Cadmium and heat response of the fungus Heliscus lugdunensis isolated from highly polluted and unpolluted areas.

Induction of heat shock protein (Hsp) 70 and distinct metallothionein-like proteins (MTLPs) in response to Cd and heat treatment were studied in two strains of the aquatic hyphomycete Heliscus lugdunensis: Hl-H4, isolated from a heavy metal polluted site, and Hl-BB taken from an unpolluted area. Upon Cd-exposure, Hsp70 was actively synthesized in the strain Hl-H4, and to a much lower degree in the strain Hl-BB. The Hsp70-expression was time- and dose-dependent, reaching a maximum after 24 h incubation with 80 microM Cd. Upon heat-stress, a similar response was observed: a strong Hsp70-expression in Hl-H4, and only a marginal one in Hl-BB. The strains reacted to Cd-exposure by a specific, environmentally related induction of MTLPs, as shown by the highly sensitive bimane derivatisation method of SH-rich proteins. In Hl-H4, a strong expression of 11 kDa MTLP was registered, which followed strictly the induction pattern of Hsp70. This suggests interdependence of the induction mechanisms and roles of these stress proteins in metal resistance. On the contrary, in Hl-BB a weak expression of MTLP of about 20 kDa was observed, exhibiting completely different induction pattern. The results suggest that the specific induction of Hsp70 and/or distinct MTLPs in the range of 11 kDa in H. lugdunensis strain Hl-H4 are essential adaptive mechanisms to continuous heavy metal exposure.

Biosynthesis of riboflavin in vitro. Isotopic incorporation studies in Pichia guilliermondii extracts.

In the riboflavin-deficient mutant rib3 and in wild-type cells of P. guilliermondii, GTP is transformed into 2,5-diamino-6-hydroxy-4-ribitylaminopyrimidine and riboflavin, respectively. We were able to demonstrate the partial in vitro synthesis of these compounds, including a reductive conversion step of the product of GTP cyclohydrolase II action upon labelled [14C]GTP. In order to analyse the pyrimidine derivatives formed, 6,7-dimethyl-8-ribitylpterin and riboflavin were synthesized by addition of diacetyl. The data obtained indicate that the radiocarbon from the ribose moiety of GTP was transformed into the ribityl sidechain of 2,5-diamino-6-hydroxy-4-ribitylaminopyrimidine as well as riboflavin without any dilution. Therefore, the ribityl sidechain of the riboflavin formed originates from the ribose moiety of GTP.

Origin of the ribityl side-chain of riboflavin from the ribose moiety of guanosine triphosphate in Pichia guilliermondii yeast.

In wild-type cells and some riboflavin-deficient mutants of P. guilliermondii GTP is transformed to the ribitylated intermediates 2,5-diamino-6-hydroxy-4-ribitylaminopyrimidine and 5-amino-2,6-dihydroxy-4-ribitylaminopyrimidine of the riboflavin biosynthetic path. We were able to show that these compounds were formed in vitro as well as in permeabilized cells by reactions including a reductive conversion of the product of GTP cyclohydrolase II action upon GTP. In order to analyse the pyrimidine derivates, 6,7-dimethyl-8-ribitylpterin and 6,7-dimethyl-8-ribityllumazine were synthesized by reaction of pyrimidines with diacetyl. The formation of ribitylated pyrimidines was shown to be strictly dependent on the presence of NADPH2. The data obtained indicate that the reductive step is catalyzed by a 2,5-diamino-6-hydroxy-4-ribosylaminopyrimidine-reductase. 6,7-Dimethyl-8-ribitylpterin and 6,7-dimethyl-8-ribityllumazine isolated from the incubation mixtures have been identified by chromatography and by their ultraviolet and fluorescence spectra.

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.

Transport of ribitol and D-glucose in the yeast Candida guillermondii.

The uptakes of the linear polyol ribitol and of D-glucose by Candida guillermondii were found to be carrier-mediated and to require metabolic energy. In glucose-grown cells ribitol possibly enters by simple diffusion but after an induction period a specific transport system is synthesized, inhibitable by higher concentrations of arabinitols, xylitol, mannitol and sorbitol. Actidione blocks the synthesis of the inducible ribitol transport system. Two systems of different affinity for substrate were found to operate in the uptake of both glucose and of ribitol. Counter-transport experiments with ribitol, D-glucose and 3-O-methyl-D-glucose support the carrier nature of the uptake system.

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.

Production and biological activity of saponins and canavanine in alfalfa seedlings.

The saponin and canavanine concentrations and pattern were analyzed in growing alfalfa seedlings (Medicago sativa L.). Accumulation of saponins and canavanine was found to follow different time courses. During the first eight days, saponin concentration rose from zero in alfalfa seeds to 8.7% in roots and 1.8% in shoots on eighth day and then slowly decreased to 7.6% in roots and 0.8% in shoots present on the 24th day. Canavanine was found in seeds at a concentration of 1 % then increased to 3.2% in seedlings on the sixth day and rapidly decreased to 0.2% per dry mass in roots and shoots on the 24th day. The effect of saponins-medicagenic acid sodium salt and medicagenic acid glycosides-on the growth ofAmaranthus andLepidium in Petri dishes and tomato (Lycopersicon) cell growth in tissue culture also was investigated. In contrast to medicagenic acid glycosides, a very strong inhibition of plant and cell growth was found as an effect of medicagenic acid.

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.

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.

Biological effects of the dihydroorotate dehydrogenase inhibitor polyporic acid, a toxic constituent of the mushroom Hapalopilus rutilans, in rats and humans.

Inhibitors of dihydroorotate dehydrogenase (DHO-DH), such as brequinar or leflunomide, have been intensively tested for their antitumour and immunomodulating effects. Polyporic acid (PA) from the mushroom Hapalopilus rutilans (H. r.) also is a DHO-DH inhibitor (50% inhibitory concn., IC50, 10(-4)-10(-3) M). As three people had been poisoned following ingestion of H. r. we wanted to investigate the effects of PA in rats and in cell cultures. Rats given PA via probang (100-800 mg/ kg) within 24 h developed strongly reduced locomotor activity, depressed visual placing response and impaired wire manoeuvre. Laboratory investigation of blood revealed hepatorenal failure, metabolic acidosis as well as hypokalaemia and hypocalcaemia. All symptoms closely paralleled the effects seen in the poisoned people. Proliferation of cultured cells (including rat brain neurons and glia, fibroblasts, tumour cells) was depressed at 10(-4)-10(-3) M PA. We conclude that the intoxication of people poisoned with H. r. is due to the high content of the DHO-DH inhibitor PA.