Growth regulator requirement for in vitro embryogenic cultures of snowdrop (Galanthus nivalis L.) suitable for germplasm preservation.

In this study, we report on the production of bulb scale-derived tissue cultures capable of efficient shoot and plant regeneration in three genotypes of snowdrop (Galanthus nivalis L., Amaryllidaceae), a protected ornamental plant. For culture line A, high auxin and low cytokinin concentration is required for callus production and plant regeneration. The type of auxin is of key importance: α-naphthaleneacetic acid (NAA) in combination with indole-3-acetic acid (IAA) at concentrations of 2 mg L-1 or 2-10 mg L-1 NAA with 1 mg L-1 N6-benzyladenine (BA), a cytokinin on full-strength media are required for regeneration. Cultures showing regeneration were embryogenic. When lines B and C were induced and maintained with 2 mg L-1 NAA and 1 mg L-1 BA, they produced mature bulblets with shoots, without roots. Line A produced immature bulblets with shoots under the above culture condition. Amplified Fragment Length Polymorphism (AFLP) analysis showed that (i) genetic differences between line A and its bulb explants were not significant, therefore these tissue cultures are suitable for germplasm preservation, and (ii) different morphogenetic responses of lines A, B and C originated from genetic differences. Culture line A is suitable for field-growing, cultivation and germplasm preservation of G. nivalis and for the production of Amaryllidaceae alkaloids.

Protective effect of CV247 against cisplatin nephrotoxicity in rats.

CV247 (CV), an aqueous mixture of copper (Cu) and manganese (Mn) gluconates, vitamin C and sodium salicylate increased the antitumour effects of cisplatin (CDPP; cis-diamminedichloroplatinum) in vitro. We hypothesized that the antioxidant and cyclooxygenase-2 (COX-2; prostaglandin-endoperoxide synthase 2) inhibitory components of CV can protect the kidneys from CDPP nephrotoxicity in rats. CDPP (6.5 mg/kg, intraperitoneally) slightly elevated serum creatinine (Crea) and blood urea nitrogen (BUN) 12 days after treatment. Kidney histology demonstrated extensive tubular epithelial damage and COX-2 immunoreactivity increased 14 days after treatment. A large amount of platinum (Pt) accumulated in the kidney of CDPP-treated rats. Furthermore, CDPP decreased renal iron (Fe), molybdenum (Mo), zinc (Zn), Cu and Mn concentrations and increased plasma Fe and Cu concentrations. CDPP elevated plasma free radical concentration. Treatment with CV alone for 14 days (twice 3 ml/kg/day orally) did not influence these parameters. Chronic CV administration after CDPP reduced renal histological damage and slightly decreased COX-2 immunoreactivity, while failed to prevent the increase in Crea and BUN levels. Blood free radical concentration was reduced, that is, CV improved redox homeostasis. CV restored plasma Fe and renal Fe, Mo and Zn, while decreased Pt and elevated Cu and Mn concentrations in the kidney. Besides the known synergistic antitumour effects with CDPP, CV partially protected the kidneys from CDPP nephrotoxicity probably through its antioxidant effect.

Histological, cytological and biochemical alterations induced by microcystin-LR and cylindrospermopsin in white mustard (Sinapis alba L.) seedlings.

This study compares the histological, cytological and biochemical effects of the cyanobacterial toxins microcystin-LR (MCY-LR) and cylindrospermopsin (CYN) in white mustard (Sinapis alba L.) seedlings, with special regard to the developing root system. Cyanotoxins induced different alterations, indicating their different specific biochemical activities. MCY-LR stimulated mitosis of root tip meristematic cells at lower concentrations (1 µg ml-1) and inhibited it at higher concentrations, while CYN had only inhibitory effects. Low CYN concentrations (0.01 µg ml-1) stimulated lateral root formation, whereas low MCY-LR concentrations increased only the number of lateral root primordia. Both inhibited lateral root development at higher concentrations. They induced lignifications, abnormal cell swelling and inhibited xylem differentiation in roots and shoots. MCY-LR and CYN induced the disruption of metaphase and anaphase spindles, causing altered cell divisions. Similar alterations could be related to decreased protein phosphatase (PP1 and PP2A) activities in shoots and roots. However, in vitro phosphatase assay with purified PP1 catalytic subunit proved that CYN in contrast to MCY-LR, decreased phosphatase activities of mustard in a non-specific way. This study intends to contribute to the understanding of the mechanisms of toxic effects of a protein phosphatase (MCY-LR) and a protein synthesis (CYN) inhibitory cyanotoxin in vascular plants.

