A rapid,sensitive method for detection of alkaline phosphatase-conjugated anti-antibody on Western blots

A rapid, sensitive method has been developed to detect antibody-antigen complexes on “Western blots.” The methods of H. Towbin, T. Staehlin, and J. Gordon were used to separate and blot the antigens onto nitrocellulose. The remaining sites of attachment were blocked and the nitrocellulose was washed with polyoxyethylenesorbitan monolaurate (Tween 20). The blot was then reacted with the antiserum or hybridoma supernate to be tested. After the antigen-antibody reaction was completed, the blot was washed and treated with anti-antibody which has been conjugated to alkaline phosphatase. The alkaline phosphatase was detected by the reduction of the tetrazolium salt to diformazan by the hydrogen ions released in the formation of indigo by the reaction of the phosphatase on the indoxyl phosphate. The advantages of this method over previously described techniques are (1) use of Tween 20 allows the blot to be stained with Coomassie blue, (2) the substrates of the alkaline phosphatase reaction are stable for long periods of time, (3) the reaction products form an intense blue color which does not fade, (4) the resolution is extremely good with little to no band broadening, (5) the reaction is sensitive to picogram quantities of antigen, and (6) the reaction is quantitative.     

Microphytobenthic biomass assessment by pigment analysis: comparison of spectrophotometry and High Performance Liquid Chromatography methods

Samples of microphytobenthos from the Tagus estuary were analysed for photosynthetic pigments by spectrophotometry and High Performance Liquid Chromatography (HPLC). Chlorophyll a values obtained with HPLC and spectrophotometry methods presented a highly significant positive correlation for both spectrophotometric methods used (with and without the correction for pheopigments), but this relationship depended on the type of sediment. We concluded that spectrophotometric methods give reliable Chl-a values, being suited for routine analysis, when a vast number of replicates is needed. However, for the correct estimation of pheopigments, HPLC analysis is indispensable. In the literature, Chl-a estimations are expressed per content (μg g⁻¹) or concentration (mg m⁻²). We discuss the influence of sediment type on the results depending on the type of unit used, and propose a simple conversion factor based on sediment water content.     

赶黄草总黄酮树脂精制工艺与检测方法

本研究以总黄酮吸附量和解析率为检测指标,结合动态洗脱考察结果,考察11种树脂对赶黄草总黄酮的富集精制能力。优化树脂类型,选定了对总黄酮进行富集纯化较好的树脂HPD450;采用单因素及正交实验,确定最佳工艺条件:上样量10 mL,以6 mL/min的流速8 BV水、6 BV 80%甲醇溶液洗脱,pH值为6~7。以此方法得到90%以上的赶黄草总黄酮,实验结果良好,总黄酮精制工艺成效显著。用HPLC法精确测定赶黄草中槲皮苷、PGHG和ThA三个指标性黄酮成分,最佳色谱条件为:C18色谱柱,检测波长280 nm,进样量5μL,流动相:乙腈-0.05%磷酸溶液(0~25 min:12%~45%乙腈;25~40 min:45%~70%乙腈)梯度洗脱;精确测定赶黄草全草中3种黄酮成分的含量。采用分光光度法检测,指导优化总黄酮精制工艺,并以HPLC法精确测定三种黄酮代表成分。比较两种方法测定结果相一致,互为补充;故在工业生产时,可以分光光度法指导生产,以HPLC法精确定量测定。     

Spectrophotometric measurements of human tissues for the detection of subjacent blood vessels in an endonasal endoscopic surgical approach

Thin slices of human tissues are characterized concerning reflection and transmission in a wavelength range from 400 to 1700 nm. The results are primarily useful to find a wavelength for the detection of subjacent blood vessels during surgical procedures, especially neurological surgery. The measurements have been conducted using a customized measuring station, utilizing two halogen bulb lamps and two spectrometers. This paper focuses on creating a data base with the optical properties of artery, brain, bone, nasal mucosa, and nerve. The spectral distributions are compared among each other, similarities and differences are pointed out. Each tissue has got unique spectral characteristics, whereas typical absorption bands can be found in the overall tissues, especially hemoglobin and water absorption bands. The reflectivity maxima are typically located in the red or near‐infrared. All the transmission maxima are located between 1075 nm and 1100 nm. The measurements have been conducted at the Institute of Anatomy at the University of Leipzig. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

