Quantification of galactosylsphingosine in the twitcher mouse using electrospray ionization-tandem mass spectrometry.

Globoid cell leukodystrophy (Krabbe disease) is an autosomal recessive inherited neurodegenerative disorder caused by the deficiency of the lysosomal enzyme beta-galactosylceramidase. The pathogenesis of the disorder has been proposed to arise from the accumulation of the cytotoxic metabolite galactosylsphingosine (psychosine). The twitcher mouse is a naturally occurring murine model of globoid cell leukodystrophy. We have developed a rapid, sensitive, and specific mass spectrometric method for determining the galactosylsphingosine concentration in the tissues of twitcher mice. Galactosylsphingosine is extracted from the tissues in methanol, isolated using strong cation-exchange and C18 solid-phase extraction chromatography, and then directly analyzed using electrospray ionization-tandem mass spectrometry. A lactosylsphingosine internal standard has been employed for quantification. The assay demonstrated significant accumulation of galactosylsphingosine in the brain, spinal cord, and kidney of twitcher mice. It is anticipated that this method may be of use in the monitoring of experimental therapies for globoid cell leukodystrophy.     

Rapid scanning of 32 P-labeled acrylamide gels

We describe a radioisotopic assay for ornithine-δ-transaminase using precursor ornithine-U-¹⁴C. We quantitate the product Δ¹-pyrroline-5-carboxylate-¹⁴C by cation-exchange chromatography. The sensitivity of the method allows for measurement of enzyme activity in extracts prepared from 10⁵ mammalian cells.     

A radiotechnique suitable for the detection of octopine synthesis in Crown-gall tissues grown in vitro

An improved method for the detection of octopine in Crown-gall tissues and in shoots regenerated from these tissues has been devised using [¹⁴C]arginine as a precursor. By using bidimensional chromatography followed by autoradiography, very small amounts of octopine can be clearly detected in tumors grown in vitro. Using this method, octopine was detected in tumor tissues when usual techniques were not sensitive enough.     

High-quality plant DNA extraction for PCR: an easy approach

Polymerase chain reaction has found wide applications in modern research involving transformations and other genomic studies. For reproducible PCR results, however, the quantity and quality of template DNA is of considerable importance. A simple and efficient plant DNA extraction procedure for isolation of high-quality DNA from plant tissues is presented here. It requires maceration of plant tissue of about 1.0 cm² (e.g. of a leaf blade) in DNA extraction buffer (100 mM Tris-HCl, 100 mM EDTA, 250 mM NaCl) using 1.5-mL microfuge tubes, followed by cell lysis with 20% SDS, and DNA extraction with phenol: chloroform: iso-amyl alcohol (25:24:1). Hydrated ether is then used to remove polysaccharides and other contaminants from the DNA preparation. Average DNA yield is 20–30 μg cm⁻² for fresh tissues, and ratio of absorbance at 260 nm to absorbance at 280 nm is 1.5–1.8. The DNA is quite suitable for PCR using microsatellites, RAPD and specific markers for recombinant selection. Amplifications have been obtained for these markers by using template DNA extracted from fresh as well as frozen leaf tissues of various plants, including barley, oat, potato and tomato. DNA stored for more than 2 years has been successfully amplified with microsatellite markers, which shows suitability of this method after long-term storage of DNA. Besides, the ease of use and cost-effectiveness make the procedure attractive.     

A preparation technique for analysis of explosives in plant tissues

A preparation technique for analysis of explosives compounds in plant tissue has been developed. The technique utilizes acetone extraction followed by ultrafiltration clean up. Ultrafiltration removes the large quantities of chlorophyll and polar organics that interfere with UV/Vis detection used in high‐performance liquid chromatography. The procedure was tested with 14 explosives‐related compounds spiked into reed canary grass extracts and method detection limits (MDLs) were calculated. In addition, the overall extraction efficiency for RDX in parrot feather and reed canary grass tissues was determined using ¹⁴C labeled compound. Scientists may find the technique useful for plant analysis at waste sites and for phytoremediation research.     

