Actinoplanes friuliensis produces the lipopeptide antibiotic friulimicin, which is a cyclic peptide with one exocyclic amino acid linked to a branched-chain fatty acid acyl residue. The structural relationship to daptomycin and the excellent antibacterial performance of friulimicin make the antibiotic an attractive drug candidate. The complete friulimicin biosynthetic gene cluster of 24 open reading frames from A. friuliensis was sequenced and analyzed. In addition to genes for regulation, self-resistance, and transport, the cluster contains genes encoding peptide synthetases, proteins involved in the synthesis and linkage of the fatty acid component of the antibiotic, and proteins involved in the synthesis of the nonproteinogenic amino acids pipecolinic acid, methylaspartic acid, and 2,3-diaminobutyric acid. By using heterologous gene expression in Escherichia coli, we provide biochemical evidence for the stereoselective synthesis of L-pipecolinic acid by the deduced protein of the lysine cyclodeaminase gene pip. Furthermore, we show the involvement of the dabA and dabB genes in the biosynthesis of 2,3-diaminobutyric acid by gene inactivation and subsequent feeding experiments.In recent years, the sequences of numerous gene clusters involved in the synthesis of many secondary metabolites have become available for comparison and exploitation. The programmed manipulation of genes encoding enzymes in the biosynthetic pathways offers promise for redesigning existing antibiotic structures to create antibiotics with new activities and the ability to overcome bacterial resistance (50). Therefore, each newly analyzed gene cluster represents a new tool for combinatorial biosynthesis and can provide information about the synthesis of unusual building blocks, such as nonproteinogenic amino acids, acyl residues, and sugar moieties. An interesting group of secondary metabolites for such experiments seems to be bioactive lipopeptides isolated from streptomycetes. So far, only a few biosynthetic gene clusters corresponding to these lipopeptides have been isolated and characterized, such as the clusters for calcium-dependent antibiotic (CDA) from Streptomyces coelicolor (17), daptomycin from Streptomyces roseosporus (26), and A54145 from Streptomyces fradiae (24). By targeted modification and gene exchange, it is possible to generate new lipopeptide structures (12,25).Another member of this group of secondary metabolites is the antibiotic friulimicin that is produced by the actinomycete Actinoplanes friuliensis. This compound is highly active against multidrug-resistant gram-positive bacteria, such as methicillinresistant Staphylococcus aureus strains (4). The biosynthesis of this lipopeptide is catalyzed by nonribosomal peptide synthetases (NRPS) (15).The eight bioactive lipopeptides isolated from A. friuliensis (4) consist of 10 amino acids that form a ring structure with one exocyclic amino acid linked to an acyl residue of various chain lengths. The acyl residue is a branched-chain fatty acid with a ⌬cis3 double bond. The ...

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The lipopeptide antibiotic friulimicin, produced by Actinoplanes friuliensis, is an effective drug against Gram-positive bacteria, such as methicillin-resistant Staphylococcus epidermidis and Staphylococcus aureus strains. Friulimicin consists of a cyclic peptide core of ten amino acids and an acyl residue linked to an exocyclic amino acid. The acyl residue is essential for antibiotic activity, varies in length from C13 to C15, and carries a characteristic double bond at position Dcis3. Sequencing of a DNA fragment adjacent to a previously described fragment encoding some of the friulimicin biosynthetic genes revealed several genes whose gene products resemble enzymes of lipid metabolism. One of these genes, lipB, encodes an acyl-CoA dehydrogenase homologue. To elucidate the function of the LipB protein, a lipB insertion mutant was generated and the friulimicin derivative (FR242) produced by the mutant was purified. FR242 had antibiotic activity lower than friulimicin in a bioassay. Gas chromatography showed that the acyl residue of wild-type friulimicin contains a double bond, whereas a saturated bond was present in FR242. These results were confirmed by the heterologous expression of lipB in Streptomyces lividans T7, which led to the production of unsaturated fatty acids not found in the S. lividans T7 parent strain. These results indicate that the acyl-CoA dehydrogenase LipB is involved in the introduction of the unusual Dcis3 double bond into the acyl residue of friulimicin.