Cisplatin nephrotoxicity aggravated by cardiovascular disease and diabetes in lung cancer patients.

Ageing lung cancer patients may be at increased risk of Cisplatin (Cp) nephrotoxicity, because of comorbidities leading to accelerated ageing of the kidneys. Therefore, the Cp-induced impairement of renal function was compared between no comorbidity (NC) and hypertension plus ischaemic heart disease (CD) patients or others having diabetes mellitus plus ischaemic heart disease (DMIH). In a preliminary study, glomerular filtration rate (GFR) was measured by clearance of technetium 99m-labelled diethylene-thiamine penta-acetate in 38 lung cancer patients with normal serum creatinine concentration ([creat]). Then, the incidence of nephrotoxicity was analysed retrospectively over 1st-4th cycles of Cp treatment among 242 lung cancer patients with initially normal [creat]. GFR was repeatedly estimated using calculated creatinine clearance. Pre-treatment GFR was 57 ± 3 mL·min⁻¹·m⁻² in those with normal (n = 15) and 42 ± 2 mL·min⁻¹·m⁻² in those with pathologically increased (n = 23) [creat] any time following their 2nd-4th Cp cycle (p < 0.05). The retrospective analysis revealed that Cp-induced nephrotoxicity developed in 7.5% of the NC (n = 80), in 20.9% of the CD (n = 110) and in 30.8% of the DMIH (n = 52) subgroups. Within the overall dropout rate from further Cp chemotherapy, nephrotoxicity was responsible in 14% of NC, 38% in CD and 75% in DMIH patients. A major portion of our ageing lung cancer patients suffered from comorbidities leading to reduced renal resistance to Cp nephrotoxicity.

Cylindrospermopsin and microcystin-LR alter the growth, development and peroxidase enzyme activity of white mustard (Sinapis alba L.) seedlings, a comparative analysis.

This work focuses on the comparative analysis of the effects of two cyanobacterial toxins of different chemical structure cylindrospermopsin (CYN) and microcystin-LR (MC-LR) on the white mustard (Sinapis alba L.) seedlings. Both cyanotoxins reduced significantly the fresh mass and the length of cotyledons, hypocotyls and main roots of seedlings in a concentration dependent manner. For various mustard organs the 50% inhibitory concentration values (IC50) of growth were between 3-5 µg ml(-1) for MC-LR and between 5-10 µg ml-1 for CYN, respectively. Cyanotoxins altered the development of cotyledons, the accumulation of photosynthetically active pigments and anthocyanins. Low MC-LR concentrations (0.01 and 0.1 µg ml(-1)) stimulated anthocyanin formation in the cotyledons but higher than 1 µg ml(-1) MC-LR concentrations strongly inhibited it. The CYN treated chlorotic cotyledons were violet coloured in consequence of high level of anthocyanins, while MC-LR induced chlorosis was accompanied by the appearance of necrotic patches. Necrosis and increases of peroxidase enzyme activity (POD) are general stress responses but these alterations were characteristic only for MC-LR treated mustard plants. These findings provide experimental evidences of developmental alterations induced by protein synthesis and protein phosphatase inhibitory cyanotoxins (CYN and MC-LR) in a model dicotyledonous plant.

Cylindrospermopsin inhibits growth and modulates protease activity in the aquatic plants Lemna minor L. and Wolffia arrhiza (L.) Horkel.

