, their cell cycle arrest is dependent upon BrdU incorporation, as has been found previously in normal cells exposed to elevated concentrations of oxygen or paraquat, a superoxide generating compound. These results suggest that BS cells may suffer from an elevated, endogenous generation of oxygen free radicals.     

A Novel Assay for Measurement of Membrane‐Protein Surface Expression using a β‐lactamase Reporter

The trafficking of membrane proteins is dynamic and contributes to the homeostatic control of their cell surface localization and their function in signal transduction. Therefore, it is important to have sensitive techniques that allow measurement of surface expression. The current assays for such measurement are time consuming and low throughput. Here, we describe a quantitative, one‐step and potentially high‐throughput assay, using the β‐lactamase enzyme (βlac) as a reporter, for measurement of surface expression of proteins. In this assay, the βlac is fused to the extracellular portion of the plasma membrane protein of interest. To selectively measure surface expression, a cell‐impermeable substrate of βlac, nitrocefin, is used. We demonstrate the utility of the βlac assay using well‐established paradigms of internalization and molecular chaperoning, applied to two G‐protein‐coupled receptors and a monoamine transporter. Considering its simplicity and low cost, this assay could become a standard technique in the measurement of protein surface expression .      

Development of a fluorescent probe for the study of nucleosome assembly and dynamics

To develop a probe for use in real-time dynamic studies of nucleosomes, core histones (from Drosophila) were conjugated to a DNA-intercalating dye, thiazole orange, by a reaction targeting Cys 110 of histone H3. In the absence of DNA, the conjugated histones are only very weakly fluorescent. However, upon reconstitution into nucleosomes by standard salt dialysis procedures, the probe fluoresces strongly, reflecting its ability to intercalate into the nucleosomal DNA. The probe is also sensitive to the nature of the DNA-histone interaction. Nucleosomes reconstituted by stepwise salt dialysis give a fluorescence signal quite different from that of the species formed when DNA and histones are simply mixed in low salt. In addition, changing either the DNA length or the type of sequence (nucleosome positioning sequences versus random DNA of the same size) used in the reconstitution alters the resulting fluorescence yield. The results are all consistent with the conclusion that a more rigid, less flexible nucleosome structure results in less fluorescence than a looser structure, presumably due to structural constraints on dye intercalation. This probe should be well suited to analyzing nucleosome dynamics and to following factor-mediated assembly and remodeling of nucleosomes in real time, particularly at the single-molecule level.     

A new xanthene‐based two‐photon fluorescent probe for the imaging of 1,4‐dithiothreitol (DTT) in living cells

1,4‐Dithiothreitol (DTT) has wide applications in cell biology and biochemistry. Development of effective methods for monitoring DTT in biological systems is important for the safe handling and study of toxicity to humans. Herein, we describe a two‐photon fluorescence probe (Rh‐DTT) to detect DTT in living systems for the first time. Rh‐DTT showed high selectivity and sensitivity to DTT. Rh‐DTT can be successfully used for the two‐photon imaging of DTT in living cells, and also can detect DTT in living tissues and mice.