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.

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.

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.

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.

Classification of Arabidopsis thaliana gene sequences: clustering of coding sequences into two groups according to codon usage improves gene prediction.

While genomic sequences are accumulating, finding the location of the genes remains a major issue that can be solved only for about a half of them by homology searches. Prediction methods are thus required, but unfortunately are not fully satisfying. Most prediction methods implicitly assume a unique model for genes. This is an oversimplification as demonstrated by the possibility to group coding sequences into several classes in Escherichia coli and other genomes. As no classification existed for Arabidopsis thaliana, we classified genes according to the statistical features of their coding sequences. A clustering algorithm using a codon usage model was developed and applied to coding sequences from A. thaliana, E. coli, and a mixture of both. By using it, Arabidopsis sequences were clustered into two classes. The CU1 and CU2 classes differed essentially by the choice of pyrimidine bases at the codon silent sites: CU2 genes often use C whereas CU1 genes prefer T. This classification discriminated the Arabidopsis genes according to their expressiveness, highly expressed genes being clustered in CU2 and genes expected to have a lower expression, such as the regulatory genes, in CU1. The algorithm separated the sequences of the Escherichia-Arabidopsis mixed data set into five classes according to the species, except for one class. This mixed class contained 89 % Arabidopsis genes from CU1 and 11 % E. coli genes, mostly horizontally transferred. Interestingly, most genes encoding organelle-targeted proteins, except the photosynthetic and photoassimilatory ones, were clustered in CU1. By tailoring the GeneMark CDS prediction algorithm to the observed coding sequence classes, its quality of prediction was greatly improved. Similar improvement can be expected with other prediction systems.

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.

Effect of stereoisomerism on the cellular pharmacology of beta-enantiomers of cytidine analogs in Hep-G2 cells.

The beta-L enantiomers of 2',3'-dideoxycytidine (beta-L-ddC) and its 5-fuoro derivative, 2',3'-dideoxy-5-fluorocytidine (beta-L-FddC), were demonstrated to be active against human immunodeficiency virus (HIV) and hepatitis B virus (HBV) replication in vitro. In the present study, we investigated the cellular pharmacology of beta-L-ddC and beta-L-FddC and compared it with that of beta-D-2',3'-dideoxy-5-fluorocytidine (beta-D-FddC). Beta-L-FddC (10 microM) was found to be phosphorylated rapidly in Hep-G2 cells to its 5'-mono-, di-, and triphosphate derivatives with intracellular triphosphate levels achieving 26.6 +/- 10.9 pmol/10(6) cells after 72 hr. In contrast, the active 5'-phosphorylated derivative of beta-D-FddC achieved lower levels with triphosphate levels of only 2.3 +/- 0.5 pmol/ (10(6) cells under the same conditions. Beta-L-ddC was also phosphorylated rapidly. A 5'-diphosphocholine (18 +/- 5.8 pmol/10(6) cells) and a 5'-diphosphoethanolamine (13.6 +/- 0.9 pmol/10(6) cells) derivative were detected in beta-D-FddC-treated cells after 72 hr, whereas in beta-L-FddC- and beta-L-ddC-treated cells, only the 5'-diphosphocholine derivative (10.9 +/- 2.8 and 60.4 +/- 5.7 pmol/10(6) cells, respectively) was detected. Beta-L-FddC-5'-triphosphate (beta-L-FddCTP), beta-D-FddC-5'-triphosphate (beta-D-FddCTP), and beta-L-ddC-5'-triphosphate (beta-L-ddCTP) followed a single phase elimination process with an intracellular half-life (T1/2) of 10.5, 5.7, and 12.3 hr, respectively. Furth ermore, beta-L-FddCTP, beta-D-FddCTP, and beta-L-ddCTP levels of 6.7 +/- 2.3, 0.3 +/- 0.1, and 12.0 pmol/10(6) cells, respectively, were still detectable 24 hr following drug removal. The higher intracellular 5'-triphosphate levels of beta-L-FddC and the extended T1/2 of its 5'-triphosphate are consistent with the more potent in vitro antiviral activity of beta-L-FddC in Hep-G2 cells when compared with its beta-D enantiomer, beta-D-FddC.

Inhibition of glucagon and insulin secretion from the perfused rat pancreas by a B-cell-selective somatostatin analog.

The isolated perfused rat pancreas was used to study the effects of somatostatin and the analog des-Asn5-[D-Trps, D-Ser13]-somatostatin on arginine-stimulated insulin and glucagon secretion. Even though the analog was found to inhibit glucagon secretion at concentrations of 10 and 100 ng/ml, it had a relatively more inhibitory effect on the B cell than on the A cell than did somatostatin itself. These data suggest that the A- and B-cell receptors for these two peptides differ.

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.

[The model of French medicine and its structural requirements for the year 2000]. / Le modèle de la médecine française et ses besoins structurels pour l'an 2000.

The authors review the quality and defects of the French medicine model regarding its structural, hygienic, human, budgetary, geographic, educational and scientific aspects. They suggest several solutions concerning the necessary separation between hospitals, which should be reserved for patients, and institutions for healthy subjects, because of microbial infection dangers. They recommend the division of the latter into centers for family planning and maternity, centers for the detection, prevention and health education and centers for medical adaptation and education. They stress the advantages and the necessity of maintaining two structures, public and private, and the need for a European health policy to produce medical appliances in the field of imaging and automation, which France lacks so much that the short and long term future of its medicine is threatened.

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.

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.

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.

Synthesis and antiviral evaluation of the beta-L-enantiomers of some thymine 3'-deoxypentofuranonucleoside derivatives.

3'-Deoxy-beta-L-erythro- (3), 3'-deoxy-beta-L-threo- (6), 2'-fluoro- (7) and 2'-azido-2',3'-dideoxy-beta-L-erythro- (10) pentofuranonucleoside derivatives of thymine have been synthesized and their antiviral properties examined. All these derivatives were stereospecifically prepared by glycosylation of thymine with a suitable peracylated 3-deoxy-L-erythro-pentofuranose sugar (1), followed by appropriate chemical modifications. The prepared compounds were tested for their activity against HIV, but they did not show an antiviral effect.

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.

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 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.

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).