An improved assay for platelet-activating factor using HPLC-tandem mass spectrometry

We describe an improved assay for platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) using HPLC-tandem mass spectrometry (LC-MS/MS). The present method can readily detect as little as 1 pg (1.9 fmol) of PAF, a significant improvement over previously described LC-MS/MS methods, and gives a linear response up to 1,000 pg of PAF. Our method also overcomes the artifacts from isobaric lipids that have limited the usefulness of certain existing LC-MS/MS assays for PAF. In the course of these studies, we detected three novel lipid species in human neutrophils. One of the novel lipids appears to be a new molecular species of PAF, and the other two have chromatographic and mass spectrometric properties consistent with stearoyl-formyl-glycerophosphocholine and oleoyl-formyl-glycerophosphocholine. These observations identify previously unknown potential interferences in the measurement of PAF by LC-MS/MS. Moreover, our data suggest that the previously described palmitoyl-formyl-glycerophosphocholine is not unique but rather is a member of a new and poorly understood family of formylated lipids.     

An improved method using densitometry for evaluating severity of laser photocoagulation induced CNV

Laser photocoagulation induced choroidal neovascularization currently is the most effective model available for the study of this disease in terms of efficacy of new drugs and therapies. Previously, evaluating the extent of choroidal neovascularization using this model was time- consuming and required the use of experienced personnel. We describe a new method for simple and rapid evaluation of laser induced choroidal neovascularization using densitometry. Fluorescein angiograms of a laser photocoagulated rat eye were scanned into a computer. Densitometry software subsequently was used to calculate the severity of the laser lesions. The densitometry method proved effective for calculating the extent of laser induced choroidal neovascularization. In addition, this method was more rapid than visual evaluations and less likely to produce errors.     

An improved method using densitometry for evaluating severity of laser photocoagulation induced CNV

Laser photocoagulation induced choroidal neovascularization currently is the most effective model available for the study of this disease in terms of efficacy of new drugs and therapies. Previously, evaluating the extent of choroidal neovascularization using this model was time- consuming and required the use of experienced personnel. We describe a new method for simple and rapid evaluation of laser induced choroidal neovascularization using densitometry. Fluorescein angiograms of a laser photocoagulated rat eye were scanned into a computer. Densitometry software subsequently was used to calculate the severity of the laser lesions. The densitometry method proved effective for calculating the extent of laser induced choroidal neovascularization. In addition, this method was more rapid than visual evaluations and less likely to produce errors.     

An improved method using densitometry for evaluating severity of laser photocoagulation induced CNV.

Laser photocoagulation induced choroidal neovascularization currently is the most effective model available for the study of this disease in terms of efficacy of new drugs and therapies. Previously, evaluating the extent of choroidal neovascularization using this model was time-consuming and required the use of experienced personnel. We describe a new method for simple and rapid evaluation of laser induced choroidal neovascularization using densitometry. Fluorescein angiograms of a laser photocoagulated rat eye were scanned into a computer. Densitometry software subsequently was used to calculate the severity of the laser lesions. The densitometry method proved effective for calculating the extent of laser induced choroidal neovascularization. In addition, this method was more rapid than visual evaluations and less likely to produce errors.     

An improved method for evaluating hardwood animal bedding products

Technological improvements have led to the development of higher quality hardwood bedding products with lower dust content. Nevertheless, testing procedures used to evaluate the quality of laboratory animal bedding products have not kept pace, resulting in the continued use of flawed and outdated test methods. The present study was conducted to develop an improved method for evaluating the quality of hardwood animal bedding products.     

An improved method for real-time monitoring of membrane capacitance in Xenopus laevis oocytes

