Interaction of sulfur and nitrogen nutrition in tobacco (Nicotiana tabacum) plants: significance of nitrogen source and root nitrate reductase.

The significance of root nitrate reductase for sulfur assimilation was studied in tobacco (NICOTIANA TABACUM) plants. For this purpose, uptake, assimilation, and long-distance transport of sulfur were compared between wild-type tobacco and transformants lacking root nitrate reductase, cultivated either with nitrate or with ammonium nitrate. A recently developed empirical model of plant internal nitrogen cycling was adapted to sulfur and applied to characterise whole plant sulfur relations in wild-type tobacco and the transformant. Both transformation and nitrogen nutrition strongly affected sulfur pools and sulfur fluxes. Transformation decreased the rate of sulfate uptake in nitrate-grown plants and root sulfate and total sulfur contents in root biomass, irrespective of N nutrition. Nevertheless, glutathione levels were enhanced in the roots of transformed plants. This may be a consequence of enhanced APR activity in the leaves that also resulted in enhanced organic sulfur content in the leaves of the tranformants. The lack of nitrate reductase in the roots in the transformants caused regulatory changes in sulfur metabolism that resembled those observed under nitrogen deficiency. Nitrate nutrition reduced total sulfur content and all the major fractions analysed in the leaves, but not in the roots, compared to ammonium nitrate supply. The enhanced organic sulfur and glutathione levels in ammonium nitrate-fed plants corresponded well to elevated APR activity. But foliar sulfate contents also increased due to decreased re-allocation of sulfate into the phloem of ammonium nitrate-fed plants. Further studies will elucidate whether this decrease is achieved by downregulation of a specific sulfate transporter in vascular tissues.

The use of the aquatic moss Fontinalis antipyretica L. ex Hedw. as a bioindicator for heavy metals: 3. Cd2+ accumulation capacities and biochemical stress response of two Fontinalis species.

We studied heavy metal stress responses of two Fontinalis species, F. antipyretica and F. dalecarlica, collected from two habitats in Germany and Canada. The capacities of the two species for extracellular adsorption (biosorption) and intracellular uptake (bioaccumulation) of Cadmium (Cd2+) were investigated in the laboratory. Time-dependent Cd2+ adsorption by cell wall and intracellular uptake differed significantly between the two species. These differences were related to the number of Cd2+ binding sites, resulting from differences in leaflet surface and cell wall composition. Glutathione (GSH) levels in response to Cd2+ exposure were monitored over a 10-day period. GSH synthesis differed significantly between the two species. Both Fontinalis species appear to be suitable for heavy metal biomonitoring in aquatic habitats.

Enantioselective high-performance liquid chromatography of therapeutically relevant aminoalcohols as their fluorescent 1-naphthyl isocyanate derivatives.

A method for determining the enantiomers of 10 therapeutically relevant aminoalcohols using HPLC and precolumn derivatization was developed. Naphthyl isocyanate reacted with racemic aminoalcohols to form urea derivatives which were separated isocratically on a cellulose tris(3,5-dimethylphenylcarbamate) coated silica gel column, and detected fluorometrically in the lower ng mL(-1) range. The effluents can also be monitored at lower sensitivity, using an ultraviolet detector operated at 220 nm.

Synthesis and biological activity of canavanine hydrazide derivatives.

The canavanine derivatives L-canavanine hydrazide (CH), L-canavanine-bis-(2-chloroethyl)hydrazide (CBCH) and L-canavanine phenylhydrazide (CPH) were synthesized and evaluated for biological activity in microorganisms, plants and tumor cells using canavanine as a positive control. (1) In microbial systems, the compounds exerted activity, as assessed in 14 bacterial strains. The effect of canavanine was easily removed by equimolar concentrations of arginine or ornithine, while the effect of CBCH or CPH was abolished by 10-fold excess of arginine or 10- to 100-fold excess of ornithine. (2) In plants, the activity of CH and CBCH were relatively low, whereas the inhibitory potential of CPH was comparable or even superior to that of canavanine, resulting at 1 mM concentration in a nearly complete block of tomato cell growth, and reducing by up to 80% the length of radicles of cress, amaranth, cabbage and pumpkin. (3) In pumpkin seeds, CPH or canavanine induced the synthesis of four small heat shock proteins of hsp-17 family in the pH range of 6 to 7.5. The proteins exhibited in both cases a similar profile, but differed in the timing of their expression and/or accumulation. With canavanine, the highest hsp-17 expression was found after 48 h of drug treatment, while with CPH this maximum was shifted to 24 h. (4) CPH proved to be highly cytotoxic against Friend leukemia cells in culture, exceeding by one order of magnitude the cytotoxicity of canavanine. The effect of canavanine was completely removed in the presence of equimolar amounts of arginine, while a 20-fold excess of arginine failed to abolish the cytotoxicity of CPH. Thus, a proper hydrazide modification of canavanine may lead to a significant increase in its growth-inhibitory activity and to a change in the mode of action of the parent compound.

