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Mechanically isolated mesophyll cells of Zinnia elegans L. cv Envy differentiate to tracheary elements when cultured in inductive medium containing sufficient auxin and cytokinin. Tracheary element differentiation was induced by the three auxins (alpha-naphthaleneacetic acid, indole-3-acetic acid, and 2,4-dichlorophenoxyacetic acid) and four cytokinins (6-benzyladenine, kinetin, 2-isopentenyladenine and zeatin) tested. Tracheary element formation is inhibited or delayed if the inductive medium is supplemented with an anticytokinin, antiauxin, or inhibitor of auxin transport.

Tomato plants homozygous for the diageotropica (dgt) mutation exhibit morphological and physiological abnormalities which suggest that they are unable to respond to the plant growth hormone auxin (indole-3-acetic acid). The photoaffinity auxin analog [3H]5N3-IAA specifically labels a polypeptide doublet of 40 and 42 kilodaltons in membrane preparations from stems of the parental variety, VFN8, but not from stems of plants containing the dgt mutation. In roots of the mutant plants, however, labeling is indistinguishable from that in VFN8. These data suggest that the two polypeptides are part of a physiologically important auxin receptor system, which is altered in a tissue-specific manner in the mutant.

1. Analytical methods (isolation, TLC, UV-spectroscopic and preferably fluorimetric determination) of [1-(p-chlorobenzoyl)-5-methoxy-2-methylindole-3-acetoxy] acetic acid (acemetacin, TV 1322, Rantudil), its analogues, hydrolysis-products and metabolites from biological material are described. 2. Stability is also dealt with. Hydrolysis of the p-chlorobenzoyl group results in strongly fluorescing compounds and can be used in determination of acemetacin, indometacin and derivatives 3. Proof is given that in in vitro studies, such as e.g. Mizushima's protein turbidity test or the test for inhibition of prostaglandin-synthetase, only intact acemetacin (and none of its potential break-down products) is present and is active. 4. In in vitro studies, such as the inhibition of total complement and of the increase in activity of serum sulfhydryl groups, acemetacin and a series of its salts proved to be at least as effective as indometacin. 5. In comparing protein binding, acemetacin, like indometacin, is found to bind strongly to albumin. The portion of free, non-protein bound acemetacin, which thus remains available for the pharmacological action, is found to be approx. 60% higher than the corresponding portion of free indometacin.

We have recently reported that type II diabetic subjects with macroangiopathy have a higher activity of aldehyde dehydrogenase (ALDH) in blood than those without clinical vascular disease. ALDH activity was measured as the elimination of acetaldehyde added to a blood homogenate in vitro. We have re-examined our clinical material with another assay of ALDH which uses indole-3-acetaldehyde as substrate and measures the formation of indole-3-acetic acid. A negative correlation between the half-life of acetaldehyde and the formation of indole-3-acetic acid was found in the group of subjects free from vascular disease (r = -0.55, p less than 0.01). Thus, a rapid elimination of acetaldehyde corresponded to a rapid formation of indole-3-acetic acid. No such correlation was found in subjects with macroangiopathy. These results suggest that the 2 groups, with and without clinical vascular disease, have differences in isoenzyme composition, in the kinetic properties of the enzyme, or in the non-enzymatic binding of acetaldehyde.

A simple and sensitive method using high performance liquid chromatography with fluorometric detection has been developed for the identification and quantitation of the endogeneous tryptamine metabolites, indoleacetic acid (IAA) and tryptophol (TOL) in the normal mouse brain. The limits of sensitivity are 5pg for both IAA and TOL. The extract procedure from the brain is only to deproteinize samples. The mean concentrations of IAA and TOL in the mouse brain are 8.99 +/- 0.31 ng/g and 3.56 +/- 0.21 ng/g respectively. The effects of pargyline and tryptamine on the levels of IAA and TOL were also studied.

Performance of the apparatus was evaluated in terms of stationary phase retention, partition efficiency and sample loading capacity. Preliminary studies with short coils revealed high retention of the stationary phase under a proper combination of the head-tail elution and planetary motion. Preparative capability of the apparatus was successfully demonstrated on efficient multigram separations of 2,4-dinitrophenyl amino acids, indole auxins, and bacitracin in a pair of large multilayer coils with a total capacity of 1.5 l.