Measurements of membrane capacitance (C(m)) in Xenopus laevis oocytes offer unique experimental possibilities but are difficult to perform with current methods. To improve C(m) measurements in the two-electrode voltage clamp (TEVC) mode, we developed a paired-ramp protocol and tested its performance in a model circuit (with tunable C(m), membrane resistance R(m), and series resistance R(s)) and in Xenopus oocytes. In the cell model and with R(s) = 0 Omega, inaccuracy of C(m) estimates was <1% under widely varying conditions (R(m) ranging from 100 to 2000 kOmega, and C(m) from 50 to 1000 nF). With R(s) > 0 Omega, C(m) was underestimated by a relative error epsilon closely approximated as epsilon approximate 2 x R(s)/(R(s) + R(m)), in keeping with the theoretical prediction. Thus, epsilon may be neglected under standard conditions or, under extreme conditions, corrected for if R(s) is known. Relative imprecision of C(m) estimates was small, independent of R(s), and inversely related to C(m) (<1.5% at 50 nF, <0.4% at 200 nF). Averaging allowed reliable detection of C(m) deviations from 200 nF of 0.1 nF, i.e., 0.05%. In Xenopus oocytes, we could resolve C(m) changes that were small (e.g., DeltaC(m) approximate 2 nF upon 100 muM 8-Br-cAMP), fast (e.g., DeltaC(m)/Deltat approximate 20nF/30s upon 1 muM phorbol myristate acetate (PMA)) or extended and complex (e.g., fast increase, followed by prolonged C(m) decrease upon 1 muM PMA). Rapidly alternating between paired ramps and a second, step protocol allowed quasi-simultaneous monitoring of additional electrical parameters such as R(m), slope conductance g(m), and reversal potential E(rev). Taken together, our method is suited to monitor C(m) in Xenopus oocytes conveniently, with high temporal resolution, accuracy and precision, and in parallel with other electrical parameters. Thus, it may be useful for the study of endo- and exocytosis and of membrane protein regulation and for electrophysiological high-throughput screening.     

An improved procedure for the isolation of glia maturation factor

A procedure for the bulk isolation of glia maturation factor (GMF) in high yield and high purity from bovine brains is outlined. The method involves extraction by homogenization and centrifugation, followed by ammonium sulfate precipitation and column chromatography with diethylaminoethyl (DEAE) Sephacel, Sephadex G-75, and hydroxylapatite. The method results in a 10,000-fold purification, a purity exceeding that of previously published procedures, and enables us to handle as much as 2.8 kg brain tissue or eight brains/week. The ability to mass-produce GMF with this method greatly facilitates its biological studies, further purification, and chemical characterization. The isolated GMF shows a molecular weight of 13,000 on Bio-gel P-30 column and an isoelectric point of about 5.4 on isoelectric focusing. The isolated GMF is heat labile and susceptible to papain and ficin but relatively resistant to trypsin, neuraminidase, and endoglycosidase.     

Non-destructive estimation of root mass using electrical capacitance on ten herbaceous species

Background and Aims

Characteristically baseline levels of Sb in the environment are low, but problematic local elevation trends arise from anthropogenic activities such as mining and incineration. Arsenic (analog of Sb) accumulation by rice can be reduced by iron (Fe) plaque. A hydroponic experiment was conducted to investigate whether Fe plaque could reduce the uptake and translocation of different Sb species in different rice cultivars.

Methods

After Fe plaque on rice roots was induced in solution containing 0, 0.2, 0.4, 0.7, 1.2, 2.0?mM Fe²⁺ for 24?h, seedlings were transferred into nutrient solution with 20?μM Sb(V) or Sb(III) for 3?d.

Results

About 60–80% (Sb(III) treatment) and 40–60% (Sb(V) treatment) of the total Sb accumulated in Fe plaque. There was a significant correlation between the concentrations of Sb and Fe on the root surface. A similar relationship was observed in roots and shoots. Cultivar (Jiahua 1) formed the most Fe plaque, had the highest Fe associated Sb sequestration but the lowest Sb concentration in the root interior.

Conclusions

Fe plaque may act as a ‘buffer’ for Sb(V) and Sb(III) in the rhizosphere, and cultivars played an important role in the different species Sb uptake and translocation.     

A comparative study of cell death using electrical capacitance measurements and dielectrophoresis

The changes in the dielectric properties of cells that occur during their exposure to various lethal environmental stresses were measured using both dielectric spectroscopy and dielectrophoresis. It is shown that the dielectric properties of both dying and dead yeast cells were strongly dependent on the method used to induce cell death. Methods which directly affected the membrane permeability, and consequently the membrane conductivity and internal conductivity, resulted in large changes in the suspension capacitance and dielectrophoretic behaviour, whilst methods which affected the cell interior but had little effect on the cell membrane resulted in few or no changes in the dielectric properties of the cells. The findings indicate that, depending on the method by which cell death is induced, dielectric spectroscopy may not always be able to observe differences between viable and non-viable cells, and that dielectrophoresis will not always be able to separate viable from non-viable cells.     

