Transinfection and growth discrepancy of Drosophila Wolbachia strain wMel in cell lines of the mosquito Aedes albopictus.

AIM: The Wolbachia strain wMel can protect Drosophila melanogaster against pathogenic RNA viruses. To analyse the potential of this inhibitory effect against arboviruses vectorized by these mosquitoes, we here first transinfected the Aedes albopictus Aa23 and C6/36 cell lines with the Wolbachia strain wMel and then monitored their infection dynamics. METHODS AND RESULTS: Wolbachia strain wMel was transferred into A. albopictus Aa23 and C6/36 cell lines using the shell vial technique. The presence of the bacterium in the transinfected cells was monitored by quantitative PCR and fluorescence in situ hybridization. Bacteria could be detected in the cytoplasm of both the Aa23 and C6/36 cell lines. However, the dynamics and stability of the bacterial infection differed depending on the initial cell background. The Aa23 cell line, which had been treated with a tetracycline antibiotic 2 years previously to eliminate its natural Wolbachia wAlbB-infecting strain, lost the introduced Wolbachia wMel strain after 12 passages postinfection. In contrast, the C6/36 cell line, which had originally been aposymbiotic, displayed a stable infection with Wolbachia wMel. The bacterial density in C6/36 was greater than that of the A. albopictus RML12 cell line from which the wMel strain had originated. CONCLUSIONS: Transient or persistent transinfection of A. albopictus Aa23 and C6/36 cell lines with Wolbachia wMel strain was achieved. The results indicate the influence of the genetic background of mosquito cells in maintaining Wolbachia originating from a distant dipteral host. SIGNIFICANCE AND IMPACT OF THE STUDY: The cell model built here can now be used to investigate the viral inhibitory effect of the Wolbachia wMel strain against arboviruses such as dengue and chikungunya, which are transmitted by the mosquito A. albopictus.

Miss rate for colorectal neoplastic polyps: a prospective multicenter study of back-to-back video colonoscopies.

BACKGROUND AND STUDY AIM: Polyp miss rates during colonoscopy have been calculated in a few tandem or back-to-back colonoscopy studies. Our objective was to assess the adenoma miss rate while limiting technique or operator expertise biases, i. e. by performing a large multicenter study, with same-day back-to-back video colonoscopy, done by two different operators in randomized order and blinded to the other examination. PATIENTS AND METHODS: 294 patients at 11 centers were included. Among the 286 analyzable tandem colonoscopies, miss rates were calculated in both a lesion- and patient-based analysis. Each of these rates was determined for polyps overall, for adenomas, and then for lesions larger than 5 mm, and for advanced adenomas. Univariate and logistic regression analysis were performed to define independent variables associated with missed polyps or adenomas. RESULTS: The miss rates for polyps, adenomas, polyps > or = 5 mm, adenomas > or = 5 mm, and advanced adenomas were, respectively, 28 %, 20 %, 12 %, 9 % and 11 %. None of the masses with a carcinomatous (n = 3) or carcinoid component (n = 1) was missed. The specific lesion miss rates for patients with polyps and adenomas were respectively 36 % and 26 % but the corresponding rates were 23 % and 9.4 % when calculated for all 286 patients. The diameter (1-mm increments) and number of polyps (> or = 3) were independently associated with a lower polyp miss rate, whereas sessile or flat shape and left location were significantly associated with a higher miss rate. Adequacy of cleansing, presence of diverticula, and duration of withdrawal for the first procedure were not associated with adenoma miss rate. CONCLUSIONS: We confirm a significant miss rate for polyps or adenoma during colonoscopy. Detection of flat polyps is an issue that must be focused on to improve the quality of colonoscopy.

Oncostatic effects of Alangium vitiense extracts (ICIG-EORTC 1131, 1186 and 1207) on lymphoid murine tumors.

A total alkaloid and two purified alkaloid extracts of Alangium vitiense were found to be oncostatic for L1210 leukemia; the total alkaloid exerted a noticeable activity, and the purified extracts exerted a borderline activity. These two purified extracts are noticeably oncostatic for two other lymphoid neoplasias in mice, P388 leukemia and Gardner lymphosarcoma. These compounds are not active on Warner myelomonocytic leukemia WEH13 or on B16 melanoma.

Purification of the dissimilative nitrate reductase of Pseudomonas fluorescens and the cloning and sequencing of its corresponding genes.

