A DESEEDED AVENA TEST METHOD FOR SMALL AMOUNTS OF AUXIN AND AUXIN PRECURSORS

The main results presented in this article may be summarized as follows: 1. A test method with deseeded Avena seedlings for small concentrations of auxin and precursors of auxin has been described. 2. This method makes possible quantitative determinations of about ten times as low concentrations of hormone as can be obtained with the standard method, (a) Through an increase in the time of the test, so that nearly all the hormone applied can be utilized. (b) Through an increase in sensitivity of deseeded plants to unilaterally applied small concentrations of hormone. 3. The effect of deseeding in relation to curvature growth is primarily the prevention of auxin regeneration through the removal of the material for auxin synthesis, and in addition the prevention of physiological aging. 4. The mechanism of auxin synthesis in the tip of the coleoptile and the mechanism of auxin regeneration in the new physiological tip have been shown to be identical. 5. The application of the deseeded method is illustrated by determinations of auxin in primary leaves and coleoptile sections of Avena seedlings. 6. The deseeded method has been used as a test method for precursors of auxin obtainable from the coleoptile and from other sources. The method further makes possible a distinction between auxins and these substances which may become activated by the plant. 7. Evidence for the existence of a precursor of auxin in the plant is given (a) indirectly by determinations of the decrease in auxin synthesis in deseeded plants. (b) Directly by its isolation from the plant. 8. Precursors of hetero-auxin are demonstrated; their chemical nature and activation are briefly considered.     

Fluorescence titration with apoflavodoxin: a sensitive assay for riboflavin 5'-phosphate and flavin adenine dinucleotide in mixtures.

A method is described for determining riboflavin 5′-phosphate (FMN) and flavin adenine dinucleotide (FAD) in mixtures by fluorimetric titration with the FMN-specific apoprotein of flavodoxin from Peptostreptococcus elsdenii. Accurate determinations can be carried out in the presence of a variety of compounds that decrease the fluorescence yield of FMN; the method may therefore be especially useful in the analysis of crude protein-free extracts of biological materials.     

Sesquiterpene and diterpene lactones from Melampodium leucanthum and the molecular structure of 4(5)-dihydromelampodin B

The aerial parts of Melampodium leucanthum afforded a novel diterpene-γ-lactone, 1,6,7-trihydroxy-17-acetoxymelcantholide, and two new sesquiterpene dilactones, 4(5)-dihydrocinerenin and 4(5)-dihydromelampodin C. The known melampolides, leucanthin A, melampodin A acetate, melampolidin and leucanthinin were also isolated. The structure determinations involved spectral and chemical methods. The molecular structure of 4(5)-dihydromelampodin B, determined by X-ray diffraction, is described.     

Analysis of polyamines in higher plants by high performance liquid chromatography

A sensitive (0.01-1 nmol) method has been developed for the analysis of polyamines in higher plant extracts based on high performance liquid chromatography (HPLC) of their benzoyl derivatives (Redmond, Tseng 1979 J Chromatogr 170: 479-481). Putrescine, cadaverine, agmatine, spermidine, spermine, and the less common polyamines nor-spermidine and homospermidine can be completely resolved by reverse phase HPLC, isocratic elution with methanol:water (64%, v/v) through a 5-μm C₁₈ column, and detection at 254 nm. The method can be directly applied to crude plant extracts, and it is not subject to interference by carbohydrates and phenolics. A good quantitative correlation was found between HPLC analysis of benzoylpolyamines and thin layer chromatography of their dansyl derivatives. With the HPLC method, polyamine titers have been reproducibly estimated for various organs of amaranth, Lemna, oat, pea, Pharbitis, and potato. The analyses correlate well with results of thin layer chromatography determinations.     

Determination of reaction rate constants in the mammillary system

It is shown that the individual rate constants can be determined for the composite chemical system: $$A + B_i \rightleftarrows C_i ; i = 1...N$$ with only measurements of the unbound species,A(t), required. The dissociation rate constants can be determined by direct analysis of a single steady state tracer study. The association constants then follow from the analysis of stable equilibrium determinations reported earlier (Hart, 1965). An approximate solution when tracer methods are in-applicable is also given.     

Automated fluorometric amino acid analysis: the determination of proline and hydroxyproline.