A highly sensitive method for the estimation of pyrimidine dimers in DNA by high-pressure liquid cation-exchange chromatography

We have developed a rapid, highly sensitive method for the separation of thymine-dimers from thymine on a cation-exchange resin, which is capable of detecting one dimer out of 5 × 10³ to 10⁴ thymine molecules (depending on the specific activity of the used material) by the means of high-pressure liquid chromatography. The assay has been very useful in DNA-hybridization studies using $\text{(}\text{TT}\text{)}$-containing DNA-strands and there is evidence that the method will be valuable for photoreactivation studies of uv-damaged DNA and for the enzymes involved in dimer-excision studies.     

Cation exchange properties and pectin content of storage-tissue disks

Summary The cation-exchange capacity (C.E.C.) of disks of storage tissue of sugar beet, red beet, potato, carrot, and swede turnip kept in running tap water at constant temperature, has been measured using an acid-washing technique. The pectin content of the disks has been estimated by measuring the CO₂ evolved on decarboxylation.There is quantitative agreement between exchange capacity and pectin content of the disks provided the former is measured on a tissue volume basis, and also provided that in assessing the latter, the substances yielding CO₂ yet not participating in cation exchange are first removed from the tissues by boiling in water. The C.E.C. and pectin content of the disks increase with time and reach a maximum 3 to 4 days after the disks are cut from the parent tissue.     

A radiometric microassay for choline acetyltransferase. Some observations on the spinal cord of the chicken embryo

This paper describes cation-exchange methods for separating acetyl[³H] coenzyme A from [acetyl-³H]choline. Blanks for the routine method were approximately 0.05% of the substrate radioactivity; product recoveries were approximately 97%. The cation-exchange method was moreefficient than the standard methods using either anion-exchange chromatography or periodide precipitation. The cation-exchange method was also morespecific than either of the other two standard methods for estimating choline acetyltransferase (ChAT) activity. ChAT activity was detected in the chicken lumbar spinal cord on embryonic day (E) 2 1/4 with the cation-exchange method. This developmental stage is about 6 hours before the final mitosis of any neuroblast in the ventral horn. Total ChAT activity per lumbar spinal cord increased more than 10,000-fold between E 3 and E 18. Changes in ChAT activity in the lumbar spinal cord following limb-bud extirpation appeared to mirror (with a phase lag) the changes in the number of motoneurons in the lateral motor column.     

13CO2示踪不同化学形态氮素添加对高寒草甸植物光合碳分配的影响

外源氮素(N)输入陆地生态系统后会引起植物和土壤各碳库的变化,但是对不同化学形态氮素的长期输入如何影响光合碳在植物组织、土壤、土壤呼吸中的分配及转运知之甚少,尤其是对于氮输入引起光合碳分配变化进而作用于植物和土壤碳库的机制的认识还非常匮乏。基于在青藏高原矮嵩草草甸开展的不同化学形态氮素添加的长期实验,利用~(13)C示踪方法揭示了光合碳在植物地上、地下组织的分配,及其随时间在土壤中的滞留和随土壤呼吸的释放。研究结果表明,外源氮素添加10年后,与对照未添加氮素处理相比,氨态氮处理下的地上生物量增加了49.5%,氨态氮处理下的地下生物量增加了111.3%。土壤中滞留的~(13)C整体呈下降趋势,氨态氮处理下的土壤碳库显著高于硝态氮处理下的值。不同处理下的土壤呼吸中~(13)C的滞留量随时间呈指数衰减的变化趋势,其中,硝态氮处理下的~(13)C衰减最快。~(13)C同位素标记后第1天测定植物茎和叶内的~(13)C约占刚刚标定完茎和叶内~(13)C的80%,不同处理之间没有显著性差异。直至标记后的第30天,茎和叶内~(13)C的滞留量约占初始量的30%。硝态氮处理下的值在第21天和第30天显著低于对照和氨态氮处理下的值,表明硝态氮处理下,植物光合固定的碳在短期内迅速输入地下组织和土壤中。这些结果从机理上阐明了植物光合碳分配对不同化学形态氮素长期输入的响应,进而影响到土壤呼吸CO_2的释放,以及对土壤碳库动态的贡献。加深了对高寒草甸土壤有机碳库稳定性维持机制的认识,能够为高寒草地的科学管理以及资源的可持续利用提供理论指导。     