The toxic effects of cylindrospermopsin (cyanobacterial toxin) on animals have been examined extensively, but little research has focused on their effects on plants. In this study cylindrospermopsin (CYN) caused alterations of growth, soluble protein content and protease enzyme activity were studied on two aquatic plants Lemna minor and Wolffia arrhiza in short-term (5 days) experiments. For the treatments we used CYN containing crude extracts of Aphanizomenon ovalisporum (BGSD-423) and purified CYN as well. The maximal inhibitory effects on fresh weight of L. minor and W. arrhiza caused by crude extract were 60% and 54%, respectively, while the maximum inhibitory effects were 30% and 43% in the case of purified CYN at 20 µg ml(-1) CYN content of culture medium. In CYN-treated plants the concentration of soluble protein showed mild increases, especially in W. arrhiza. Protease isoenzyme activity gels showed significant alterations of enzyme activities under the influence of CYN. Several isoenzymes were far more active and new ones appeared in CYN-treated plants. Treatments with cyanobacterial crude extract caused stronger effects than the purified cyanobacterial toxins used in equivalent CYN concentrations.

Analysis of genetic diversity in crocuses with Carpathian Basin origin using AFLP-markers.

Crocus taxonomy has until now been based primarily on morphology, taking chromosome numbers into consideration. The genetics and genome structure of the genus, the relationships and diversity within the genus are not well known. Amplified fragment length polymorphism (AFLP) is a whole genome approach to study genetic variation that is gaining in popularity for lower-level systematics. The present study employed the AFLP technique for analyzing relationships among taxa of the Crocus genus (particularly the Crocus vernus aggregate) with Carpathian Basin origin. The molecular variance obtained was based on amplification, separation and detection of EcoRI and Tru1I double-digested Crocus spp. genomic DNAs. Our results confirm the relatedness of C. tommasinianus, C. vittatus and C. heuffelianus at the Verni series of the Crocus genus. C. banaticus is taxonomically isolated as the sole member of the subgenus Crociris based on unique morphological features, but the difference is not convincing from AFLP data. The second interesting AFLP analysis result is the position of C. scepusiensis which separated it from the Crocus vernus aggregate.

Annotation of a 95-kb Populus deltoides genomic sequence reveals a disease resistance gene cluster and novel class I and class II transposable elements.

Poplar has become a model system for functional genomics in woody plants. Here, we report the sequencing and annotation of the first large contiguous stretch of genomic sequence (95 kb) of poplar, corresponding to a bacterial artificial chromosome clone mapped 0.6 centiMorgan from the Melampsora larici-populina resistance locus. The annotation revealed 15 putative genetic objects, of which five were classified as hypothetical genes that were similar only with expressed sequence tags from poplar. Ten putative objects showed similarity with known genes, of which one was similar to a kinase. Three other objects corresponded to the toll/interleukin-1 receptor/nucleotide-binding site/leucine-rich repeat class of plant disease resistance genes, of which two were predicted to encode an amino terminal nuclear localization signal. Four objects were homologous to the Ty1/ copia family of class I transposable elements, one of which was designated Retropop and interrupted one of the disease resistance genes. Two other objects constituted a novel Spm-like class II transposable element, which we designated Magali.

Synthesis and antiviral evaluation of C-4-hydrazide derivatives of 2',3'-dideoxycytidine.

Syntheses of three hitherto unknown derivatives of 2',3'-dideoxycytidine, namely C-4-(salicylic hydrazide)-ddC, C-4-(N-butyloxycarbonyl-isoleucine hydrazide)-ddC and its N-unprotected chlorhydrate salt have been carried out. These compounds do not induce inhibition of HIV-1 replication in cell culture experiments. Nevertheless, the modifications on the base moiety increased in all cases the lipophilicity of the parent molecule with an acceptable water solubility compared to ddC.

Synthesis and in vitro activity of D- and L-enantiomers of 5-(trifluoromethyl)uracil nucleoside derivatives.

Recently, beta-L-nucleoside analogues have emerged as a new class of sugar modified nucleosides with potential antiviral and/or antitumoral activity. As a part of our ongoing research on this topic, we decided to synthesize 5-CF3-beta-L-dUrd (7), the hitherto unknown L-enantiomer of Trifluridine, an antiherpetic drug approved by FDA but only used in topical applications due to concomitant cytotoxicity. 5-CF3-beta-L-dUrd (7) as well as some other related L-nucleoside derivatives were stereospecifically prepared and tested in vitro against viral (HSV-1 and HSV-2) and human thymidine kinases (TK).

