Jean-Jacques Sanglier

LEPTOMYCIN B IS AN INHIBITOR OF NUCLEAR EXPORT : INHIBITION OF NUCLEO-CYTOPLASMIC TRANSLOCATION OF THE HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 (HIV-1) REV PROTEIN AND REV-DEPENDENT MRNA

BACKGROUND The human immunodeficiency virus type 1 (HIV-1) regulatory protein Rev is required for unspliced and incompletely spliced viral mRNAs to appear in the cytoplasm and thus for viral replication. Translocation of Rev from the nucleus to the cytoplasm is essential if Rev is to function. We wanted to identify inhibitors of this transport process because they would be potential antiviral agents. RESULTS The Streptomyces metabolite, leptomycin B, and other antibiotics of the leptomycin/kazusamycin family were identified as inhibitors of the nucleo-cytoplasmic translocation of Rev at nanomolar concentrations. Rev-dependent export of mRNA into the cytoplasm is also blocked by leptomycin B, which inhibits Rev-dependent, but not Rev-independent gene expression in a short-term transfection assay. In primary human monocytes, leptomycin B suppresses HIV-1 replication. CONCLUSIONS Leptomycin B is the first low molecular weight inhibitor of nuclear export to be identified. Although it cannot be used therapeutically, it should serve as a valuable tool for dissecting nuclear export pathways.

Sanglifehrin A, a novel cyclophilin-binding compound showing immunosuppressive activity with a new mechanism of action.

We report here on the characterization of the novel immunosuppressant Sanglifehrin A (SFA). SFA is a representative of a class of macrolides produced by actinomycetes that bind to cyclophilin A (CypA), the binding protein of the fungal cyclic peptide cyclosporin A (CsA). SFA interacts with high affinity with the CsA binding side of CypA and inhibits its peptidyl-prolyl isomerase activity. The mode of action of SFA is different from known immunosuppressive drugs. It has no effect on the phosphatase activity of calcineurin, the target of the immunosuppressants CsA and FK506 when complexed to their binding proteins CypA and FK binding protein, respectively. Moreover, its effects are independent of binding of cyclophilin. SFA inhibits alloantigen-stimulated T cell proliferation but acts at a later stage than CsA and FK506. In contrast to these drugs, SFA does not affect IL-2 transcription or secretion. However, it blocks IL-2-dependent proliferation and cytokine production of T cells, in this respect resembling rapamycin. SFA inhibits the proliferation of mitogen-activated B cells, but, unlike rapamycin, it has no effect on CD154/IL-4-induced Ab synthesis. The activity of SFA is also different from that of other known late-acting immunosuppressants, e.g., mycophenolate mofetil or brequinar, as it does not affect de novo purine and pyrimidine biosynthesis. In summary, we have identified a novel immunosuppressant, which represents, in addition to CsA, FK506 and rapamycin, a fourth class of immunophilin-binding metabolites with a new, yet undefined mechanism of action.

Planotetraspora thailandica sp. nov., isolated from soil in Thailand.

A Gram-positive-staining, filamentous bacterial strain that developed cylindrical sporangia containing four oval- to rod-shaped spores at the ends of short sporangiophores on branched aerial mycelium was isolated from tropical rainforest soil near a hot spring. The cell-wall peptidoglycan contained meso-diaminopimelic acid, glutamic acid and alanine as cell-wall amino acids; the whole-cell hydrolysate contained rhamnose, madurose, glucose, galactose and 3-O-methylmannose as whole-cell sugars. The predominant menaquinone was MK-9(H(4)). Mycolic acids were not detected. The diagnostic phospholipid was phosphatidylethanolamine. The predominant cellular fatty acids were iso-C(16 : 0) and 10-methylated C(17 : 0). The G+C content of the DNA was 71.1 mol%. The phenotypic and chemotaxonomic analyses showed that the isolate had characteristics typical of members of the genus Planotetraspora. Furthermore, 16S rRNA gene sequence analysis also indicated that this strain belongs to the genus Planotetraspora, but as a putative novel species. Following phenotypic, chemotaxonomic and genotypic studies, the isolate is proposed to be a representative of a novel species, to be named Planotetraspora thailandica sp. nov. The type strain is BCC 21825(T) (=NBRC 104271(T)). An emended description of the genus Planotetraspora is also presented.