The pattern of ontogenetic development of tryptophan (TP), tryptamine (T), indole-3-acetic acid (IAA), 5-hydroxytryptamine (5-HT; serotonin), and 5-hydroxyindole-3-acetic acid (5-HIAA) in the brains of rats aged 1-45 days is presented. Analysis of the five components in each brain allows the calculation of the acid/amine and amine/amino acid ratios. These metabolic indexes are a useful tool to study and compare the metabolic origins and fates of both amines. The ontogenetic patterns of TP, T, and IAA are very similar, especially during the first week postpartum. The highest and lowest levels found for T were 2.2 ng/g and 0.1 ng/g at the 1st and 5th day, respectively. The temporal relationship between the T/TP and IAA/T ratios suggests the existence of mechanisms protecting T against monoamine oxidase (MAO) which develop in parallel to synaptogenesis. Significant correlations were found between TP and IAA during the whole period studied and between TP and T during the first week after birth. The 5-HT peak found during the first postpartum week could be due to a non-neuronal pool of 5-HT protected against MAO and possibly contained in mast cells. Preliminary determinations on leptomeningeal membranes suggest the existence of such a pool.

When indole-3-acetaldehyde was incubated with rat brain tissue, an aldehyde dehydrogenase-independent irreversible disappearance of the aldehyde was found. This was accompanied by an increase in absorbance at 240-400 nm, with a peak at 310 nm. The results suggested that this change in absorbance was caused by a membrane-bound nonenzymatic reaction between indole-3-acetaldehyde and phospholipids. A similar reaction occurred between indole-3-acetaldehyde and pure preparations of phosphatidylethanolamine and phosphatidylserine, but not phosphatidylcholine. Indole-3-acetaldehyde levels also decreased slightly when incubated with albumin but absorbance at 310 nm was unaltered. It is suggested that nonenzymatic reactions between indole-3-acetaldehyde (or other biogenic aldehydes) and membrane components might occur in vivo, and could be involved in the effects of drugs such as ethanol and barbiturates.

The cross-axis synchronous flow-through coil planet centrifuge with a 20-cm revolutional radius and a total capacity of 1600 ml was successfully applied to preparative counter-current chromatography of various biological samples, which include sea buckthorn extract, steroid reaction mixture, indole plant hormones, and dinitrophenylamino acids. The present system offers advantages of stable balance of the centrifuge, a large column capacity, and high resolution.

A method for the separation of substituted indole derivatives has been developed by the use of ion suppression-reversed-phase high-performance liquid chromatography (IS-HPLC). Signal response, selectivity (alpha), retention times (tR), and capacity factors (k') were monitored by varying the mobile phase with respect to methanol composition and pH. Chromatographic parameters including tR, k', K (distribution coefficients), alpha, number of theoretical plates, height equivalent to theoretical plates, and column resolution were calculated and assessed in regard to the suitability of four stationary phases in the analysis of 20 substituted indole derivatives. This work has established the chromatographic foundation needed to analyze over 10 specific enzyme reactions involved in the microbial and plant metabolism of auxins. Quantitative studies on the stability of auxins were possible by employing IS chromatography (ISC). The application of the developed IS chromatographic technique was employed to detect indole-3-acetic acid derived from L-tryptophan in a fluorescent pseudomonad culture.

Indoleacetic acid (IAA) is produced in cancer tissue and promotes the growth of cancer cells and of the surrounding cells which become cancerous. It was shown that the urine of cancer patients contains larger amounts of IAA than that of healthy persons (except diabetics). This increased IAA level may be derived from the cancer tissue. The measurement of urinary IAA can thus be used to screen cancer patients. Various assay methods for IAA were tested, but 15 min. are required even with the modified HPLC used in this laboratory directly with unpurified urine. Hence it is unsuitable for measuring large numbers of samples within short period. The present work reports on a simple, easy and rapid method for determining urinary IAA in large numbers of samples by using Avena coleoptile sections. Ten sections are floated for 3 hr. on 1/50 diluted urine with 0.5 ppm gibberellic acid (GA). Up to a certain limit their elongation is proportional to the IAA concentration in the diluted urine. Persons whose urine causes coleptile section to elongate more than 1.3 mm require detailed clinical examination to determine whether or not they suffer from cancer.