Synthesis and antiviral evaluation of 3'-C-trifluoromethyl nucleoside derivatives bearing adenine as the base.

3'-deoxy-3'-C-trifluoromethyl- (3), 2',3'-dideoxy-3'-C-trifluoromethyl- (5) and 2',3'-dideoxy-2',3'-didehydro-3'-C-trifluoromethyladenosine (6) derivatives have been synthesized and their antiviral properties examined. All these derivatives were stereospecifically prepared by glycosylation of adenine with a trifluoromethyl sugar precursor (1), followed by appropriate chemical modifications. The prepared compounds were tested for their activity against HIV, but they did not show an antiviral effect.

Stereospecific synthesis and biological evaluations of beta-L-pentofuranonucleoside derivatives of 5-fluorouracil and 5-fluorocytosine.

In the search for new chemotherapeutic agents, we have focused our work on the synthesis and the study of several unnatural beta-L-nucleoside analogues. In this paper, we report on the synthesis of beta-L-pentofuranonucleosides (and their 2'-deoxy derivatives) of 5-fluorouracil and their inhibitory effects on the proliferation of several murine and human tumor cells. The corresponding 5-fluorocytosine derivatives were also synthesized and their anti-HIV and anti-HBV activities have been evaluated.

5-(Trifluoromethyl)-beta-l-2'-deoxyuridine, the L-enantiomer of trifluorothymidine: stereospecific synthesis and antiherpetic evaluations.

As a part of our ongoing work on beta-L-nucleoside analogues as potential antiviral drugs, we have synthesized 5-(trifluoromethyl)-beta-L-2'-deoxyuridine (L-TFT), the hitherto unknown L-enantiomer of trifluorothymidine (CF(3)dUrd, TFT). We have also studied the effect of L-TFT on human and herpes simplex virus (HSV) type 1 and 2 thymidine kinases, and human thymidine phosphorylase, as well as its anti-HSV-1 and anti-HSV-2 activities in cell cultures. L-TFT has been found: (i) to inhibit HSV-1 TK with activity comparable to TFT, with no effect on human TK, (ii) to be phosphorylated by the viral enzyme with similar efficiency to TFT, (iii) to be resistant, in contrast to TFT, to hydrolysis by human thymidine phosphorylase. Unfortunately, when evaluated in cell cultures, L-TFT did not show any anti-HSV-1 and anti-HSV-2 activities.

Translation conditional models for protein coding sequences.

A coding sequence is defined as a DNA sequence coding the primary structure of a protein (a polypeptide). Such a sequence must satisfy a specific constraint, which consists in coding a functional protein. As the genetic code is degenerated, there exists, for a given polypeptide, a set of synonymous sequences which would code the same polypeptide. Translation conditional models are being defined on such sets. The aim of this paper is to give a common formalism. Besides the codon bias model, a few other conditional models will be defined. Statistical estimators and comparison methods will be briefly presented. These models can be used for gene classification, or to find out, in a real sequence, remarkable features. An example will be presented on Escherichia coli genes.

1,2-di-O-acetyl-5-O-benzoyl-3-deoxy-L-erythro-pentofuran ose, a convenient precursor for the stereospecific synthesis of nucleoside analogues with the unnatural beta-L-configuration.

The title compound 1,2-di-O-acetyl-5-O-benzoyl-3-deoxy-L-erythro-pentofuranose (5), a useful precursor for the stereospecific synthesis of beta-L-nucleoside analogues as potential antiviral agents, has been synthesised by a multi-step reaction sequence from L-xylose with a 38% overall yield. The preparation involved conversion of L-xylose to 1,2-O-isopropylidene-alpha-L-xylofuranose which, upon selective 5-O-benzoylation and subsequent radical deoxygenation, provided the protected 3-deoxy sugar derivative. Finally, cleavage of the acetonide group gave the resulting 5-O-benzoyl-3-deoxy-L-erythro-pentose which was acetylated to afford crystalline alpha,beta-5.