Whereas cation transport by the electrogenic membrane transporter Na⁺,K⁺-ATPase can be measured by electrophysiology, the electroneutrally operating gastric H⁺,K⁺-ATPase is more difficult to investigate. Many transport assays utilize radioisotopes to achieve a sufficient signal-to-noise ratio, however, the necessary security measures impose severe restrictions regarding human exposure or assay design. Furthermore, ion transport across cell membranes is critically influenced by the membrane potential, which is not straightforwardly controlled in cell culture or in proteoliposome preparations. Here, we make use of the outstanding sensitivity of atomic absorption spectrophotometry (AAS) towards trace amounts of chemical elements to measure Rb⁺ or Li⁺ transport by Na⁺,K⁺- or gastric H⁺,K⁺-ATPase in single cells. Using Xenopus oocytes as expression system, we determine the amount of Rb⁺ (Li⁺) transported into the cells by measuring samples of single-oocyte homogenates in an AAS device equipped with a transversely heated graphite atomizer (THGA) furnace, which is loaded from an autosampler. Since the background of unspecific Rb⁺ uptake into control oocytes or during application of ATPase-specific inhibitors is very small, it is possible to implement complex kinetic assay schemes involving a large number of experimental conditions simultaneously, or to compare the transport capacity and kinetics of site-specifically mutated transporters with high precision. Furthermore, since cation uptake is determined on single cells, the flux experiments can be carried out in combination with two-electrode voltage-clamping (TEVC) to achieve accurate control of the membrane potential and current. This allowed e.g. to quantitatively determine the 3Na⁺/2K⁺ transport stoichiometry of the Na⁺,K⁺-ATPase and enabled for the first time to investigate the voltage dependence of cation transport by the electroneutrally operating gastric H⁺,K⁺-ATPase. In principle, the assay is not limited to K⁺-transporting membrane proteins, but it may work equally well to address the activity of heavy or transition metal transporters, or uptake of chemical elements by endocytotic processes.     

A simple,cheap but reliable method for evaluation of zinc chelating properties

BackgroundZinc is an essential trace element. Both its lack and excess are associated with pathological states. The former is more common and can ensue from the excessive treatment with clinically used iron/copper chelators.Aim and methodThe aim of this work was to prepare a reliable, rapid and cheap method for the screening of zinc chelation. Spectrophotometric assessment using a known zinc indicator dithizone was selected.ResultsInitial screening performed by comparison of spectra of dithizone and its complex with zinc suggested 530 and 570 nm as suitable wavelengths for determination of zinc at pH 4.5 while 540 and 590 nm for pH 5.5–7.5. Additional research showed the lower wavelengths to be more suitable. The sensitivity of the method was always bellow 1 μM with good linearity relationship between absorbance and zinc concentration. The method suitability was confirmed by use of two known zinc chelators, ethylenediaminetetraacetic acid (EDTA) and N,N,N′,N′-tetrakis(2-pyridylmethyl)-1,2-ethylenediamine (TPEN).ConclusionThis method represents a sufficiently precise method for zinc chelation screening usable at pathophysiologically relevant pH conditions. Such method can be employed for both screening of novel zinc chelators and for testing affinity of other metal chelators for zinc.     

DNA binding and nuclease activity of a one-dimensional heterometallic nitrosyl complex

The interaction of a structurally characterized Sr–Fe nitrosyl complex with DNA has been studied by UV–vis and fluorescence spectroscopy, viscometric, and gel electrophoresis techniques. From the absorption titration studies the intrinsic binding constant of the complex with DNA was calculated to be 1.6 × 10⁴ M⁻¹. Fluorimetric studies indicate that the complex compete with EB in binding to DNA. The complex shows nuclease activity on pUC19 supercoiled DNA in presence of H₂O₂.     