The dissimilative membrane-bound nitrate reductase from Pseudomonas fluorescens strain AK15 was purified and the alpha subunit of the enzyme partially sequenced. On the basis of this partial amino acid sequence and of conserved stretches of amino acids between Escherichia coli and Bacillus subtilis, degenerate primers were design to amplify the narG gene and part of the narH gene in a PCR approach. The deduced amino acid sequence of narG shows 72% and 52% and narH 78% and 62% identity to the homologous subunit of E. coli and B. subtilis, respectively.

[New aspects of diabetes]. / Nouveaux aspects du diabète.

Type II diabetes is a serious, insidious disease which is growing at an impressive rate, with 200 million diabetics worldwide and as many who ignore their state. Having been seriously studied over more than a century and a half, an enormous quantity of knowledge regarding this disease has been accumulated. The research we are conducting has allowed us to identify the most important actors responsible for diabetes. These are glucose which leads to glyoxal and to methylglyoxal which in turn reacts with innumerable targets in the organism (including insulin) unless prevented from doing so by detoxifying mechanisms (e.g., glyoxalases). The role of microorganisms in the occurrence and development of diabetes has also to be seriously examined.

Structure-activity relationships of Taxol and Taxotere analogues.

From Taxol, Taxotere, and 10-deacetyl baccatin III, a number of compounds have been prepared. Their biological activity was evaluated on microtubule disassembly at 0 degrees C. The conformation of these Taxol and Taxotere analogues was determined by molecular modeling experiments and nuclear magnetic resonance spectroscopy and compared with the structure of Taxotere in the crystal, obtained by an x-ray analysis. The results of these studies given information on the crucial parts of the active molecules involved in the binding to tubulin.

NO, thiols and disulfides.

The chemical nature of the messenger molecule, nitric oxide (NO), and especially its reactivity towards thiol groups and disulfides, could explain, at least partly, its intervention in so many different biological processes. NO can be regarded as the smallest molecule suitable for electron transport in biological systems. The S-nitrosation reaction and its reverse reaction represent the most convenient general way to store, to transport and finally to release NO. Nitric oxide is also particularly convenient for playing a role in interconversions of thiol groups and disulfides in chain radical or oxidation-reduction processes, and to be subsequently engaged in complex sequences of reactions accounting for different biological situations.

Hybrid molecules: growth inhibition of Leishmania donovani promastigotes by thiosemicarbazones of 3-carboxy-beta-carbolines.

The thiosemicarbazones of beta-carboline-3-carboxaldehyde (compound 2) and 3-acetyl-beta-carboline (compound 3) were found to effectively inhibit the in vitro growth of the promastigote form of Leishmania donovani, 50% inhibition being obtained at concentrations of 5.0 and 2.5 microM, respectively, while irreversible growth inhibition was achieved at 40 (compound 2) and 17.5 microM (compound 3). The thiosemicarbazone of pyridine-2-carboxyaldehyde (compound 4) was considerably less active while both methyl beta-carboline-3-carboxylate (compound 1) and the thiosemicarbazone of ethyl 5-formyl-6-azaindole-2-carboxylate (compound 5) were inactive at the highest concentrations tested. At concentrations provoking approximately 50% growth inhibition of promastigotes, compound 2 was observed to preferentially block DNA rather than RNA synthesis, but for compound 3, the reverse was true. Compound 3, the most active analogue studied, may thus act, at least partially, via a novel, though as yet unelucidated, mechanism.

NAcSDKP analogues resistant to angiotensin-converting enzyme.

Two series of analogues of the tetrapeptide NAcSDKP, an inhibitor of hematopoietic stem cell proliferation, were prepared, and their enzymatic stability toward rabbit lung angiotensin-converting enzyme (ACE) was evaluated as well as their capacity to inhibit NAcSDKP hydrolysis by this enzyme. In the first series, each of the peptide bonds has been successively replaced by an aminomethylene bond. In the second one, the C-terminus of the peptide has been modified by decarboxylation or amidation. The results reported here indicate that all of these molecules but one have good stability toward the enzyme but none of the compounds is able to inhibit NAcSDKP hydrolysis by ACE.

Synthesis of beta-carboline-benzodiazepine hybrid molecules: use of the known structural requirements for benzodiazepine and beta-carboline binding in designing a novel, high-affinity ligand for the benzodiazepine receptor.