A fluorometric method for the automated determination of the imino acids proline and hydroxyproline has been developed. The assay is based on the postcolumn reaction of the imino acids with alkaline sodium hypochlorite, which yields oxidation products amenable to detection with fluorogenic amine reagents. The method is simple and can be adapted readily to high-sensitivity amino acid analyzers which use o-phthalaldehyde for detection. As little as 10 pmol proline and 20 pmol hydroxyproline can be determined accurately. Thus the full array of natural imino and amino acids can now be determined on a high-sensitivity amino acid analyzer using o-phthalaldehyde.     

Temperature Effects on Race Determination in Heterodera glycines

Currently there are 16 possible races for Heterodera glycines, and these are differentiated based on ability of a nematode population to develop on a set of four differential soybean genotypes. Because results are based on numbers of nematode females that develop to a specific stage rather than on the reproductive capability of these females, race determinations based on female indices may not represent results obtained after several reproductive cycles of H. glycines. Counting numbers of eggs and juveniles, and then developing corresponding indices, would allow reproduction to be considered in making race determinations. Our objectives were to compare the present race identification scheme for H. glycines based on female indices with those using egg and juvenile indices and to examine the effect of temperature on race designations using female, egg, and juvenile indices. Race designations for H. glycines populations from two locations in Illinois were determined at 20, 27, and 30 °C in a water bath. The numbers of females, eggs, and juveniles (at 19 days) were recorded, and an index based on each life stage was calculated. Race determinations based on female, egg, or juvenile indices were inconsistent when conducted at 20 °C, which demonstrates that this temperature is not suitable for identifying races of H. glycines. However race designations at 27 and 30 °C were consistent for all three indices. This indicates that counting females, eggs, or juveniles should be equally reliable when race determinations are conducted at these two temperatures, and choice of method would depend on investigator preference or research objective.     

Gas chromatographic assay of total and O-acetyl groups in bacterial lipopolysaccharides

An improved gas chromatographic method for the determination of total, i.e., amide- and ester-linked, acetyl groups in hydrolysates of bacterial lipopolysaccharides has been developed. After hydrolysis with 0.2 n HCl overnight at 100°C and adjustment of the hydrolysate to pH 3–4, 2 μl of the samples contalning 0–6 μg of acetic and 1–2 μg of propionic acid are directly injected into a gas chromatograph fitted with a 1.5-m glass column packed with Porapak QS. O-Acetyl groups can selectively be determined after hydrolysis with 0.05 n NaOH for 3–4 hr at room temperature and adjustment of the sample to pH 3–4. N-Acetyl groups can be calculated as the difference between total and O-acetyl. The above-mentioned phase allows quantitation of short-chain volatile fatty acids (S-C VFAs) up to n-valeric acid in a range of 0–6.0 μmol using an appropriate internal standard.     

Lowry protein determination on membrane preparations: need for standardization by amino acid analysis

The protein content of three membrane protein preparations has been determined by the Lowry method with bovine serum albumin as a standard and also by quantitative amino acid analysis as an absolute method. The results differ considerably, the Lowry method giving 29–42% higher values. This implies that many published data for such proteins, based on Lowry protein determinations with bovine serum albumin as the generally applied standard, are in error. Suggestions are made on how to standardize the Lowry method so that reliable values can be obtained for membrane protein.     

A simple determination of hexosamines in column effluents

A simple method of determinations of three hexosamines is presented. The solutions of hexosamines heated in 1 n NaOH at 60°C for 60 min, absorb ultraviolet light with the maximum at 302 nm. A linear relationship exists between the concentration of hexosamine up to 100 μg and the absorbance at 302 nm. N-acetylated hexosamines, having been deacetylated, may be determined in this way. The characteristic absorption curve ranging from 225 to 360 nm with the maximum at 302 nm may be utilized as a qualitative criterion for identification of hexosamines and N-acetylated hexosamines after their deacetylation.     

Syntheses,properties, and use of fluorescent N-(5′-phospho-4′-pyridoxyl)amines in assay of pyridoxamine (pyridoxine) 5′-phosphate oxidase

A sensitive and simple fluorometric assay has been developed for detection of pyridoxamine (pyridoxine) 5′-phosphate oxidase. This technique utilizes fluorescent N-(5′-phospho-4′-pyridoxyl)amines as substrates that, upon incubation with the oxidase, release the free fluorescent amine. The substrates were prepared by condensation of pyridoxal 5′-phosphate with fluorescent amines and subsequent hydrogenation of the Schiff bases. Since N-(1-naphthyl)ethylenediamine is 15 times less fluorescent in the intramolecularly quenched substrate than the product amine, the direct increase of fluorescence, as well as selective extraction of more fluorescent product, can be utilized for assay. The apparent K_m value for this substrate is 8 μm, which is slightly less than that of pyridoxamine 5′-phosphate; V is larger than the natural substrate value. The greater sensitivity gained by this fluorimetric method allows detection of the oxidase in smaller quantities than can be determined by the conventional colorimetric assay.     