An improved bioassay for cytokinins using cucumber cotyledons

The cucumber cotyledon greening bioassay is frequently used for detecting cytokinins. Beneficial modifications of the original technique included using 5-day-old cucumber (Cucumus sativus L., cv. National Pickling) cotyledons treated with combinations of 40 millimolar KCl and various concentrations of cytokinins. A dark incubation period of 20 hours was followed by an exposure to light for 3.5 hours. Under these conditions, extremely low (0.0001 milligram per liter) concentrations of N⁶-benzyladenine, zeatin, kinetin, or zeatin riboside can be detected. Of the four cytokinins tested, kinetin appeared to be the least active. With these improvements, the assay is 10 times more sensitive than is the previously described cucumber cotyledon greening bioassay for cytokinins.     

一种改进的小鼠局灶性脑缺血神经症状定量评价方法

本文旨在建立一种客观评价小鼠局灶性脑缺血神经症状的定量方法。在大脑中动脉阻塞诱导局灶性脑缺血后24h,采用悬挂试验分别测定转动的平均角和优势角以及转动次数,并用爬板试验测定小鼠攀爬角度;分析定量测定指标与脑梗死体积、神经元密度的相关性,并与经典的行为学评价方法比较。还以此法观察抗脑缺血药{pranlukast,4-氧-8-[对-(4-苯丁氧基)苯甲酰氨基]-2-(5-四氮基)-4H-1-苯并吡喃半水化合物}(ONO-1078)的作用。结果显示,脑缺血小鼠各项指标均有显著改变,定量评价总分与脑梗死体积、神经元密度减少密切相关,与经典行为学评分之间也密切相关。ONO-1078可显著抑制缺血损伤,并降低定量评价总分。因此,本方法可反映局灶性脑缺血神经症状变化,具有客观、定量、操作简便、无损伤的优点,并能用于评价抗脑缺血药物的作用。     

An improved technique for hyaluronan histochemistry using microwave irradiation

Summary A hyaluronan binding protein (HABP), extracted from cartilage, was biotin-labelled and used for histochemical localization of hyaluronan (HA) in tissue sections. Various tissues were fixed for a mixture of formaldehyde and glutaraldehyde during microwave irradiation. The microwave oven when set at 700 W and 45°C yielded an intense and specific staining of HA. Under these conditions the relative proportion of the two aldehydes did not influence the staining intensity. Aldehyde fixation during microwave irradiation for HA histochemistry, (1) saves time, (2) eliminates the use of cetylpyridinium chloride (CPC), and (3) improves the reproducibility.     

An improved method for the study of apoptosis-related genes using the tet-on system

Inducible gene expression systems are particularly useful for the functional characterization of genes with putative toxic properties. In the course of studying the role of hypoxia-regulated gene expression on cell survival using the tetracycline-inducible (tet-on) system, the author noted that exposure to the inducing ligand doxycycline (dox) inhibited caspase-3 cleavage in control samples. To limit this confounding off-target effect, he devised an in vitro pulse dose, delayed-injury protocol testing both dox and a novel tetracycline analog 9-t-butyl doxycycline (9-TB). Although 9-TB induced higher transgene levels compared to matched concentrations of dox, continuous exposure to both drugs inhibited caspase-3 cleavage in hypoxic samples. Conversely, a 6-h pulse dose of 9-TB followed by a 40-h washout period prior to hypoxic challenge activated robust transgene expression and lessened the inhibitory effects on caspase-3 processing. It is anticipated that these protocol modifications will improve the performance of tet-regulated genetic screens, particularly in situations where cell death is used as a primary end point.     

An optimized approach to membrane capacitance estimation using dual-frequency excitation.

We present an optimized solution to the problem of membrane impedance estimation when a patch-clamped cell is stimulated by a dual-frequency, sinusoidal excitation. The complete data set of raw whole-cell current samples is typically reduced, via digital lock-in detection, to measurements of the complex cell model admittance at the two stimulus frequencies. We describe a statistical model of both data sets and demonstrate that the admittance data adequately represent the essential features obtained from the raw data. The parameter estimates obtained by a nonlinear weighted least-squares solution (NWLS), which under normal recording conditions is equivalent to the maximum likelihood solution, essentially obtain the theoretical lower bound on variance established by the Cramér-Rao bound. Our software implementation of the NWLS solution produces estimates of the cell model parameters that are less noisy than other dual-frequency systems. Our system can be used 1) to measure slow changes in membrane capacitance-in the face of large, slow changes in membrane resistance, 2) to detect with confidence capacitance changes expected from the exocytosis of moderate-sized dense core granules, and 3) to reduce the cross-talk between transient changes in membrane conductance and membrane capacitance.