Hybrid molecules incorporating pharmacologically important structural features of both 3-carboxy-beta-carbolines and 1,4-benzodiazepines were synthesized, and their affinities for the benzodiazepine receptor were determined in vitro. One of these hybrids, 8,14-dioxo-13,14-dihydro-8H-indolo[3',2':4,5]pyrido[2,1-c] [1,4]benzodiazepine (13), demonstrated high affinity for the receptor, displacing both benzodiazepines (IC50 = 23 nM) and beta-carbolines (IC50 = 47 nM) from their binding sites. Of the compounds synthesized, 13 also most closely satisfied the structural requirements that generally ensure a high affinity of both beta-carbolines and benzodiazepines for the receptor (e.g., aromaticity of the beta-carboline, presence of a carbonyl at C-3 of the beta-carboline and of a pi 2-region on the benzodiazepine). The hybrids not fulfilling these requirements had no affinity for the receptor. In vivo pharmacological properties of 13 could not be demonstrated because of its metabolic instability and/or its poor transport into the brain. The results are discussed in terms of a possible overlapping of beta-carboline binding sites with those of benzodiazepines on the receptor.

Synthesis and activity of NAcSerAspLysPro analogues on cellular interactions between T-cell and erythrocytes in rosette formation.

Analogues of NAcSerAspLysPro (AcSDKP), a natural regulator of hematopoiesis isolated from fetal calf bone marrow, were synthesized. The biological activity of these molecules were evaluated in vitro in the rosette assay, which measures the interaction between human Jurkat T-cells and sheep red blood cells. In this test, the tripeptide SerAspLys was the most efficient. Inhibitory activity was detected at the concentration 10(-14) M for the analogue and at 10(-9) M for the parent tetrapeptide. The dipeptide NAcSerAsp still showed activity but at much higher doses (10(-6) M). Substitution of polar amino acids led mostly to inactive molecules. Thus, replacement of Ser by Ala, or Lys by Orn yielded completely inactive compounds and replacement of Asp by Glu decreased the activity (10(-6) M). The present study gives an insight into the role of individual amino acids of AcSDKP in the inhibition of the rosette formation which implicates interactions with T-cell CD2 glycoprotein.

Relationships between the structure of taxol analogues and their antimitotic activity.

A variety of synthetic analogues of taxol, a naturally occurring antitumor diterpene, were examined for their potency to inhibit microtubule disassembly. For some of the compounds, the in vitro cytotoxic properties showed a good correlation with the tubulin assay. This structure-activity relationship study shows that inhibition of microtubule disassembly is quite sensitive to the configuration at C-2' and C-3'. A correlation between the conformation of the side chain at C-13 and the activity is suggested. Of all the compounds examined, one of the most potent in inhibiting microtubule disassembly and in inhibiting murine P388 leukemic cells, N-debenzoyl-N-tert-(butoxycarbonyl)-10-deacetyltaxol, named taxotere, was selected for evaluation as a potential anticancer agent.

Synthetic routes to 4-amino-3-carboxy-beta-carboline derivatives: incidental formation of novel furo[3,4-c]-beta-carbolin-2-ones displaying high affinities for the benzodiazepine receptor.

The synthesis of the first 4-amino-3-carboxy-beta-carboline derivative (35) is described. This synthesis is based on ozonolysis of the 4-vinyl-beta-carboline-3-carboxamide 17 to give the 4-aldehyde 20 and potassium permanganate oxidation of the latter to the 4-carboxylic acid 34 followed by a DPPA-promoted Curtius rearrangement. During the course of these transformations, a number of furo[3,4-c]-beta-carbolin-2-ones, differing in substituents at the C-10 position, were formed. While these beta-carboline lactones (15,25,26,33) generally displayed good affinities for the central type benzodiazepine receptor in vitro (IC50's in the 10-50 nM range), one compound, 29, demonstrated an exceptionally high binding affinity (IC50 = 0.2 nM). Compound 29 was shown in electrophysiological and behavioral studies to act as a benzodiazepine receptor antagonist. The unusually high binding affinity of compound 29 corroborates the hypothesis that the benzodiazepine receptor preferentially recognizes the C-3 carbonyl function of 3-carboxy-beta-carbolines in an s-cis conformation (i.e., the carbonyl oxygen on the same side as the pyridinyl nitrogen).