New clerodanes from Symphiopappus itatiayensis

The isolation and structure determinations of two new ent-cis-clerodane dilactones from Symphiopappus itatiayensi are reported.     

A Proposal for the Use of Standardized Methods for Chlorophyll Determinations in Ecological and Eco-physiological Investigations

The various methods for chlorophyll determination, found in the literature, may give chlorophyll values differing by as much as 20%. Not only the choice of solvent and equations but also the routines and conditions for processing of samples can be large sources of errors. In the present paper some recommendations are given concerning sampling and storage of plant material, extraction and determination of chlorophylls. A simple and fast method for chlorophyll determinations is described and recommended as a suitable method for most ecological and eco-physiological investigations in which chlorophylls are determined. The principle of the method is that the total amount of chlorophylls is determined in a colorimeter calibrated for chlorophyll measurements. Results from a prototype of the “Chlorophyllometer” are presented and compared with spectrophotometric determinations.     

Larval development,growth and age determination in the sharpnose pufferfishCanthigaster valentini (Teleostei: Tetraodontidae)

The eggs, early larvae and juveniles of the sharpnose pufferfishCanthigaster valentini are described, based on material collected in Great Barrier Reef waters. Eggs were obtained in the field by divers and reared in the laboratory. The eggs are spherical, strongly adhesive, 0.68–0.72 mm in diameter, possess a dense cluster of small oil droplets, and hatch around sunset 3 to 5 days after fertilization. Newly hatched larvae have a small yolk sac, pectoral fin folds, 17 myomeres (6 pre-anal, 11 post-anal) and measure 1.30–1.40 mm in notochord (standard) length. The eggs ofC. valentini differ from those of other tetraodontids in being much smaller and having a longer incubation time. The larvae can be distinguished from other tetraodontid larvae by pigmentation, myomere count and size at hatching. Growth is most rapid during the first day of larval life. Age determinations (based on otolith microstructure) of field collected juveniles, both pelagic and newly settled, indicate a pelagic phase of between 64 and 113 days for this species. This estimate appears consistent with the extended pelagic juvenile stages observed in other tetraodontiform fishes and could indicate thatC. valentini can delay settlement for some time after becoming competent to settle at a minimum age of 64 days.     

A sensitive, precise, and convenient method for determination of 1,25-dihydroxyvitamin D in human plasma.

A new, highly sensitive and relatively convenient method has been developed for the determination of 1,25-dihydroxyvitamin D₃ and 1,25-dihydroxyvitamin D₂ in blood plasma. The method involves a simplified and more specific extraction procedure, new rapid and effective methods of purification, and a competitive binding assay using intestinal cytosol from rachitic chicks. The method also includes a procedure for stabilizing the cytosol binding protein and a convenient procedure for the separation of bound from free 1,25-dihydroxyvitamin D₃ with the use of polyethylene glycol. The recovery of 1,25-dihydroxyvitamin D₃ during extraction and purification is 68% and triplicate determinations can be made on a 5-ml plasma sample. With this method, rachitic chick plasma, plasma from anephric patients, and plasma from patients suffering severe endstage renal failure show no detectable 1,25-dihydroxyvitamin D, while normal human values have been found to be 29 ± 2 pg/ml.     

Determination of asparagine and glutamine in polypeptides using bis(I,I-trifluoroacetoxy)iodobenzene

A simple yet accurate method is described by which the numbers of asparagine and glutamine residues in polypeptides can be determined. The method involves difference analysis of the aspartic acid and glutamic acid contents of the polypeptide after acid hydrolysis (in 6 n HCl), without and with prior treatment of the sample with bis(I,I-trifluoroacetoxy)iodobenzene. Under the conditions described, this reagent quantitatively converts the carboxamide residues to the corresponding amines, which are eluted near (and interfere with the estimation of) the lysine peak on conventional ion-exchange amino acid analysis. During the carboxamide conversion, certain amino acid residues sensitive to oxidation are partially or completely destroyed and cannot be accurately determined.     