Gene prediction and gene classes in Arabidopsis thaliana.

Gene prediction methods for eukaryotic genomes still are not fully satisfying. One way to improve gene prediction accuracy, proven to be relevant for prokaryotes, is to consider more than one model of genes. Thus, we used our classification of Arabidopsis thaliana genes in two classes (CU(1) and CU(2)), previously delineated according to statistical features, in the GeneMark gene identification program. For each gene class, as well as for the two classes combined, a Markov model was developed (respectively, GM-CU(1), GM-CU(2) and GM-all) and then used on a test set of 168 genes to compare their respective efficiency. We concluded from this analysis that GM-CU(1) is more sensitive than GM-CU(2) which seems to be more specific to a gene type. Besides, GM-all does not give better results than GM-CU(1) and combining results from GM-CU(1) and GM-CU(2) greatly improve prediction efficiency in comparison with predictions made with GM-all only. Thus, this work confirms the necessity to consider more than one gene model for gene prediction in eukaryotic genomes, and to look for gene classes in order to build these models.

Inhibition of the ras-dependent mitogenic pathway by phosphopeptide prodrugs with antiproliferative properties.

Phosphopeptide prodrugs bearing two S-acyl-2-thioethyl (SATE) biolabile phosphate protections were developed. They are capable to inhibit the Shc/Grb2 interaction and MAP kinases (ERK1 and ERK2) phosphorylation in cellular assay. The S-acetyl-2-thioethyl (MeSATE) analogue showed an IC50 of 1 microM in the inhibition of the colony formation of tumor cell line NIH3T3/HER2.

Synthesis and antiviral evaluation of unnatural beta-L-enantiomers of 3'-fluoro- and 3'-azido-2',3'-dideoxyguanosine derivatives.

3'-fluoro-2',3'-dideoxy- (3) and 3'-azido-2',3'-dideoxy- (4) beta-L-ribofuranonucleoside derivatives of guanine have been synthesized and their antiviral properties examined. All these derivatives were regioselectively and stereospecifically prepared by glycosylation of 2-N-acetyl-6-O-(diphenylcarbamoyl)guanine 5 with a suitable peracylated L-xylo-furanose sugar 6, followed by appropriate chemical modifications. The prepared compounds were tested for their activity against HIV and HBV viruses, but they did not show significant activity.

Unnatural enantiomers of 5-azacytidine analogues: syntheses and enzymatic properties.

Although 2'-deoxy-beta-D-5-azacytidine (Decitabine) and beta-D-5-azacytidine display potent antileukemic properties, their therapeutic use is hampered by their sensitivity to nucleophiles and to deamination catalysed by cytidine deaminase. As shown earlier [Shafiee M., Griffon J.-F., Gosselin G., Cambi A., Vincenzetti S., Vita A., Erikson S., Imbach J.-L., Maury G., Biochem. Pharmacol. 56 (1998) 1237-1242], beta-L-enantiomers of cytidine derivatives are resistant to cytidine deaminase. We thus synthesized several 5-azacytosine beta-L-nucleoside analogues to evaluate their enzymatic and biological properties. 2'-Deoxy-beta-L-5-azacytidine (L-Decitabine), beta-L-5-azacytidine, 1-(beta-L-xylo-furanosyl)5-azacytosine, and 1-(2-deoxy-beta-L-threo-pentofuranosyl)5-azacytosine were stereospecifically prepared starting from L-ribose and L-xylose. D- and L-enantiomers of 2'-deoxy-beta-5-azacytidine were weak substrates of human recombinant deoxycytidine kinase (dCK) compared to beta-D-deoxycytidine, whereas both enantiomers of beta-5-azacytidine or the L-xylo-analogues were not substrates of the enzyme. As expected, none of the presently reported derivatives of beta-L-5-azacytidine was a substrate of human recombinant cytidine deaminase (CDA). The prepared compounds were tested for their activity against HIV and HBV and they did not show any significant activity or cytotoxicity. In the case of L-Decitabine, this suggests that the enantioselectivities of concerned enzymes other than dCK and CDA might not be favourable.