Induction of an extracellular cyclic nucleotide phosphodiesterase as an accessory ribonucleolytic activity during phosphate starvation of cultured tomato cells.

During growth under conditions of phosphate limitation, suspension-cultured cells of tomato (Lycopersicon esculentum Mill.) secrete phosphodiesterase activity in a similar fashion to phosphate starvation-inducible ribonuclease (RNase LE), a cyclizing endoribonuclease that generates 2':3'-cyclic nucleoside monophosphates (NMP) as its major monomeric products (T. Nürnberger, S. Abel, W. Jost, K. Glund [1990] Plant Physiol 92: 970-976). Tomato extracellular phosphodiesterase was purified to homogeneity from the spent culture medium of phosphate-starved cells and was characterized as a cyclic nucleotide phosphodiesterase. The purified enzyme has a molecular mass of 70 kD, a pH optimum of 6.2, and an isoelectric point of 8.1. The phosphodiesterase preparation is free of any detectable deoxyribonuclease, ribonuclease, and nucleotidase activity. Tomato extracellular phosphodiesterase is insensitive to EDTA and hydrolyzes with no apparent base specificity 2':3'-cyclic NMP to 3'-NMP and the 3':5'-cyclic isomers to a mixture of 3'-NMP and 5'-NMP. Specific activities of the enzyme are 2-fold higher for 2':3'-cyclic NMP than for 3':5'-cyclic isomers. Analysis of monomeric products of sequential RNA hydrolysis with purified RNase LE, purified extracellular phosphodiesterase, and cleared -Pi culture medium as a source of 3'-nucleotidase activity indicates that cyclic nucleotide phosphodiesterase functions as an accessory ribonucleolytic activity that effectively hydrolyzes primary products of RNase LE to substrates for phosphate-starvation-inducible phosphomonoesterases. Biosynthetical labeling of cyclic nucleotide phopshodiesterase upon phosphate starvation suggests de novo synthesis and secretion of a set of nucleolytic enzymes for scavenging phosphate from extracellular RNA substrates.

Simultaneous determination of diclofenac and oxybuprocaine in human aqueous humor with HPLC and electrochemical detection.

A sensitive and selective bioanalytical method for simultaneous determination of diclofenac and oxybuprocaine in human aqueous humor using reversed-phase HPLC and electrochemical detection is described. Chromatographic separation was achieved by using a Regis SPS 100 RP-8 column (5 microns; 150 x 4.6 mm I.D.). This support is coated with a hydrophilic polyoxyethylenepolymer. It allows protein-containing samples to be injected directly onto the column. The electrochemical detector permit a detection limit of 500 pg diclofenac per ml (daily relative standard deviation 6.3%) and 50 ng oxybuprocaine per ml (daily R.S.D. 2.6%), respectively. Results of administered and measured drug-concentrations in time dependent decrease are presented.

Tolbutamide attenuates diazoxide-induced aggravation of hypoxic cell injury.