EPC Syntheses of Trifluorocitronellol and of Hexafluoropyrenophorin – A Comparison of Their Physiological Properties with the Nonfluorinated Analogs

The natural products pyrenophorin (1a) and citronellol (2a), in which CH3 groups are replaced by CF3, were synthesized in enantiomerically pure form from simple four-carbon trifluorohydroxy acids (obtained by resolution). The cyclizations of analogous CH3 and CF3 seco acids (cf. 9) to give pyrenophorin derivatives require different methodologies; the F6 derivative 10a could be obtained in only very poor yield; in contrast to pyrenophorin. Most surprisingly, F6-pyrenophorin (1d) has an extremely poor solubility in common organic solvents, and has essentially no antimicrobial activity (see Table 2). The synthesis of F3-citronellol is the first application of an enantiopure F3-Roche acid (12) as a synthetic builiding block (see its derivatives 17–23). An olfactory comparison of F3-citronellol [(R)-(+)-2b] with citronellol and ent-citronellol (Scheme 6) shows that the fluorine derivative has a “very metallic, aggressive” character and lacks totally the “sweetness” of (R)-(+)- and (S)-(–)-2a. A number of generally useful, CF3-substituted electrophilic (iodides 4, 18, 37, tosylates 19, 33, aldehydes 5, 29, 39) and nucleophilic (Li dithiane precursor of 5, Li compounds 20, 38) reagents are described for the first time.

Microbial conversion of rapamycin

Abstract In order to obtain derivatives of rapamycin, a total of 28 bacterial and 72 fungal strains were screened for their ability to transform rapamycin. In the course of this screening, the already known derivatives 39-o-demethylrapamycin, 27-o-demethylrapamycin, 16-o-demethylrapamycin, and the 40-o-phosphoric ester of rapamycin were detected and isolated out of fermentations with Streptomyces rimosus ATCC 28893 or Thamnidium elegans ATCC 8997. Biotransformation of rapamycin using Syncephalastrum racemosus ATCC 1332B, Gliocladium deliquescens ATCC 10097, or Bacillus subtilis ATCC 55060 yielded the conversion products 24-hydroxyrapamycin, secorapamycin A, B, and C, and 16-o-demethylsecorapamycin B. None of these derivatives exhibited a stronger immunosuppressive effect than the parent compound; however, in the case of 24-hydroxyrapamycin and 40-o-phosphoric ester of rapamycin, a FKBP-binding affinity comparable to rapamycin was observed.

Exobiopolymer production of Ophiocordyceps dipterigena BCC 2073: optimization, production in bioreactor and characterization

BackgroundBiopolymers have various applications in medicine, food and petroleum industries. The ascomycetous fungus Ophiocordyceps dipterigena BCC 2073 produces an exobiopolymer, a (1→3)-β-D-glucan, in low quantity under screening conditions. Optimization of O. dipterigena BCC 2073 exobiopolymer production using experimental designs, a scale-up in 5 liter bioreactor, analysis of molecular weight at different cultivation times, and levels of induction of interleukin-8 synthesis are described in this study.ResultsIn order to improve and certify the productivity of this strain, a sequential approach of 4 steps was followed. The first step was the qualitative selection of the most appropriate carbon and nitrogen sources (general factorial design) and the second step was quantitative optimization of 5 physiological factors (fractional factorial design). The best carbon and nitrogen source was glucose and malt extract respectively. From an initial production of 2.53 g·L-1, over 13 g·L-1 could be obtained in flasks under the improved conditions (5-fold increase). The third step was cultivation in a 5 L bioreactor, which produced a specific growth rate, biomass yield, exobiopolymer yield and exobiopolymer production rate of 0.014 h-1, 0.32 g·g-1 glucose, 2.95 g·g biomass-1 (1.31 g·g-1 sugar), and 0.65 g.(L·d)-1, respectively. A maximum yield of 41.2 g·L-1 was obtained after 377 h, a dramatic improvement in comparison to the initial production. In the last step, the basic characteristics of the biopolymer were determined. The molecular weight of the polymer was in the range of 6.3 × 105 - 7.7 × 105 Da. The exobiopolymer, at 50 and 100. μg·mL-1, induced synthesis in normal dermal human fibroblasts of 2227 and 3363 pg·mL-1 interleukin-8 respectively.ConclusionsHigh exobiopolymer yield produced by O. dipterigena BCC 2073 after optimization by qualitative and quantitative methods is attractive for various applications. It induced high IL-8 production by normal dermal fibroblasts, which makes it promising for application as wound healing material. However, there are still other possible applications for this biopolymer, such as an alternative source of biopolymer substitute for hyaluronic acid, which is costly, as a thickening agent in the cosmetic industry due to its high viscosity property, as a moisturizer, and in encapsulation.

Kutzneria buriramensis sp. nov., isolated from soil, and emended description of the genus Kutzneria