Culture and fusion of pollen protoplasts of Brassica oleracea L. var. italica with haploid mesophyll protoplasts of B. rapa L. ssp. pekinensis

Hybrid callus was formed from the successful protoplast fusion between pollen protoplasts of Brassica oleracea var. italica and haploid mesophyll protoplasts of Brassica rapa. The pollen protoplast isolation frequency in broccoli was highly related to the ratio of trinucleate pollens in the male gametophyte population. Large quantities of pollen protoplasts with high vigor could be isolated, and the isolation frequency reached up to 90% in 6.0-7.0 mm long flower buds with about 94.7% trinucleate-stage pollens. Pollen protoplasts could be collected and purified by discontinuous gradient centrifugation. In 1% Na-alginate embedding culture, cell divisions were observed but no further development was found. The haploid mesophyll protoplasts were isolated from in vitro haploid plants of B. rapa. Results strongly showed the variability in culturability of mesophyll protoplasts from different haploid lines. Both pollen protoplasts and haploid mesophyll protoplasts retained a stable round shape in the designed prefusion solution with an osmotic pressure of 0.74 osmol/kg. Polyethylene glycol was used for the protoplast fusion, and 40% polyethylene glycol 4000 enabled the highest fusion frequency of about 20%. Some postfusion protoplasts showed cell divisions up to callus proliferation. Calli were screened by random amplified polymorphic DNA analysis for their hybrid character. Results revealed the existence of the hybrid calli. Some of the hybrid calli grew well with green color and shoot primordia. According to our knowledge, this is the first report about a hybrid formation between two haploid protoplasts. Potential comprehensive applications, as well as problems of this technique, are discussed.     

Caffeine, pentoxifylline and theophylline form stacking complexes with IQ-type heterocyclic aromatic amines

Methylxanthines (MTX), in particular caffeine (CAF), are known as the most widely consumed alkaloids worldwide. Many accumulated statistical data indicate the protective effect of CAF intake against several types of cancer. One of the possible explanations of this phenomenon is direct non-covalent interaction between CAF and aromatic mutagen/carcinogen molecules through stacking (π-π) complexes formation. Here we demonstrate that CAF and other MTX, pentoxifylline (PTX) and theophylline (TH), form stacking complexes with carcinogenic imidazoquinoline-type (IQ-type) food-borne heterocyclic aromatic amines (HCAs). We estimated neighborhood association constants (K_AC of the order of magnitude of 10² M⁻¹) in neutral and acidic environment and enthalpy changes (ΔH values between −15.1 and −39.8 kJ/mol) for these interactions using UV-Vis spectroscopy, calculations based on thermodynamical model of mixed aggregation and titration microcalorimetry. Moreover, using Ames test with Salmonella typhimurium TA98 strain and recently developed mutagenicity assay based on bioluminescence of Vibrio harveyi A16 strain, we demonstrated a statistically significant reduction in HCAs mutagenic activity in the presence of MTX.     

Towards a new attenuating compound: a potentiometric, spectrophotometric and NMR equilibrium study on Fe(III), Al(III) and a new tetradentate mixed bisphosphonate-hydroxypyridinonate ligand

Coordination properties toward Fe(III) and Al(III) of a mixed bisphosphonate-hydroxypyridinonate ligand are presented. Potentiometric, spectrophotometric and NMR results allowed to conclude that Fe(III) and Al(III) coordination takes place on the pyridinone moiety. The high steric hindrance prevents the possibility of simultaneous coordination of both groups to the same metal ion. Quantum mechanical calculations confirm this finding allowing to determine the minimal length of the linker necessary for a stable conformation of complexes in which Fe(III) is coordinated both by pyridinone and bisphosphonate groups.