Quantitative and rapid estimation of h fluxes in membrane vesicles : software for analysis of fluorescence quenching and relaxation

Proton transport is often visualized in membrane vesicles by use of fluorescent monoamines which accumulate in acidic intravesicular compartments and undergo concentration-dependent fluorescence quenching. Software for an IBM microcomputer is described which permits logging and editing of changes in fluorescence monitored by a Perkin-Elmer LS-5 luminescence spectrometer. An accurate estimate of the instantaneous rate of fluorescence quenching or recovery is then facilitated by least squares fitting of fluorescence data to a nonlinear function. The software is tested with tonoplast vesicles from Beta vulgaris. Quenching of acridine orange fluorescence by ATP-driven (primary) transport and relaxation of quenching by Na⁺/H⁺ antiport can both be fitted with single exponential functions. Initial rates of ATP- and Na⁺ -dependent fluorescence changes are derived and can be used for K_m determinations. The method constitutes a simple and efficient alternative to manual analysis of analog fluorescence traces and results in a reliable quantitative measurement of the relative rate of proton transport in membrane vesicle preparations.     

A ‘simple clock’ approach of circadian rhythms: An easy way to predict the clock's singularity

A graphical method is presented which allows the prediction of phase resetting curves of circadian rhythms for both type 1 and type 0 resetting, starting from one experimentally determined phase resetting curve. Calculations were based on literature data for the pupal eclosion rhythm of Drosophila pseudoobscura. The method is based on the assumption that for all practical purposes the rhythm may be approached as a “simple clock”, i.e. an oscillator with only one state variable, namely its phase or circadian time, CT. Besides predicting both “types” of phase resetting the method is capable to locate the “position” of the phase singularity and thus the transition from type 1 to type 0 resetting. This graphical method is an elaboration of the “transformation method”, developed in 1972 by A. Johnsson and H. G. Karlsson, which was effective in predicting phase resetting by “strong” stimuli, but failed in the case of “weak” stimuli. Predictions made using the extended transformation method are in good agreement with experimental results obtained with Drosophila. Also for the flesh fly, Sarcophaga argyrostoma, a prediction is made of the position of the phase singularity of the eclosion rhythm and compared with experimental results.     

A sensitive analytical method for the detection and quantitation of adenosine in biological samples

A new method for the measurement of adenosine in biological materials has been developed. The method is based on the combined principles of isotope dilution and enzymatic catalysis using a highly specific adenosine kinase isolated from rat heart. By differential centrifugation and gel filtration, this adenosine kinase was obtained free of adenosine deaminase and other enzymes which would have been a source of error in the use of this enzyme in the adenosine assay. The cardiac adenosine kinase was shown to be highly specific and to exhibit an apparent K_m for adenosine of 0.35 μM. Using this enzyme, unknown quantities of adenosine could be detected by measuring the effect of their addition on the conversion of radioisotopic adenosine to 5′-AMP in the kinase reaction. In this procedure, as little as 20 pmoles of adenosine could be detected. To test the applicability of the assay, measurements of the tissue content of this nucleoside were made in samples of dog and rat hearts frozen in situ under control, hypoxic, or ischemic conditions. The assay has several advantageous features when compared to other existing methods used to measure adenosine: a minimum of sample preparation is required before the actual assay procedure; many samples can be processed per day by a single operator; single determinations can be done on as little as 5 μl of sample, and the specificity of the assay can be readily checked by treatment of samples with adenosine deaminase.     

高效液相色谱法分析猪屎豆种子胶多糖中的单糖组成

建立了一种采用SUGAR SP-G 0810(Pb型)糖分析色谱柱,以纯水为流动相,利用示差折光检测器的高效液相色谱外标法直接分离分析半乳甘露聚糖胶中单糖组成的方法。木糖、葡萄糖、半乳糖和甘露糖的分离在20 min内完成,检出限分别达到2.0μg,20μg,1.0μg和20μg,线性范围为2～10 mg/mL。该方法简便、快速、重现性好,用于猪屎豆种子胶多糖中单糖组分的测定,并进行回收率试验,结果5次测定的半乳糖和甘露糖的回收率分别为95.83%和103.68%,RSD分别为1.53%和1.50%。