High-efficiency liquid-scintillation counting of 14C-labelled material in aqueous solution and determination of specific activity of labelled proteins

1. Inexpensive scintillation mixtures are described which enable the detection of as little as 40μμc of ¹⁴C in aqueous solution with an efficiency of counting of over 80%. 2. A rapid method for the counting of alkaline, acidic and neutral aqueous solutions of up to 1ml. volume is described. Ethanol or 2-ethoxyethanol is used as blending agent. 3. The scintillation counting of alkaline solutions is applied to the accurate determination of the specific activity of ¹⁴C-labelled proteins from plant tissues. 4. Attention has been paid to the importance of a standardized washing procedure for the removal of all traces of radioactive material from glassware.     

Strongly acidic auxin indole-3-methanesulfonic Acid: synthesis of [C]indole-3-methanesulfonic Acid and studies of its chromatographic, spectral, and biological properties

A radiochemical synthesis is described for [¹⁴C]indole-3-methanesulfonic acid (IMS), a strongly acidic auxin analog. Techniques were developed for fractionation and purification of IMS using normal and reverse phase chromatography. In addition, the utility of both Fourier transform infrared spectrometry and fast atom bombardment mass spectrometry for analysis of IMS has been demonstrated. IMS was shown to be an active auxin, stimulating soybean hypocotyl elongation, bean first internode curvature, and ethylene production. IMS uptake by thin sections of soybean hypocotyl was essentially independent of solution pH and, when applied at a 100 micromolar concentration, IMS exhibited a basipetal polarity in its transport in both corn coleoptile and soybean hypocotyl sections. [¹⁴C]IMS should, therefore, be a useful compound to study fundamental processes related to the movement of auxins in plant tissues and organelles.     

Measurement of Bremsstrahlung radiation for in vivo monitoring of 14C tracer distribution between fruit and roots of kiwifruit (Actinidia arguta) cuttings

In vivo measurements of ¹⁴C tracer distribution have usually involved monitoring the β^? particles produced as ¹⁴C decays. These particles are only detectable over short distances, limiting the use of this technique to thin plant material. In the present experiments, X-ray detectors were used to monitor the Bremsstrahlung radiation emitted since β^? particles were absorbed in plant tissues. Bremsstrahlung radiation is detectable through larger tissue depths. The aim of these experiments was to demonstrate the Bremsstrahlung method by monitoring in vivo tracer-labelled photosynthate partitioning in small kiwifruit (Actinidia arguta (Siebold &; Zucc.) Planch. ex Miq.) plants in response to root pruning. A source shoot, consisting of four leaves, was pulse labelled with ¹⁴CO₂. Detectors monitored import into a fruit and the root system, and export from a source leaf. Repeat pulse labelling enabled the comparison of pre- and post-treatment observations within an individual plant. Diurnal trends were observed in the distribution of tracer, with leaf export reduced at night. Tracer accumulated in the roots declined after approximately 48 h, which may have resulted from export of ¹⁴C from the roots in carbon skeletons. Cutting off half the roots did not affect tracer distribution to the remaining half. Tracer distribution to the fruit was increased after root pruning, demonstrating the higher competitive strength of the fruit than the roots for carbohydrate supply. Increased partitioning to the fruit following root pruning has also been demonstrated in kiwifruit field trials.     

