Non-radioactive detection of ß-glucuronidase and chloramphenicol acetyltransferase activities in co-transformed protoplasts by HPLC.

The use of transient gene expression assays for the study of natural or engineered plant promoters is affected by a considerable degree of inter-experiment variability. As a means of obtaining interpretable data from a limited number of experiments, we worked out conditions for the simultaneous determi nation of the activity of two reporter genes, a "sample" and a "reference", ona single extract of co-transformed protoplasts. ß-glucuronidase (GUS) and chloramphenicol acetyl transferase (CAT) genes, both under the control of the CaMV 35S promoter, were transferred into tobacco (Nicotiana tabacum L.) protoplasts on two independent plasmids. The parallel expression of the two reporter genes in several independent co-transformation experiments was verified. Conditions for the use of a single protoplast extraction buffer and for the simultaneous assay of both reporter gene activities were set up. A HPLC method for the non-radioactive determination of both enzyme activities on a single aliquot of the reaction mixture was developed. The resulting procedure was tested using the GUS gene as "reference" and the CAT gene, under the control of either wild type or upstream-deleted (-90) CaMV 35S promoter, as "sample". The protocol is simple and allows the fast analysis of plant promoters in the presence of a true internal standard under conditions in which assay manipulations are reduced to a minimum and both reporter gene activities are subjected to the same experimental treatments.

Ion trap detector--capillary gas chromatography of valproic acid and its mono-unsaturated metabolites in serum using methyl ester derivatives.

A quantitative method was developed for valproic acid and five of its mono-unsaturated metabolites using capillary gas chromatography-mass spectrometry with selected ion monitoring. The method was applied to serum and all metabolites were measured in a single run. Methyl esters were synthesized as the derivatives suitable for gas chromatography. Calibration curves were found to be linear and the sensitivities in the order of 0.1 micrograms ml-1. Patients' data are presented. By this method it is possible to separate the stereoisomers of 2-n-propyl-2-pentenoic acid and of 2-n-propyl-3-pentenoic acid.

Determination of oxalate in urine by flow injection analysis.

A method is described for the determination of oxalate in urine using flow injection analysis and fluorimetry. Oxalate is precipitated with calcium chloride at pH 4.5, redissolved in H2SO4 and measured by flow injection analysis. The minimum detection limit is 6 mumol/l. The coefficient of variation is 7%. Results are in good accordance with normal values found with traditional oxalate analysis.

[Biological activity of 1,2-benzylisothiazoles. Relationship between the structure and the auxinlike activity of the 1,2-benzylisothiazol-3-ylacetic acid class]. / Attività biologica di composti 1,2-benzisotiazolici. Relazione fra struttura ed attivià auxinica nella classe degli acidi 1,2-benzisotiazol-3-ilacetici.

The LFER model was used to study quantitatively the interrelations between chemical structure and auxinic activity of some methyl esters of 1,2-benzisothiazol-3-ylacetic acids, using as biological response the cell enlargement induced in etiolated stems of Pisum sativum, var. Alaska. Having demonstrated that activity, expressed as log 1/C, is correlated, by Eq. 8, with factionary charge (-- Qs) calculated for the sulfur atom and with the logarithm of retention time (log t), determined by reverse phase HPLC, the AA. deal with the influences of the latter parameters on the effect registered.

[High pressure liquid-liquid chromatography: correlation between retention times and biological activity of 1,3,5-triazine derivatives]. / Cromatografia liquido-liquido ad alta pressione. Correlazione fra tempi di ritenzione ed attività biologica di derivati 1,3,5,-trazinici

An investigation has been made of the possibility of using the retention times t obtained by reversed phase high pressure liquid-liquid chromatography in the study of quantitative structure-activity relatiionships. Taking as an example a group of herbicides which inhibit the Hill reaction, it was found that log t is additive and can be used as the hydrophobic parameter in regression analyses. The characteristics, advantages and possible developments of the method are discussed and the analytical interest is highlighted.