铽离子与RNA的荧光反应及RNA测定的研究

核糖核酸（ＲＮＡ）与稀土铽离子在ｐＨ５．０～６．５条件下能形成具有较强荧光的络合物,其激发峰在２８８ｎｍ处,发射峰为４９４ｎｍ及５４５ｎｍ处．ＲＮＡ在０．１～１０ｍｇ／Ｌ范围内与其荧光强度呈线性关系,其检出限为６．０×１０￣（－８）ｍｏｌ／Ｌ．腺苷酸,尿苷酸,胞苷酸对ＲＮＡ的干扰比较小,因此可在其存在下选择性地测定ＲＮＡ．     

Feasibility of using GFP-expressing Escherichia coli, coupled with fluorimetry, to determine protozoan ingestion rates

The feasibility of using a live Escherichia coli population, which had been engineered to express the green fluorescent protein (GFP), coupled with fluorimetry, was tested as a means for determining protozoan ingestion rates. Its potential use was based on evidence that once cells are acidified, e.g. in a food vacuole, the fluorescence is lost. Of the 29 protozoa tested, over 85% ingested the GFP-expressing E. coli and a detailed experiment with the ciliate Tetrahymena pyriformis was carried out, principally to assess the performance of the live bacterium against two commonly used surrogate prey, i.e. fluorescently labelled bacteria (FLB) and fluorescently labelled microspheres (FLMs). A decrease in GFP-expressing E. coli fluorescence and, hence, concentration, was recorded by fluorimetry and epifluorescence microscopy, with calculated ingestion rates being equivalent. A higher ingestion rate was determined by counting the number of fluorescent E. coli within the ciliate over 120 s, but this was equivalent to that obtained for the stained E. coli using the same direct method of analysis. However, the ciliate was shown to process the stained and unstained E. coli cells differently, with only the latter resulting in an increase in ciliate abundance.     

Effects of Somatostatin on Intracellular Calcium Concentration in PC12 Cells

Abstract: Somatostatin (SS) is a neuropeptide that is distributed in various regions of the CNS, where it may act as a neurotransmitter or neuromodulator. SS produces multiple effects in the CNS through interactions with membrane receptors. In particular, SS inhibits various secretory responses in different cell types. In the present study, we have investigated the effects of exogenous application of SS on the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in PC12 cells, a rat pheochromocytoma cell line. SS did reduce the magnitude of the secondary, maintained Ca²⁺ influx brought about by K⁺ depolarization. Similar effects were obtained with the application of SS analogues, such as d -Trp⁸-SS, d -Trp⁸- d -Cys¹⁴-SS, CGP-23996, and SMS-201995. In addition, treatment with cyclo-SS, a SS antagonist, did not alter [Ca²⁺]_i. Experiments with selective blockers of different voltage-dependent Ca²⁺ channels, such as methoxyverapamil (D600) and Ω-conotoxin GVIA, demonstrated that the effects of SS on [Ca²⁺]_i were mediated by voltage-dependent Ca²⁺ channels of the L type. Control experiments with a membrane potential indicator, i.e., the fluorescent dye bisoxonol, excluded that SS influenced the level of the membrane potential. SS effects on PC12 cells suggest the possibility that this neuropeptide plays a role in the modulation of cell functional activity by altering Ca²⁺ influx.     

Determination of the DNA-binding characteristics of ethidium bromide, proflavine, and cisplatin by flow injection analysis: usefulness in studies on antitumor drugs

Flow injection analysis was used to study the reactions occurring between DNA and certain compounds that bind to its double helix, deforming this and even breaking it, such that some of them (e.g., cisplatin) are endowed with antitumoral activity. Use of this technique in the merging zones and stopped-flow modes afforded data on the binding parameters and the kinetic characteristics of the process. The first compound studied was ethidium bromide (EtdBr), used as a fluorescent marker because its fluorescence is enhanced when it binds to DNA. The DNA-EtdBr binding parameters, the apparent intrinsic binding constant (0.31+/-0.02 microM(-1)), and the maximum number of binding sites per nucleotide (0.327+/-0.009) were determined. The modification introduced in these parameters by the presence of proflavine (Prf), a classic competitive inhibitor of the binding of EtdBr to the DNA double helix, was also studied, determining the value of the intrinsic binding constant of Prf (K(Prf) = 0.119+/-9x10(-3) microM(-1)). Finally, we determined the binding parameters between DNA and EtdBr in the presence of the antitumor agent cisplatin, a noncompetitive inhibitor of such binding. This provided information about the binding mechanism as well as the duration and activity of the binding of the compound in its pharmacological use.     

Heterometallic complex formation on p-sulfonatothiacalix[4]arene platform resulting in pH- and redox-modification of [Ru(bpy)3] luminescence

The pH-dependent heterometallic complex formation with p-sulfonatothiacalix[4]arene (TCAS) as bridging ligand in aqueous solutions was revealed by the use of spectrophotometry, nuclear magnetic relaxation and fluorimetry methods. The novelty of the structural motif presented is that the appendance of emission metal center ([Ru(bpy)₃]²⁺) is achieved through the cooperative non-covalent interactions with the upper rim of TCAS. The second metal block (Fe(III), Fe(II) and Mn(II)), bound with the lower rim of TCAS in the inner sphere coordination mode is serving as quencher of [Ru(bpy)₃]²⁺ emission. The difference between the complex ability of Fe(III) and Fe(II) ions provides pH conditions for redox-dependent emission of [Ru(bpy)₃]²⁺.     

