Jan Hanuš

Distribution, biological activities, metabolism, and the conceivable function of cis-zeatin-type cytokinins in plants

Cytokinins (CKs) are plant hormones affecting numerous developmental processes. Zeatin and its derivatives are the most important group of isoprenoid CKs. Zeatin occurs as two isomers: while trans-zeatin (transZ) was found to be a bioactive substance, cis-zeatin (cisZ) was reported to have a weak biological impact. Even though cisZ derivatives are abundant in various plant materials their biological role is still unknown. The comprehensive screen of land plants presented here suggests that cisZ-type CKs occur ubiquitously in the plant kingdom but their abundance might correlate with a strategy of life rather than with evolutionary complexity. Changing levels of transZ and cisZ during Arabidopsis ontogenesis show that levels of the two zeatin isomers can differ significantly during the life span of the plant, with cisZ-type CKs prevalent in the developmental stages associated with limited growth. A survey of the bioassays employed illustrates mild activity of cisZ and its derivatives. No cis↔trans isomerization, which would account for the effects of cisZ, was observed in tobacco cells and oat leaves. Differences in uptake between the two isomers resulting in distinct bioactivity have not been detected. In contrast, cisZ and transZ have a different metabolic fate in oat and tobacco. Analysis of a CK-degrading enzyme, cytokinin oxidase/dehydrogenase (CKX), reveals that Arabidopsis possesses two isoforms, AtCKX1 expressed in stages of active growth, and AtCKX7, both of which have the highest affinity for the cisZ isomer. Based on the present results, the conceivable function of cisZ-type CKs as delicate regulators of CK responses in plants under growth-limiting conditions is hypothesized.

Biological activity of cytokinins derived from Ortho- and Meta-Hydroxybenzyladenine

To determine the structure-activity relationships of natural aromatic cytokinins, the activity of 6-benzylaminopurine (BAP) and its hydroxylated derivatives was compared in three bioassays based on stimulation of tobacco callus growth, retention of chlorophyll in excised wheat leaves, and dark induction of betacyanin synthesis in Amaranthus cotyledons. The aromatic cytokinins 6-(2-hydroxybenzylamino)purine (ortho-topolin) and 6-(3-hydroxybenzylamino)purine (meta-topolin), their 9-ribosides and 9-glucosides, were synthesized by the condensation of 6-chloropurine and its 9-glycosides with the appropriate hydroxybenzylamine. The activity of free bases, 9-ribosides and 9-glucosides was compared with that of BAP, trans-zeatin and their 9-glycosides. Hydroxylation of the benzyl ring in the meta position increased the activity of BAP and its riboside in tobacco callus and chlorophyll retention bioassays, whereas ortho-hydroxylation decreased the activity. In contrast, in the Amaranthus bioassay meta-hydroxylation of BAP substantially decreased its activity. Ribosylation at position 9 had no significant effect on the activity of zeatin, BAP and both topolins. The activity of 9-glucosides of all cytokinins tested was near zero. The biological activity of meta-topolin and its riboside is comparable to that of the most active isoprenoid cytokinin, zeatin, in tobacco callus growth and senescence bioassays. The results establish the existence of a family of endogenous aromatic cytokinins centered around the highly active compound, meta-topolin. We also report here an improved chlorophyll retention bioassay based on incubation of 2.5 cm long detached wheat leaf segments in microtiter plate wells containing 150 µl of cytokinin solution. The consumption of cytokinin to be tested is 0.1 µmol per assay only. The amount as small as 1.5 pmol of substance can be estimated using this biotest.

Aromatic cytokinins in micropropagated potato plants

Endogenous cytokinins were studied in three micropropagated Solanum tuberosumL. cultivars (Kennebec, Turia and Jaerla) differing in survival after transplanting. Leaf and stem cytokinins were determined both in vitro and 10 d after being transferred to ex vitro conditions by a combination of high-performance liquid chromatography and enzyme-linked immunosorbent assay. Nine aromatic and nine isoprenoid type cytokinins were identified. Higher levels of total cytokinins mainly aromatics (92%) were detected in Kennebec, the cultivar showing better in vitro growth and 99% survival. On the contrary, a predominance of isoprenoid cytokinins (up to 57%) was observed after transplanting in Jaerla, the cultivar showing lower viability. Significant survival improvement was obtained in the Jaerla cultivar after addition to the culture medium of the aromatic cytokinin meta-topolin riboside (mTR). We also report here isolation and identification of this cytokinin by several sophisticated techniques including mTR-specific immunoaffinity chromatography, diode-array high-performance liquid chromatography (HPLC), and gas chromatography–mass spectrometry of permethylated HPLC fractions. The occurrence of the aromatic cytokinins in potato plants is described for the first time.