3-Amino-beta-carboline derivatives and the benzodiazepine receptor. Synthesis of a selective antagonist of the sedative action of diazepam.

Seven 3-N-substituted derivatives of 3-amino-beta-carboline were synthesized and their affinities for the benzodiazepine receptor were assessed in vitro. Two compounds, 3-(ethylamino)-beta-carboline and 3-[(methoxycarbonyl)amino]-beta-carboline (beta-CMC), showing IC50 values of 460 and 71 nM, respectively, were selected for in vivo studies. The former compound showed long-lasting proconvulsant activity in Papio papio baboons while beta-CMC was shown in mice to selectively antagonize the sedative effects of diazepam without exhibiting convulsant, proconvulsant, or anxiogenic activity by itself.

Synthesis and benzodiazepine receptor affinities of rigid analogues of 3-carboxy-beta-carbolines: demonstration that the benzodiazepine receptor recognizes preferentially the s-cis conformation of the 3-carboxy group.

1H-Indolo[3',2':4,5]pyrido[3,2-b]-2-penten-5-olide (6) and 1H,5H-indolo[3',2'-c]-6,7-dihydro-2-pyridone (7), rigid analogues of methyl 4-ethyl-beta-carboline-3-carboxylate (8) and N-methyl-4-ethyl-beta-carboline-3-carboxamide (9), respectively, were synthesized and their in vitro binding affinities to the central type benzodiazepine receptors were compared. The IC50 values of 6 and 8 were approximately equivalent (42 and 27 nM, respectively). The amide derivative 9, for which theoretical energy calculations indicate that the s-trans carbonyl conformation is the preferred one, displayed very low affinity (IC50 greater than 10(4) nM). However, when the carbonyl group of 9 was forced to adopt the s-cis conformation as in lactam 7, binding to the benzodiazepine receptor was largely restored (IC50 = 150 nM), indicating that the s-cis carboxy conformation at C-3 of beta-carbolines is preferentially recognized by this receptor. In vivo, compound 6 showed neither convulsant, proconvulsant, nor anticonvulsant activity in mice. Moreover, 6 did not antagonize methyl beta-carboline-3-carboxylate induced convulsions in mice. This lack of activity of 6 was attributed to its inability to cross the blood-brain barrier since no significant displacement of [3H]Ro 15-1788 from mouse brain benzodiazepine receptors by 6 could be observed in vivo.

A new synthetic flavonoid protects endothelium-derived relaxing factor-induced relaxation in rabbit arteries in vitro: evidence for superoxide scavenging.

A new synthetic flavone derivative, 6,7-dimethoxy-8-methyl-3',4',5-trihydroxyflavone, was studied for its capacity to protect the acetylcholine-induced relaxation of rabbit ear and cerebral arteries from inhibition by superoxide anion. This property was evaluated via two types of in vitro experiments, using rabbit ear or basilar arteries mounted in organ baths equipped for isometric tension measurement. When a high level of superoxide anion was generated by adding 3 x 10(-4) M pyrogallol to the bath, the relaxation to acetylcholine was substantially inhibited. This inhibition was significantly reversed by both superoxide dismutase (25 and/or 50 U/mL) and the flavonoid (3 x 10(-6) M and/or 10(-5) M) in both types of arteries. In the presence of the basal level of superoxide anion, the responses to acetylcholine were significantly potentiated by the flavonoid (10(-5) M) in the ear but not the basilar artery. Thus this flavonoid protects endothelium-dependent relaxation from high levels of superoxide anion possibly by scavenging superoxide anion and may have a certain therapeutic value as an agent capable of promoting natural vasodilatation.

Influence of the antioxidant quercetin in vivo on the level of nitric oxide determined by electron paramagnetic resonance in rat brain during global ischemia and reperfusion.