Free sphingoid bases in normal murine tissues

Free sphingoid bases, which have been considered not to occur naturally, were detected in murine tissues by derivatization with o-phthalaldehyde and the use of high-performance liquid chromatography. The concentrations were 10-30 pmol/mg tissue. The lung contained the largest amounts of sphingoid bases. In the molecular species of sphingoid bases, the most abundant was C18-sphingenine followed by C18-sphinganine, 4-hydroxysphinganine and C20-sphingenine, in that order. The central nervous tissues contained relatively high amounts of C20-sphingenine and there was a high concentration of 4-hydroxysphinganine in the kidney. In addition, galactosylsphingenine was detected simultaneously in the spinal cord and sciatic nerve. Sphingoid bases were purified from normal murine lungs using lipid-extraction, cation-exchange and silicic acid column chromatographies, alkaline saponification and preparative thin-layer chromatography. In the purified sphingoid bases, erythro-C18-sphingenine and erythro-C18-sphinganine were identified using thin-layer chromatography, high-performance liquid chromatography and fast-atom-bombardment mass spectrometry. Free sphingoid bases occurring in normal tissues may be metabolic intermediates required for the synthesis or be products of degradation of the sphingolipids and function to regulate cellular metabolism.     

Sink to source translocation in soybean

The possibility that phloem loading may occur in the reproductive sink tissues of soybeans (Glycine max Merr. cv Chippewa 64) was examined. When [¹⁴C]sucrose was applied to seed coat tissues from which the developing embryo had been surgically removed, 0.1% to 0.5% of the radioactivity was translocated to the vegetative plant parts. This sink to source translocation was largely unaffected by destroying a band of phloem with steam treatment on the stem above and below the labeled pod. The same steam treatment, however, completely abolished translocation of [¹⁴C]sucrose between mature leaves and developing fruits. These results indicate that the movement of nutrients from developing seed coats to the vegetative plant parts occur in the xylem and that phloem loading does not occur in this sink tissue.     

Determination of monosaccharides and sugar alcohols in tissues from diabetic rats by high-performance liquid chromatography with pulsed amperometric detection.

A sensitive and simple high-performance liquid chromatographic method has been developed to determine the concentration of monosaccharides and sugar alcohols in animal tissues. Five neutral monosaccharides (D-glucose, D-galactose, D-mannose, D-fructose, and D-ribose) and three neutral sugar alcohols (myo-inositol, glycerol, and D-sorbitol) predominate in the renal cortices and sciatic nerves of rats. These monosaccharides and sugar alcohols were extracted with distilled water, purified by deproteinization with ethanol, a Sep-Pak C18 cartridge, and columns of Dowex 50W-X8 and Amberlite CG-400, then separated on Ca2+ and Pb2+ cation-exchange columns, eluted with deionized distilled water at 80 degrees C, and detected using integrated pulsed amperometry. About 10 pmol of each sugar was detectable with a signal-to-noise ratio of 10:1. D-Glucose, D-fructose, D-sorbitol, and D-mannose were higher in both the renal and sciatic tissues of diabetic rats than in those of normal animals. D-Ribose and glycerol were higher in the renal cortex of diabetic animals.     

Improved Method for HPLC Analysis of Polyamines, Agmatine and Aromatic Monoamines in Plant Tissue

The high performance liquid chromatographic (HPLC) method of Flores and Galston (1982 Plant Physiol 69: 701) for the separation and quantitation of benzoylated polyamines in plant tissues has been widely adopted by other workers. However, due to previously unrecognized problems associated with the derivatization of agmatine, this important intermediate in plant polyamine metabolism cannot be quantitated using this method. Also, two polyamines, putrescine and diaminopropane, also are not well resolved using this method. A simple modification of the original HPLC procedure greatly improves the separation and quantitation of these amines, and further allows the simulation analysis of phenethylamine and tyramine, which are major monoamine constituents of tobacco and other plant tissues. We have used this modified HPLC method to characterize amine titers in suspension cultured carrot (Daucus carota L.) cells and tobacco (Nicotiana tabacum L.) leaf tissues.     