Induction of apoptosis and regression of spontaneous dog melanoma following in vivo application of synthetic cyclin-dependent kinase inhibitor olomoucine.

This case report describes a dog with spontaneous melanoma of the orofacial region which was treated by a synthetic inhibitor of cyclin-dependent kinases, i.e. olomoucine (OC). The drug was applied i.v. in a single dose of 8 mg/kg/day for 7 days in succession. Repeated bioptic examinations of metastatic cervical lymph nodes showed rapid induction of apoptosis in tumor cells as early as on the third day of treatment. Standard clinical and laboratory examinations did not reveal side effects of the therapy. There were no detectable manifestations of myelosuppression, hepatotoxicity, nephrotoxicity or neurotoxicity. However, transient anemia developed following bleeding from a devitalized tumor mass. For this reason, the dog underwent surgery to minimize tumor load as well as to eliminate the source of bleeding. Two kilograms of primary tumor were extirpated in the course of surgery, including cervical node metastases. Unfortunately, the dog died soon after surgery due to respiratory depression. Histological examinations of the tumor tissue showed marked apoptosis of melanoma cells in both the primary tumor and metastases. The induction of programmed cell death of cancer cells by OC resulted in rapid eradication of at least 68% of the tumor cells. The remaining melanoma cells retained at least equally well in vitro sensitivity to OC as to drugs currently used in clinical practice.

Ortho-topolin-9-glucoside, an aromatic cytokinin from Populus x canadensis cv Robusta leaves

Abstract A new natural cytokinin was isolated from Populus x canadensis Moench cv Robusta leaves. Its structure was established as N6-(o-hydroxybenzylamino-9-O-β- d -glucopyranosylpurine (termed ortho-topolin-9-glucoside) by UV and mass spectrometry and confirmed by synthesis. This cytokinin exhibited comparatively low biological activity in three common bioassays. It was quantified in the leaf extract by combined high performance liquid chromatography—enzyme linked immunoassay. Amounting to 50 pmol g−1 fresh weight, ortho-topolin-9-glucoside was the dominant aromatic cytokinin in poplar leaves collected 2 hr after sunrise.

2,6,8,9-Tetrasubstituted purines as new CDK1 inhibitors

Purine inhibitors of cyclin-dependent kinases attract attention as potential anticancer drugs because their first representative roscovitine recently entered clinical trials. Although well described in terms of structure-activity relationships, we still present here a novel modification of the purine scaffold influencing their inhibitory properties. The introduced C-8 substituents, however, lowered the CDK inhibitory activity of roscovitine, whereas the antiproliferative potential of several derivatives remained high.

Analysis of 2-methylthio-derivatives of isoprenoid cytokinins by liquid chromatography-tandem mass spectrometry

A sensitive and reliable high-performance liquid chromatographic method with tandem mass spectrometric detection has been developed and used for the determination of 2-methylthio-cytokinin derivatives produced by the phytopathogenic actinomycete Rhodococcus fascians. The cultivation medium containing secreted cytokinins was concentrated and subjected to a solid-phase extraction (C18 and ion-exchange). The purified samples were further separated and analyzed by HPLC-ESI-MS/MS. This allowed to achieve chromatographic resolution of six highly hydrophobic cytokinin species including 2-methylthio-isopentenyladenine, 2-methylthio-isopentenyladenosine, 2-methylthio-trans-zeatin and 2-methylthio-trans-zeatin riboside and their cis-isomers when a reversed-phase chromatographic column (C4) and a mobile phase consisting of acetonitrile and 20 mM ammonium formate, pH 5, were used. Quantification was performed by a standard isotope dilution method using a multiple-reaction monitoring (MRM) mode. In the MRM mode, limits of detection reached 20-30 fmol and linear ranges spanned four orders of magnitude. Recovery values were between 35% and 65% and the analytical accuracy between 95% and 149%. The proposed bioanalytical method, which takes advantage of effective chromatographic separation of six 2-methyltio-derivatives (including isomers of zeatin-type cytokinins) and sensitive mass spectrometric detection, may become useful for plant biologists studying the significance of these substances in plant-microbe interactions.