ATP-dependent potassium (KATP) channels of neurons are closed in the presence of physiological levels of intracellular ATP and open when ATP is depleted during hypoxia or metabolic damage. The present study investigates hypoxic alterations of purine and pyrimidine nucleotide levels supposed to intracellularly modulate KATP channels. In addition, the effects of the KATP channel activator diazoxide and its antagonist tolbutamide were investigated on ATP, GTP, CTP and UTP levels in slices of the parietal cortex. Hypoxia was evoked by saturation of the medium with 95% N2-5% CO2 instead of 95% O2-5% CO2 for 5 min. Nucleotide contents were measured by anion-exchange HPLC in neutralized perchloric acid extracts obtained from slices frozen immediately at the end of incubation. Hypoxia per se decreased purine and pyrimidine nucleoside triphosphate contents. Thus, ATP and GTP contents were reduced to 69.9 and 77.6% of the respective normoxic levels. UTP and CTP contents were even more decreased (to 60.9 and 41.6%),, probably because the salvage pathway of these pyrimidine nucleotides is less effective than that of the purine nucleotides ATP and GTP. While tolbutamide (30 microM) had no effect on the hypoxia-induced decrease of nucleotides, diazoxide at 300, but not 30 microM aggravated the decline of ATP, UTP and CTP to 51.8, 37.5 and 28.5% of the contents observed at normoxia; GTP levels also showed a tendency to decrease after diazoxide application. Tolbutamide (300 microM) antagonized the effects of diazoxide (300 but not 30 microM aggravated the decline of ATP, UTP and CTP to 51.8, 37.5 and 28.5% of the contents observed at normoxia; GTP levels also showed a tendency to decrease after diazoxide application. Tolbutamide (300 microM) antagonized the effects of diazoxide (300 MicroM). Nucleoside diphosphate (ADP, GDP and UDP) levels were uniformly increased by hypoxia. There was no hypoxia-induced increase of ADP contents in the presence of tolbutamide (300 microM). The ATP/ADP, GTP/GDP and UTP/UDP ratios uniformly declined at a low pO2. However, only the ATP/ADP ratio was decreased further by diazoxide (300 microM). The observed alterations in nucleotide contents may be of importance for long- and short-term processes related to acute cerebral hypoxia. Thus, hypoxia-induced alterations of purine and pyrimidine nucleotide levels may influence the open state of KATP-channels during the period of reversible hypoxic cerebral injury. Furthermore, alterations during the irreversible period of cerebral injury may also arise, as a consequence of decreased pyrimidine nucleotide contents affecting cell survival viaprotein and DNA synthesis.

Crocheted ETFE-reactor for on-line post-column photoderivatization of diclofenac in high-performance liquid chromatography.

A sensitive and selective bioanalytical method for diclofenac using reversed-phase HPLC and fluorescence detection is described. Diclofenac was detected as its fluorescent derivative after on-line post-column photoderivatization. Irradiation with UV light of diclofenac in aqueous solutions leads to the sequential loss of both chlorine substituents and ring closure. The major product, carbazole-1-acetic acid, was detected by a fluorescence detector using an excitation wavelength of 286 nm and an emission wavelength of 360 nm. The self-made reactor was a crocheted ethylene and tetrafluoroethylene (ETFE, named TEFZEL) capillary, 20 m in length, wound directly around a 253.7 nm UV lamp. The capillary was crocheted in order to overcome peak widening. Chromatographic separation was achieved by using a Regis SPS 100 RP-8 column (5 microm; 150 mm x 4.6 mm i.d.) and a LiChrospher 100 RP-18 (5 microm) guard column from E. Merck. The detection limit was 1 ng ml(-1) at an injection volume of 20 microl. Daily relative standard deviations (RSD) were 5.5%, (73 ng diclofenac/ml, n = 9), and 5.1% (405 ng diclofenac/ml, n = 6), respectively. Chromatograms of human aqueous humor and human serum containing diclofenac, and figures showing the time dependent increase/decrease of the photoderivatization product, are shown.

High-performance liquid chromatographic separation of cis-trans isomers of proline-containing peptides. II. Fractionation in different cyclodextrin systems.

beta-Cyclodextrin-bonded silica is demonstrated to be a suitable stationary phase for high-performance liquid chromatography of conformational isomers of proline-containing peptides. In contrast to reversed-phase chromatography, the principle of inclusion complexation shows significant selectivities in conformer resolution based on a variety of interactions. New results of inclusion HPLC of biologically active oligopeptides related to beta-casomorphin on stationary phases containing bonded cyclodextrins of different internal diameters indicate a steric discrimination process during the conformer separation. beta-Cyclodextrin used as a mobile phase additive in reversed-phase systems is shown to offer the opportunity to investigate conformational changes using commercially available reversed-phase columns.

Metabolism and exudation of canavanine during development of alfalfa (Medicago sativa L. cv. verko).