A Gram-staining-positive, filamentous bacterium, which developed large globose sporangia at the ends of long sporangiophores on aerial mycelium, was isolated from dry soil collected in a deciduous forest in Thailand. The cell-wall peptidoglycan of the novel bacterium, which was designated strain A-T 1846(T), contained meso-diaminopimelic acid and the whole-cell sugars comprised rhamnose, ribose, mannose, glucose and galactose. The predominant menaquinone was MK-9(H(4)). Mycolic acids were not detected. The identified phospholipids were phosphatidylmethylethanolamine, phosphatidylethanolamine, hydroxyphosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol and diphosphatidylglycerol. The predominant cellular fatty acids were iso-C(16:0), iso-C(16 : 0) 2-OH and C(16 : 0) 10-methyl. The G+C content of the genomic DNA was 72 mol%. The phenotypic and chemotaxonomic characteristics of the novel strain resembled those of established members of the genus Kutzneria. Phylogenetic analyses based on 16S rRNA gene sequences also indicated that this strain belonged to the genus Kutzneria but as a representative of a putative novel species. In hybridization experiments, the DNA-DNA relatedness values recorded between the novel strain and established members of the genus Kutzneria all fell well below 70 %. Based on the phenotypic, chemotaxonomic and genotypic evidence and the results of the DNA-DNA hybridizations, strain A-T 1846(T) represents a novel species in the genus Kutzneria, for which the name Kutzneria buriramensis sp. nov. is proposed. The type strain is A-T 1846(T) (=BCC 29373(T) =NBRC 107931(T)). An emended description of the genus Kutzneria is provided.

Sphaerisporangium krabiense sp. nov., isolated from soil.

A Gram-staining-positive, filamentous bacterial strain, designated A-T 0308(T), was isolated from soil of a tropical mangrove forest in Thailand. Strain A-T 0308(T) developed spherical sporangia containing non-motile spores on aerial mycelium. The novel strain contained meso-diaminopimelic acid, N-acetyl-type peptidoglycan and madurose, mannose, ribose, galactose and glucose as whole-cell sugars. The predominant menaquinones were MK-9(H(4)) and MK-9(H(6)); a small amount of MK-9(H(2)) and MK-9 was also detected. Mycolic acids were not detected. The diagnostic phospholipids were phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside and phosphoglycolipid. The predominant cellular fatty acids were iso-C(16:0) and 10-methylated C(17:0). The G+C content of the DNA was 72 mol%. Phenotypic and chemotaxonomic analyses showed that the novel isolate had characteristics typical of members of the genus Sphaerisporangium. 16S rRNA gene sequence analysis also indicated that the strain belongs to the genus Sphaerisporangium and that it represents a clade distinct from other members of the genus with sequence similarities ranging from 96.3 to 97.8% between the novel strain and its closest relatives. Based on the results of phenotypic, chemotaxonomic and phylogenetic studies, strain A-T 0308(T) (=BCC 21702(T) =NBRC 107571(T)) represents a novel species of the genus Sphaerisporangium, for which the name Sphaerisporangium krabiense sp. nov. is proposed.

Bioconversion of ascomycin and its 6-alkoxyderivatives

Ascomycin is known to be O-demethylated to 32-O-desmethyl-ascomycin by bioconversion using the strain Actinoplanes sp. ATCC 53771. Using modified screening media, the conversion rate could be improved from previously published 15% to up to 35%. Together with 32-O-desmethyl-ascomycin, two further metabolites, the already known 32-O-desmethyl-19-hydroxymethylascomycin and the new 32-O-desmethyl-32-epi-ascomycin were isolated and characterized. Prompted by the observed demethylation capability of ATCC 53771, we studied whether this strain was also able to dealkylate 6-alkoxy derivatives of ascomycin to 6-hydroxy-ascomycin. Although the 6-alkoxy derivatives were demethylated at various positions of the molecule, production of the hypothetical metabolite 6-hydroxy-ascomycin by biotransformation could not be shown. However, work up of the fermentation broth allowed the isolation and characterization of a new metabolite, the 6-ethoxy-15-desmethyl-ascomycin.

Planobispora siamensis sp. nov., isolated from soil.

A novel actinomycete strain, A-T 4600(T), which developed cylindrical sporangia containing a longitudinal pair of motile spores forming singly or in bundles on short ramifications of the aerial mycelium, was isolated from soil collected from an evergreen forest in Thailand. The cell-wall peptidoglycan contained meso-diaminopimelic acid. The whole-cell sugars contained ribose, madurose, mannose and glucose. The predominant menaquinones were MK-9(H2). Mycolic acids were not detected. The diagnostic phospholipids were phosphatidylethanolamine, phosphatidylmethylethanolamine, phosphatidylinositol, phosphatidylglycerol, phosphatidylinositol mannoside, diphosphatidylglycerol and aminophosphoglycolipid. The predominant cellular fatty acids were unsaturated C17 : 1, C18 : 1, saturated C16 : 0, and C17 : 0. The G+C content of the DNA was 70.8 mol%. Phenotypic and chemotaxonomic analyses showed that the isolate had the typical characteristics of members of the genus Planobispora. Furthermore, 16S rRNA gene sequence analysis also indicated that this strain belonged to the genus Planobispora but as a putative novel species. DNA-DNA relatedness values that differentiate the isolate from previously described members of the genus Planobispora were significantly below 70 %. Following an evaluation of phenotypic, chemotaxonomic and genotypic studies, it is proposed that the isolate represents a novel species, Planobispora siamensis sp. nov.; the type strain is A-T 4600(T) ( = BCC 39469(T) = NBRC 107568(T)).