Endogenous Release of Neuronal Serotonin and 5-Hydroxyindoleacetic Acid in the Caudate-Putamen of the Rat as Revealed by Intracerebral Dialysis Coupled to High-Performance Liquid Chromatography with Fluorimetric Detection

Extracellular levels of endogenous serotonin (5-HT) and its major metabolite, 5-hydroxyindoleacetic acid (5-HIAA), were measured in the caudate-putamen of anesthetized and awake rats using intracerebral microdialysis coupled to HPLC with fluorimetric detection. A dialysis probe (of the loop type) was perfused with Ringer solution at 2 microliters/min, and samples collected every 30 or 60 min. Basal indole levels were followed for up to 4 days in both intact and 5,7-dihydroxytryptamine (5,7-DHT) lesioned animals. Immediately after the probe implantation, the striatal 5-HT levels were about 10 times higher than the steady-state levels that were reached after 7-8 h of perfusion. The steady-state baseline levels, which amounted to 22.5 fmol/30 min sampling time, remained stable for 4 days. In 5,7-DHT-denervated animals, the steady-state levels of 5-HT, measured during the second day after probe implantation, were below the limit of detection (less than 10 fmol/60 min). However, during the first 6 h post-implantation, the 5-HT output was as high as in intact animals, which suggests that the high 5-HT levels recovered in association with probe implantation were blood-derived. As a consequence, all other experiments were started after a delay of at least 12 h after implantation of the dialysis probe. In awake, freely moving animals, the steady-state 5-HT levels were about 60% higher than in halothane-anesthetized animals, whereas 5-HIAA was unaffected by anesthesia. KCl (60 and 100 mM) added to the perfusion fluid produced a sharp increase in 5-HT output that was eight-fold at the 60 mM concentration and 21-fold at the 100 mM concentration. In contrast, 5-HIAA output dropped by 43 and 54%, respectively. In 5,7-DHT-lesioned animals, the KCl-evoked (100 mM) release represented less than 5% of the peak values obtained for the intact striata. Omission of Ca2+ from the perfusion fluid resulted in a 70% reduction in baseline 5-HT output, whereas the 5-HIAA levels remained unchanged. High concentrations of tetrodotoxin (TTX) added to the perfusion medium (5-50 microM) resulted in quite variable results. At a lower concentration (1 microM), however, TTX produced a 50% reduction in baseline 5-HT release, whereas the 5-HIAA output remained unchanged. The 5-HT reuptake blocker, indalpine, increased the extracellular levels of 5-HT sixfold when added to the perfusion medium (1 microM), and threefold when given intraperitoneally (5 mg/kg). By contrast, the 5-HIAA level remained unaffected during indalpine infusion.(ABSTRACT TRUNCATED AT 400 WORDS)     

Long-term exposition of cells to beta-amyloid results in decreased intracellular calcium concentration

The ubiquitously present β-amyloid peptide plays an important role in the pathogenesis of Alzheimer’s disease. Its neurotoxicity has been blamed on its mal-activity to increase calcium-levels. In the present study, we demonstrate that treatment of fibroblasts with β-amyloid has, in deed, resulted in a transient rise in the calcium-concentration. Chronic exposition of cultures to the peptide, however, caused a fall in the calcium-level. Apparently, β-amyloid has biphasic effects: acutely, it increases the calcium-concentration of cells; in contrast, on the long-run, β-amyloid peptide acts as a calcium-antagonist. Therefore, the idea that β-amyloid peptide leads to neural degeneration solely by increasing cells’ calcium concentration must be replaced with a more complex view of its dual function in intracellular ionic homeostasis.     

Insights into the reaction of beta-lactam antibiotics with copper(II) ions in aqueous and micellar media: kinetic and spectrometric studies

Degradation of beta-lactam antibiotics by means of metallic cations seems to have a very complex chemistry, involving not only the catalytic effect of the metal ion but also complex formation. Many different compounds, such as methylpyrazines, oxazolones, penicilloic, penicillenic, and penicillonic acids, have been reported as degradation products of such antibiotics, although not many details about the progress of the reaction can be found in the literature. Two novel fluorimetric and spectrophotometric methods previously published by the authors, as well as kinetic studies, have been used to propose a possible reaction mechanism for the ampicillin degradation in the presence of copper(II) ions. Likewise, we have proposed the chemical structure required by the beta-lactam antibiotics to develop absorption or fluorescence properties. Kinetics in micellar and aqueous media shows that the copper-ampicillin reaction proceeds through different pathways depending on the reaction medium.     

Simple fluorimetric method for quantification of sialic acids in glycoproteins

A simple and rapid fluorimetric method was developed for detection and quantitative analysis of sialic acids in glycoproteins. Sialic acid residues in glycoproteins were specifically oxidized with periodate at 0 degrees C for 45 min. Formaldehyde generated from carbon 9 (C-9) of sialic acid was converted specifically to fluorescent dihydropyridine derivative with acetoacetanilide and ammonia at room temperature for 10 min. The reaction products indicate intense fluorescence with excitation and emission maxima at 388 and 471 nm, respectively. When the reaction was conducted in approximately a 1-ml volume, the linearity of the calibration exhibited between 2 and 180 microg of bovine fetuin, or between 0.3 and 27 nmol of N-acetylneuraminic acid, as a model glycoprotein. The limit of detection, based on three times the standard deviation of the reagent blank, was 0.5 microg of fetuin. The proposed method was applied to determination of sialic acids in various glycoprotein samples. This proposed method is simple and obviates the heating and extraction steps. It is highly specific to sialic acids in glycoproteins and indicates no fluorescence of neutral glycoproteins.     

富硒生物样品中硒的价态和形态分析

本文利用2,3—二氨基萘(DAN)荧光法测定了富硒玉米粉、硒酵母蛋白等样品中四价硒、六价硒、有机硒含量及总硒量。进一步验证了差减法测定不同价态硒含量的实验方法