The structural analog of amino acidL-arginine,L-canavanine (2-amino-4-guanidinooxybutyric acid), is found in 26 cultivars of alfalfa. Its concentration ranges from 6 to 16 mg/g of dry seeds. Canavanine represented more than 70% of the total soluble nitrogen in seeds. Practically all of the canavanine was stored in the cotyledons. Comparison is made of the canavanine content in the cultivars Verko and Europa harvested in different years. During sprouting, 29% of the guanidinooxy compound was translocated into the hypocotyl and radicle in 24 hr. In this early stage of seedling development, the level of the nonprotein amino acid, canavanine, increased threefold whereas the protein amino acid, arginine, as well as asparagine increased 11- and 35-fold, respectively. Two-day-old seedlings are capable of synthesizing canavanine derived from canaline up to 25%. Contrary to this finding in seedlings grown in the time range of 24 days, the guanidino compounds canavanine and arginine were metabolized rapidly, whereas asparagine increased. Furthermore, the toxic canavanine got into the environment of swelled seeds or into the rhizosphere of young seedlings and increased in the milieu to concentrations at 3-57µM. In a biotest, this inhibited the growth of a tomato cell suspension culture as well as the growth of cabbage radicle.

Biological activity and mode of action of some dihydroorotic and dihydroazaorotic acid derivatives.

The conversion of six dihydroorotate analogues by the dihydroorotate dehydrogenase (DHO-DH) of plant and animal mitochondria was studied. In the case of plant DHO-DH the dehydrogenation of analogues was as follows: Dihydroorotic acid (DHO) (control, 100%), DHO-hydrazide (40%), azaDHO (13%), azaDHO-ethyl ester (23%), azaDHO-hydrazide (11%), dihydrouracil (0%), dihydrothymine (0%). When animal DHO-DH was used the analogues were practically not dehydrogenated. These compounds were also tested as inhibitors of DHO-dehydrogenation. AzaDHO, azaDHO-hydrazide and azaDHO-ethylester inhibited this reaction by 75, 70% and 60%, respectively, for plant DHO-DH. AzaDHO and azaDHO-ethylester inhibited this reaction to 90% and 70%, respectively, for animal enzyme. The other analogues had no effect. The compounds showed a moderate antibacterial activity. AzaDHO was more active than azaDHO-ethyl ester and azaDHO-hydrazide. A considerable inhibitory effect of azaDHO and azaDHO-ethyl ester was observed on the growth of St. aureus mutant UV-2 and S. lutea. The analogues were little active against the experimental mouse tumors leukemia L 1210, Lewis lung carcinoma (LLC) and B-16 melanoma. AzaDHO-ethyl ester and azaDHO-hydrazide inhibited the growth of LLC by 59% and 56%, respectively. In addition, the effect of analogues on the growth of plant cells was studied. AzaDHO and azaDHO-ethyl ester inhibited the growth of tomato cells in suspension culture by 10% and 41%, respectively.

Inhibitory effects of pyruvic acid semi- and thiosemicarbazones on the growth of bacteria, yeasts, experimental tumours and plant cells.

Pyruvic acid semi- and thiosemicarbazones (1 and 2, respectively) were tested as inhibitors of bacterial, fungal, experimental tumour and plant cell growth. 1 and 2 displayed a growth-inhibitory effect in vitro against different bacterial strains, and especially against St. aureus mutant UV-2 and S. lutea. The compounds proved to have low activity in vivo against L 1210 and P 388 leukemia, adenocarcinoma 755 and melanoma B 16. 2 inhibited strongly the growth of cultured cells of Lycopersicon esculentum (100% inhibition at a concentration of 1,5 mumol/ml) while 1 was not active.

Metabolic conversion of orotic acid hydrazide into a nucleotide in mouse and rat liver.

When [2-14C] orotic acid hydrazide (OAH) was injected i.p. in mice the bulk of the radioactivity of the acid-soluble fraction of liver was found in a metabolite obviously not identical with natural pyrimidines known so far as elucidated by chromatographic methods. This compound is also formed in vitro by the cytosolic fraction of mouse liver or by purified orotate phosphoribosyltransferase/orotidine-5'-phosphate decarboxylase (OPRTase/ODCase) provided that PRPP is present in the medium. Using alkaline hydrolysis and snake venom to split off the hydrazine- and the phosphate group, respectively, the metabolic product of OAH was identified as a nucleotide, orotic acid hydrazide nucleoside monophosphate. The identity of OMP and orotidine thus formed was confirmed by thin-layer chromatography.