Cells from metazoan organisms are eliminated in a variety of physiological and pathophysiological processes by apoptosis. In this report, we describe the cloning and characterization of molecules from the marine sponges Geodia cydonium and Suberites domuncula, whose domains show a high similarity to those that are found in molecules of the vertebrate Bcl-2 superfamily and of the death receptors. The Bcl-2 proteins contain up to four Bcl-2 homology regions (BH). Two Bcl-2-related molecules have been identified from sponges that are provided with two of those regions, BH1 and BH2, and are termed Bcl-2 homology proteins (BHP). The G. cydonium molecule, BHP1_GC, has a putative size of 28,164, while the related sequence from S. domuncula, BHP1_SD, has a M(r) of 24,187. Phylogenetic analyses of the entire two sponge BHPs revealed a high similarity to members of the mammalian Bcl-2 superfamilies and to the Caenorhabditis elegans Ced-9. When the two domains, BH1 and BH2, are analyzed separately, again the highest similarity was found to the members of the Bcl-2 superfamily, but a clearly lower relationship to the C. elegans BH1 and BH2 domains in Ced-9. In unrooted phylogenetic trees the sponge BH1 and BH2 are grouped among the mammalian sequences and are only distantly related to the C. elegans BH domains. The analysis of the gene structure of the G. cydonium BHP showed that the single intron present is located within the BH2 domain at the same position as in C. elegans and rat Bcl-x(L). In addition, a sponge molecule comprising two death domains has been characterized from G. cydonium. The two death domains of the potential proapoptotic molecule GC_DD2, M(r) 24,970, share a high similarity with the Fas-FADD/MORT1 domains. A death domain-containing molecule has not been identified in the C. elegans genome. The phylogenetic analysis revealed that the sponge domain originated from an ankyrin building block from which the mammalian Fas-FADD/MORT1 evolved. It is suggested that the apoptotic pathways that involve members of the Bcl-2 superfamily and of the death receptors are already present in the lowest metazoan phylum, the Porifera.

Spontaneous remission in patients with acute myeloid leukemia (AML) is a rarely reported phenomenon of usually short duration. The etiology remains unclear, but an association with preceding blood transfusions or bacterial infections has been reported. Triggered immune responses are suggested to play a potential role in the development of spontaneous remission. Acute monocytic leukemia was diagnosed in a 61-yr-old male patient. Cytogenetic analysis revealed a sole translocation (9;11) (q22;q23) and RT-PCR the MLL/AF9 fusion gene. As a result of the patient's reduced performance status and septic condition, cytostatic therapy was withheld. No microorganisms could be detected. Hematologic and molecular remission occurred after initiating antibiotic therapy without any cytostatic treatment; 29 months after the initial diagnosis, he is in complete remission, and excellent physical condition. Our report includes a review of the literature since 1985, reporting cases of patients with AML and spontaneous remission together with informative cytogenetics. Balanced translocations such as in core binding factor (CBF) leukemias appear somewhat overrepresented. We speculate that AML-specific T cells might be relevant for induction of spontaneous remission and need to be further investigated.

Differential methylation of CpG islands is a regulatory mechanism for promoter activity of different classes of genes, including tissue-specific genes. These CpG islands are targets for transformation-associated, aberrant hypermethylation activity during leukemogenesis. Therefore the pharmacological reversion of this methylator phenotype (e.g. by reactivation of tumor suppressor gene expression) is an important rationale for development of inhibitors of DNA methyltransferase activity. In vitro, inhibition of methylation using azanucleosides results in modest differentiation of transformed myeloid cell lines. In vivo, low doses of these agents induce DNA demethylation of malignant myeloid cells. Indeed, the first drug specifically approved for the treatment of myelodysplastic syndrome (MDS) was the azanucleoside 5-azacytidine (Vidaza). The most potent DNA demethylating agent available, 5-aza-2' deoxycytidine (Decitabine, Dacogen) also has recently been approved by the U.S.A. FDA for treatment of MDS of all subtypes. About 30 % of MDS patients with an abnormal karyotype have normalization of their karyotype after receiving the drug. This activity is especially relevant in patients with high-risk karyotypic abnormalities (complex karyotype and/or abnormalities of chromosome 7) compared to patients with intermediate-risk karyotype. Both drugs offer a novel, non-intensive therapeutic approach, particularly in the older patient population who due to comorbidities and/or reduced performance status are ineligible for aggressive chemotherapies. Target genes being particularly prone to demethylation by these drugs in the aberrant cells (e.g. p15/INK4b) are under active investigation. Future translational and clinical studies will be aimed at improving the response rate and duration of response to non-intensive treatment with demethylating agents, by studying rational drug combinations e.g. with inhibitors of histone deacetylase activity.