Can cutin and suberin biomarkers be used to trace shoot and root-derived organic matter? A molecular and isotopic approach

Cutins of plant shoots and suberins, mostly present in roots could contribute to significant portions of stable soil organic matter. Their biomarker potential, residing in their unique compositions in different plant types, has been used previously to infer sources of organic matter in sediments. These aliphatic plant biopolyesters contain specific biomarkers, which may be used for tracing their fate in soils and sediments, when combined with stable ¹³C isotope labelling. In order to evaluate the potential use of cutin and suberin biomarkers as indicators of shoot and root contributions from C₃ and C₄ plant origins, the objectives of this study were to 1) identify their constitutive monomers, which are specific for shoots and roots of maize (C₄) and wheat (C₃); 2) evaluate the ¹³C differences between maize and wheat biomarkers. Mid-chain hydroxy carboxylic acids were mainly found in the aboveground biomass, while α,ω-alkanedioic acids were only present in the roots. The differences in the isotopic composition of the specific monomers between wheat and maize plants (17–18‰ for shoot markers, 19‰ for root markers) were larger than those observed for bulk plant tissues and close to those observed for lignin-derived phenols in other studies. These differences should make it possible to differentiate and quantify the different types and sources of organic matter in sediments and soils. In particular, the molecular and isotopic signatures of cutins and suberins can be used to evaluate the specific dynamics of root vs shoot tissues in soils using C₃/C₄ chronosequences.     

Double-standard isotope dilution assay: I. Quantitative assay of indole-3-acetic acid

Isotope dilution analysis for the quantitation of labile compounds has been limited in applicability by the amount of sample necessary to determine specific activity. A method is described for the analysis of radiolabeled compounds which allows the direct determination of specific activity by gas chromatography. It requires the availability of the radiolabeled internal standard, as is customarily used in an isotope dilution assay, and also requires a chemically related radiolabeled compound to serve as a second internal standard. It is this second internal standard, added in known amounts, that permits quantitation of the gas chromatography. The method is illustrated by assaying indole-3-acetic acid in plant extracts using [¹⁴C]indole-3-acetic acid as the internal standard and adding [¹⁴C]indole-3-butyric acid as the second internal standard for quantitation of the gas chromatographic procedures. Used with a nitrogen-specific thermionic detector the method is selective and is sensitive at the nanogram level. The synthesis of [2-ring-¹⁴C]indole-3-butyric acid is also described.     

A sand-zeolite culture system for simulating plant acquisition of potassium from soils

A sand-zeolite culture system simulating plant acquisition of K from soils has been developed. The system, a mixture of synthetic zeolite IE-96 and coarse-sand, provides K concentrations comparable to soils depending on the K concentration ratios of solutions loaded onto the cation-exchange sites of the zeolite and the ionic strengths of the nutrient solutions supplied during the period of plant growth. Tomato (Lycopersicon esculentum Mill.) strain 203, PI 124163, was used in this study. An increase in the amount of the loaded zeolite per culture pot did not shift solution phase K activity ratios (AR^k) of the culture system but did result in a linear increase of plant dry matter accumulation, indicating that K bioavailability is diffusion-limited in the sand-zeolite culture system as in soils.     

Development of an Efficient Protocol of RNA Isolation from Recalcitrant Tree Tissues

Isolation of RNA from recalcitrant tree tissues has been problematic due to large amounts of secondary metabolites and interfering compounds in their cells. We have developed an efficient RNA extraction method, which yielded high-quality RNA preparations from tissues of the lychee tree. The method reported here utilized EDTA, LSS, and CTAB to successfully inhibit RNase activities. It was found that a high ionic strength brought about by 2 M NaCl was necessary. In addition, secondary metabolites and other interfering compounds were effectively removed using sodium borate and PVPP under a deoxidized condition. The quality of purified RNA was tested by both RACE and Northern blotting analysis, ensuring that the RNA could be used for subsequent gene expression analysis. This method has been successfully applied to purify RNA from 15 other plant species. In conclusion, the protocol reported here is expected to have excellent applications for RNA isolation from recalcitrant plant tissues.