Hypoxia-inducible factor 1␣ (HIF-1␣) is essential for vascular development during embryogenesis and pathogenesis. However, little is known about its role in brain development. To investigate the function of HIF-1␣ in the central nervous system, a conditional knockout mouse was made with the Cre/LoxP system with a nestin promoter-driven Cre. Neural cell-specific HIF-1␣-deficient mice exhibit hydrocephalus accompanied by a reduction in neural cells and an impairment of spatial memory. Apoptosis of neural cells coincided with vascular regression in the telencephalon of mutant embryos, and these embryonic defects were successfully restored by in vivo gene delivery of HIF-1␣ to the embryos. These results showed that expression of HIF-1␣ in neural cells was essential for normal development of the brain and established a mouse model that would be useful for the evaluation of therapeutic strategies for ischemia, including hypoxia-mediated hydrocephalus.Oxygen deprivation initiates a wide range of responses to increase oxygen supply, including compensation for the loss of vital energy by alternating the expression of a variety of genes. Many of these hypoxia-inducible genes appear to have a common mode of regulation that involves activation of hypoxiainducible factor 1␣ (HIF-1␣), an oxygen-responsive subunit member of the basic helix-loop-helix PAS (PER-ARNT-SIM) family. HIF-1␣ heterodimerizes with the aryl hydrocarbon receptor nuclear translocator (ARNT; HIF-1␤) and plays a key role in maintaining oxygen homeostasis by signaling hypoxic exposure to genes that are involved in angiogenesis, erythropoiesis, glycolysis, and cell survival (10,34,37). In addition to its roles in physiological oxygen homeostasis, HIF-1␣ has also been implicated in the pathogenesis of various diseases, including ischemic heart disease, stroke, and cancer (11,29,33,36).HIF-1␣ is expressed in the developing brain (16) and modulates gene activity in response to low oxygen in a hypoxic brain in vivo. HIF-1␣ has also been implicated as a critical factor in the pathogenesis of brain tumor vascularization and stroke by regulating local brain hypoxia (8,36,38). Although these results indicate that HIF-1␣ is involved in angiogenesis in the brain and neuroprotection, it has not been established whether HIF-1␣ contributes to brain development. Systemic disruption of the Hif-1␣ gene leads to embryonic lethality by day of embryonic development 11 (E11) that is accompanied by cardiovascular malformation and defective cephalic vascularization, indicating that HIF-1␣ is essential for embryonic vascularization (14,21,32). However, the significance of HIF-1␣ in the development of the central nervous system remains unclear.To investigate the function of HIF-1␣ in the central nervous system, a conditional knockout mouse was generated with the Cre/LoxP system with a nestin promoter-based neural precursor-specific Cre recombinase (12). The nestin promoter directs gene expression specifically in neural precursor cells, so that a loxP-flanked (floxed) gene can be disr...

Polyketides form many clinically valuable compounds. However, manipulation of their biosynthesis remains highly challenging. An understanding of gene cluster evolution provides a rationale for reprogramming of the biosynthetic machinery. Herein, we report characterization of giant modular polyketide synthases (PKSs) responsible for the production of aminopolyol polyketides. Heterologous expression of over 150 kbp polyketide gene clusters successfully afforded their products, whose stereochemistry was established by taking advantage of bioinformatic analysis. Furthermore, phylogenetic analysis of highly homologous but functionally diverse domains from the giant PKSs demonstrated the evolutionary mechanism for structural diversification of polyketides. The gene clusters characterized herein, together with their evolutionary insights, are promising genetic building blocks for de novo production of unnatural polyketides.

Genome sequencing of Streptomyces species has highlighted numerous potential genes of secondary metabolite biosynthesis. The mining of cryptic genes is important for exploring chemical diversity. Here we report the metabolite-guided genome mining and functional characterization of a cryptic gene by biochemical studies. Based on systematic purification of metabolites from Streptomyces sp. SN-593, we isolated a novel compound, 6-dimethylallylindole (DMAI)-3-carbaldehyde. Although many 6-DMAI compounds have been isolated from a variety of organisms, an enzyme catalyzing the transfer of a dimethylallyl group to the C-6 indole ring has not been reported so far. A homology search using known prenyltransferase sequences against the draft sequence of the Streptomyces sp. SN-593 genome revealed the iptA gene. The IptA protein showed 27% amino acid identity to cyanobacterial LtxC, which catalyzes the transfer of a geranyl group to (؊)-indolactam V. A BLAST search against IptA revealed much-more-similar homologs at the amino acid level than LtxC, namely, SAML0654 (60%) from Streptomyces ambofaciens ATCC 23877 and SCO7467 (58%) from S. coelicolor A3(2). Phylogenetic analysis showed that IptA was distinct from bacterial aromatic prenyltransferases and fungal indole prenyltransferases. Detailed kinetic analyses of IptA showed the highest catalytic efficiency (6.13 min ؊1 M ؊1 ) for L-Trp in the presence of dimethylallyl pyrophosphate (DMAPP), suggesting that the enzyme is a 6-dimethylallyl-L-Trp synthase (6-DMATS). Substrate specificity analyses of IptA revealed promiscuity for indole derivatives, and its reaction products were identified as novel 6-DMAI compounds. Moreover, ⌬iptA mutants abolished the production of 6-DMAI-3-carbaldehyde as well as 6-dimethylallyl-L-Trp, suggesting that the iptA gene is involved in the production of 6-DMAI-3-carbaldehyde.Natural products have been an important resource for drug discovery and development. Actinomycetes have been a rich source of natural products, and a wide variety of these chemicals have been used as medicinal drugs (7, 40) and as bioprobes (56) for the elucidation of biological functions. Recently, the screening of bioactive compounds from microorganisms has often resulted in the identification of previously isolated compounds. The decreasing hit rate for new chemicals has reduced the advantage of natural product screening. However, genome sequencing of Streptomyces species highlighted numerous potential areas with metabolic diversity (4, 25, 42). The number of cryptic gene clusters was much larger than that of secondary metabolites identified from each strain. In addition, the cryptic gene clusters contained genes encoding plenty of unique modification enzymes that had the potential to expand the chemical diversity in drug seeds.To uncover cryptic gene clusters that might code for biosynthesis of secondary metabolites, genome sequence-guided metabolite identification has been performed in combination with heterologous expression, gene knockout, and complementation analyses ...

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