We characterized the changes in nitric oxide (NO) levels in the brain during global forebrain ischemia and reperfusion and tested the ability of the natural flavonoid, quercetin, and a synthetic flavonoid, FB277, to increase the amount of available NO by elimination of the superoxide radicals produced during reperfusion. In Sprague-Dawley rats, we used a four-vessel occlusion model of forebrain ischemia (15 min) and reperfusion (30 min). Brain NO was measured on samples of cerebral cortex and cerebellum ex vivo by electron paramagnetic resonance (EPR) spectroscopy. The spin trap used was diethyldithiocarbamate sodium salt (DETC) associated with ferrous citrate. The complex Fe(DETC)2NO was detected at 77 K as a triplet signal at g = 2.035. Groups of animals were treated with quercetin or FB277 (3-morpholinomethyl-3',4',5,7tetramethoxyflavone) or polyethylene glycol-conjugated superoxide dismutase (PEG-SOD). In control (intact anesthetized animals), the signal was about 3 times greater in the cortex than in the cerebellum. During ischemia, the signal rose to 110% in cortex (NS) and 283% in cerebellum (P < 0.05). In reperfusion, it fell again to 91% of control in cerebellum (NS) and 35% in cortex (P < 0.05). Treatment by quercetin (5 mg/kg i.v.) of intact and ischemia-reperfusion groups did not significantly change the signal amplitude in the cerebellum, but did double it in the cortex (to 76% of control) for the ischemia-reperfusion group (P < 0.05). In contrast, FB277 (3.75 mg/kg i.v.) did not increase the signal in the cortex during ischemia-reperfusion, but did do so in the cerebellum (to 152% of control, P < 0.05). The results obtained for PEG-SOD (10,000 U/kg i.v.) were similar to those for FB277. In separate in vitro measurements, we found that quercetin but not FB277 efficiently scavenged superoxide. We hypothesize that quercetin but not FB277 scavenged superoxide anions released in the cortex during reperfusion, thus diminishing the amount of NO removed by the formation of peroxynitrite. The lack of effect of PEG-SOD may be related to the need for chronic treatment to obtain protection.

Therapeutic response to taxol of six human tumors xenografted into nude mice.

To test the antineoplastic activity of taxol, a natural product isolated from yew (Taxus baccata L.), six human tumors transplanted into athymic mice were used (primary tumors of breast, endometrium, ovary, brain, lung and a recurrence of tongue tumor). While the growth rates varied with the histopathological characteristics of different tumor types, all mice were treated at a mean tumor volume of 200 +/- 8 mm3. Taxol was given SC at a dose level of 12.5 mg/kg per injection per day for 5 consecutive days out of 7 over a period of 3 weeks. With this schedule antitumor responses were obtained in all of the six neoplasms xenografted into nude mice. In the case of the ductal carcinoma of the breast total tumor regressions were observed in four of the five treated animals. In the five other experimental models taxol produced significant growth delays. We believe that the results of these initial tests on the nude mouse--human tumor xenograft system are convincing and justify clinical assessment of this drug.

First use of a beta-carboline as photoaffinity label for the benzodiazepine receptor.

Photolabelling of benzodiazepine receptors isolated from rat cortex with a new beta-carboline-type photoaffinity label, ethyl 6-azido-beta-carboline-3-carboxylase, at 254 nm produced a 42% decrease in the maximal number of propyl beta-carboline-3-carboxylate binding sites but practically no decrease in the number of flunitrazepam binding sites. Moreover, the binding affinity of ethyl beta-carboline-3-carboxylase was diminished 11-fold by photolabelling while that of diazepam was diminished less than 2-fold. These results provide additional evidence that beta-carbolines and benzodiazepines bind to discrete sites on the benzodiazepine receptor.

Hispidulin, a natural flavone, inhibits human platelet aggregation by increasing cAMP levels.

Hispidulin, a natural flavone, and theophylline inhibited platelet aggregation triggered by adenosine-5'-monophosphate, arachidonic acid, paf-acether and collagen. Hispidulin was 100-fold more potent than theophylline. A threshold concentration of PGE1 did not modify the anti-aggregatory effect of hispidulin but potentiated the effect of theophylline. A threshold concentration of hispidulin had no effect on the inhibitory action of theophylline. Hispidulin (100 microM) and theophylline (10 mM) increased the control cAMP level in platelets 4-fold. A threshold concentration of PGE1 had a small effect on hispidulin-induced cAMP levels but increased the theophylline-induced cAMP levels 3-fold. Theophylline (10 mM)-induced cAMP levels were not modified by hispidulin. We demonstrate a correlation between the inhibition of platelet aggregation and the increase in cAMP levels induced by hispidulin. These data suggest that hispidulin could inhibit platelet aggregation by elevating cAMP levels by a mechanism different from that of theophylline or PGE1.