Photoaffinity labelling of the mitochondrial uncoupling protein by [3H]azido fatty acid affects the anion channel

Brown adipose tissue (BAT) mitochondria were incubated with the azido derivative of fatty acid (hexadecanoic) containing four tritium atoms, [3H]AzHA, and among all mitochondrial proteins only a few proteins were photolabelled after irradiation with UV. It suggests the existence of specific fatty acid binding sites on mitochondrial proteins. It was also possible to label with [3H]AzHA the isolated uncoupling protein (UcP) of BAT mitochondria with a low stoichiometry — lower than one AzHA per dimeric UcP. These results together with the observed competition (i.e. prevention of photolabelling) of various UcP anionic substrates with [3H]AzHA and its dodecanoic acid analogue, suggest the existence of the specific fatty acid binding site on UcP identical with the anion channel or anion translocating site.

Utilization of hyperspectral image optical indices to assess the Norway spruce forest health status

The work is concerned with assessing the health status of trees of the Norway spruce species using airborne hyperspectral (HS) data (HyMap). The study was conducted in the Sokolov basin in the western part of the Czech Republic. First, statistics were employed to assess and validate diverse empirical models based on spectral information using the ground truth data (biochemically determined chlorophyll content). The model attaining the greatest accuracy ( D 718 / D 704 ∶ RMSE = 0.2055     mg / g , R 2 = 0.9370 ) was selected to produce a map of foliar chlorophyll concentrations ( C a b ). The C a b values retrieved from the HS data were tested together with other nonquantitative vegetation indicators derived from the HyMap image reflectance to create a statistical method allowing assessment of the condition of Norway spruce. As a result, we integrated the following HyMap derived parameters ( C a b , REP, and SIPI) to assess the subtle changes in physiological status of the macroscopically undamaged foliage of Norway spruce within the four studied test sites. Our classification results and the previously published studies dealing with assessing the condition of Norway spruce using chlorophyll contents are in a good agreement and indicate that this method is potentially useful for general applicability after further testing and validation.

Natural and Azido Fatty Acids Inhibit Phosphate Transport and Activate Fatty Acid Anion Uniport Mediated by the Mitochondrial Phosphate Carrier

The electroneutral Pi uptake via the phosphate carrier (PIC) in rat liver and heart mitochondria is inhibited by fatty acids (FAs), by 12-(4-azido-2-nitrophenylamino)dodecanoic acid (AzDA) and heptylbenzoic acid (∼1 μm doses) and by lauric, palmitic, or 12-azidododecanoic acids (∼0.1 mmdoses). In turn, reconstituted E. coli-expressed yeast PIC mediated anionic FA uniport with a similar pattern leading to FA cycling and H+ uniport. The kinetics of Pi/Pi exchange on recombinant PIC in the presence of AzDA better corresponded to a competitive inhibition mechanism. Methanephosphonate was identified as a new PIC substrate. Decanephosphonate, butanephosphonate, 4-nitrophenylphosphate, and other Pi analogs were not translocated and did not inhibit Pi transport. However, methylenediphosphonate and iminodi(methylenephosphonate) inhibited both electroneutral Pi uptake and FA cycling via PIC. AzDA analog 16-(4-azido-2-nitrophenylamino)-[3H4]-hexadecanoic acid (3H-AzHA) bound upon photoactivation to several mitochondrial proteins, including the 30- and 34-kDa bands. The latter was ascribed to PIC due to its specific elution pattern on Blue Sepharose and Affi-Gel. 3H-AzHA photolabeling of recombinant PIC was prevented by methanephosphonate and diphosphonates and after premodification with 4-azido-2-nitrophenylphosphate. Hence, the demonstrated PIC interaction with monovalent long-chain FA anions, but with divalent phosphonates of short chain only, indicates a pattern distinct from that valid for the mitochondrial uncoupling protein-1.