When cultured in inductive medium containing adequate auxin and cytokinin, isolated mesophyll cells of Zinnia elegans L. cv Envy differentiate into tracheary elements with lignified secondary wall thickenings. Differentiation does not occur when cells are cultured in control medium, which has reduced levels of auxin and/or cytokinin. The activities of two enzymes involved in lignin synthesis, 4-coumarate:coenzyme A ligase and peroxidase, were examined. An induction-specific cationic isoperoxidase, visualized by low pH polyacrylamide gel electrophoresis, is detectable in soluble and wall fractions of cultured Zinnia cells long before tracheary elements visibly differentiate and is thus an early marker of differentiation. Compounds (such as antiauxins, anticytokinins, and tunicamycin) that inhibit or delay differentiation alter the expression of this isoperoxidase. 4-Coumarate:coenzyme A ligase activity increases dramatically only as cells differentiate. Together, these results suggest that the onset of lignification in differentiating Zinnia cells might be controlled by the availability of precursors synthesized by way of 4-coumarate:coenzyme A ligase. These precursors would then be polymerized into lignin in the cell wall by the induction-specific isoperoxidase.

We have measured the content of the auxin, indole-3-acetic acid (IAA), in cloned, crown-gall teratoma line of Nicotiana tabacum L. cv. "Turkish" by a highly specific and sensitive radioimmunoassay. This tissue line, which does not require auxin for continuous growth in culture, exhibits two phases of growth. During the first phase, which lasts about two weeks after subculturing, growth is exponential on a fresh weight basis and the content of IAA is about 10-7-10-6 mol.kg-1, but variable. During the second phase, growth rate declines gradually and the IAA content of the tissue drops dramatically; however, this drop does not result from a net loss of auxin by the tissue. The rate of growth during the exponential phase was not correlated with the IAA content of the tissue, but was strongly correlated with the IAA content of the inoculum. We found that rapidly growing leaves of Turkish tobacco have roughly the same IAA content as cultured teratoma tissues. Moreover, both tissues exhibited a similar relationships between auxin content expressed per leaf or per tissue explant and growth. These findings do not support the hypothesis that the autonomous growth of plant tumors results from an abnormally high content of auxin in the tissue.

A method of analysis of 3-indolylacetic acid (IAA) and abscisic acid (ABA), allowing the simultaneous extraction of both regulators from plant material, has been developed. The method involves extraction with methanol, isolation of the acid fraction, diazomethane methylation, separation of the hormones through reverse-phase preparative high-performance liquid chromatography, and quantification of both compounds by gas-liquid chromatography. The recovery percentage at each step was monitored with radioactive compounds added at the beginning of the process. The final recovery was 70% for IAA and 96% for ABA. The method was applied to the analysis of the IAA and ABA content of stems of hazel (Corylus avellana L.).

We have separated and identified three endogenous, naturally fluorescent substances in uremic hemodialysate by using reversed-phase liquid chromatography with fluorescence detection. Co-chromatography with authentic standards, monitoring peak shifts after enzymic treatment, and spectrofluorescence measurements were used to confirm the identity of indican, tryptophan, and indole-3-acetic acid. Concentrations of indican were about 1.5 those of tryptophan and considerably greater than those of indole-3-acetic acid in hemodialysate samples from 12 renal patients. These three compounds, as well as eight unidentified components, were consistently present in dialysate samples from each of the 12 patients.

The authors describe a method of estimation of clometacine in biological fluids based on extraction in the pure state of a pair of ions, purification by chromatography (in liquid phase, adsorption, then exchange, methylation by diazo-methane and gas chromatography associated with detection by electron capture.

Disseminated gonococcal infection occurs in 1%-3% of patients with gonorrhea in Seattle. Most strains of Neisseria gonorrhoeae recovered from patients with disseminated gonococcal infection are susceptible to less than or equal to 0.015 mug of penicillin G/ml. Strains susceptible to less than or equal to 0.015 mug/ml, whether isolated from patients with disseminated gonococcal infection or from those with uncomplicated infection, did not grow on chemically defined medium prepared with several lots of agar, whereas less susceptible isolates grew well. Use of methanol-extracted nontoxic agar permitted growth of the penicillin-susceptible strains and demonstrated that all of them required arginine, hypoxanthine, and uracil (Arg-Hyx-Ura-) for growth. These Arg-Hyx-Ura- strains accounted for 89% of the cases of disseminated gonococcal infection in Seattle, 38% of uncomplicated infections in Seattle, 18% of infections in Milwaukee, and none of 104 uncomplicated gonococcal infections acquired in the Philippines and Taiwan, where disseminated gonococcal infection is unusual. Regional variations in the prevalence of these Arg-Hyx-Ura- strains may correlate with regional variations in the incidence of disseminated gonococcal infection. The genetic, physiologic, and immunologic bases for concurrence of penicillin and agar susceptibility. Arg-Hyx-Ura- phenotype, and association with disseminated gonococcal infection remain undefined, as do host factors that determine whether or not disseminated gonococcal infection will result in patients infected by such strains.

Indole-3-acetic acid (IAA) and 2-(indol-3-ylmethyl)indol-3-yl acetic acid were identified in lipid extracts of Sulfolobus acidocaldarius; they occurred at concentrations of 0.57 and 0.59 mumol/g (dry weight), respectively. The amount of IAA found in these cells is more than a thousand times greater than that found in a typical extract of a plant in which IAA serves as a plant growth hormone. Neither of these compounds was detected in the other archaebacteria that were analyzed; these included Sulfolobus sulfataricus, Halobacterium salinarium, and several strains of methanogenic bacteria. This is the first report of the natural occurrence of 2-(indol-3-ylmethyl)indol-3-yl acetic acid.

Methyl 4-chloroindolyl-3-acetate (MCIA), a naturally occurring auxin, has been identified in canned and frozen peas by gas-liquid chromatography (GLC)/mass spectrometry. Eight samples were analyzed for MCIA using slightly modified official AOAC multiple pesticide residue procedures employing GLC with microcoulometric detection. Levels of MCIA averaged 0.20 ppm for canned peas and 0.16 ppm for frozen peas. Recovery of MCIA from a fortified sample was 40%.

Endogenous tryptamine, 5-hydroxytryptamine, indoleacetic acid, 5-hydroxyindoleacetic and tryptophan have been recovered from urine and cerebro-spinal fluid by adsorption on XAD-2 resin (0.3 g). After adsorption of the sample on the resin, desorption with methanol provides a single fraction that contains all of these metabolites. The mass spectra of their pentafluoropropionyl derivatives show prominent ions at m/e 276 and 438 which are characteristic of indoles and 5-hydroxyindoles, respectively, a feature that allows the concurrent determination of all the components of each group by functional group analysis. A method has been developed to carry out single ion monitoring with the peak matching system of an Hitachi RMU-6H mass spectrometer. Identifications are based on the respective Kovats Indices and single ion monitoring of two characteristic ions per compound: tryptophan (m/e 276 and 347); tryptamine (m/e 276 and 289); indoleacetic acid (m/e 276 and 335); 5-hydroxytryptamine (m/e 438 and 451); 5-hydroxyindoleacetic acid (m/e 438 and 497). The method described illustrates the feasibility of assaying biogenic indoleamines and acidic metabolites, as well as their precursor amino acid on a single fraction in contrast to other standard fractionation methods. This is possible even if the mass spectrometer is not equipped with an alternating voltage accelerator provided that it has a peak matcher, although the lack of an alternating voltage accelerator requires two separate injections of the same sample, for quantification and identification; one for the indole profile and another for the 5-hydroxyindole profile. Both profiles can be verified by individual monitoring of the other confirmatory ions. With this method the use of a multiple ion detector would allow a simultaneous determination of all of these metabolites in one gas chromatograph mass spectrometer run.

Ethephon (2-chloroethylphosphonic acid) and fenoprop (2-(2,4,5-trichlorophenoxy) propionic acid) may be determined in the same apple sample. After extraction with methanol, 2 separate methylation procedures were required to quantitatively convert each compound. Ethephon was esterified with diazomethane and analyzed by a flame photometric detector in the P-mode. Fenoprop was esterified with boron trifluoride/methanol and analyzed by electron capture gas chromatography. Average recoveries were about 95% at 0.05 ppm for both compounds. The limit of detection was 0.05 ppm for ethephon and 0.01 ppm for fenoprop in a 1 g sample. The persistence of both compounds before and after harvest was studied. Ethephon and fenoprop were applied simultaneously to apple trees at the recommended concentrations of 300 and 20 ppm, respectively. Ethephon residues averaged 1.6, 0.75, and 0.4 ppm at 2 hr, 10 days, and after washing at 13 days, respectively. The corresponding fenoprop residues were 0.70, 0.025, and 0.024 ppm.