Kabirimine, a New Cyclic Imine from an Okinawan Dinoflagellate.

On our quest for new bioactive molecules from marine sources, two cyclic imines (1, 2) were isolated from a dinoflagellate extract, inhibiting the growth of the respiratory syncytial virus (RSV). Compound 1 was identified as a known molecule portimine, while 2 was elucidated to be a new cyclic imine, named kabirimine. The absolute stereochemistry of 1 was determined by crystallographic work and chiral derivatization, whereas the structure of 2 was elucidated by means of spectroscopic analysis and computational study on all the possible isomers. Compound 1 showed potent cytotoxicity (CC50 < 0.097 µM) against HEp2 cells, while 2 exhibited moderate antiviral activity against RSV with IC50 = 4.20 µM (95% CI 3.31-5.33).

Construction of a metagenomic library for the marine sponge Halichondria okadai.

Symbionts of the marine sponge Halichondria okadai are promising as a source of natural products. Metagenomic technology is a powerful tool for accessing the genetic and biochemical potential of bacteria. Hence, we established a method of recovering bacterial-enriched metagenomic DNA by stepwise centrifugation. The metagenomic DNA was analyzed by ultrafast 454-pyrosequencing technology, and the results suggested that more than three types of bacterial DNA, Alphaproteobacteria, Actinobacteria, and Cyanobacteria, had been recovered, and that eukaryotic genes comprised only 0.02% of the metagenomic DNA. These results indicate that stepwise centrifugation and real-time quantitative PCR were effective for separating sponge cells and symbiotic bacteria, and that we constructed a bacteria-enriched metagenomic library from a marine sponge, H. okadai, selectively for the first time.

Quantitative gene-expression analysis of the ligand-receptor system for classical neurotransmitters and neuropeptides in hippocampal CA1, CA3, and dentate gyrus.

We have shown quantitative expression levels of genes coding for the "ligand-receptor system" for classical neurotransmitters and neuropeptides in hippocampal subregions CA1, CA3, and dentate gyrus (DG). Using a combination of DNA microarray and quantitative PCR methods, we found that the three subregions have relatively similar expression patterns of ionotropic receptors for classical neurotransmitters. Expression of ionotropic receptors for glutamate and GABA represents more than 90% of all ionotropic receptors for classical neurotransmitters, and the expression ratio between ionotropic receptors for glutamate and GABA is constant (1.2:1-1.6:1) in each subregion. Meanwhile, the three subregions have different expression patterns of neuropeptide receptors. Furthermore, there are asymmetric expression patterns between neuropeptides and their receptors. Expression of Cck, Npy, Sst, and Penk1 represents 90% of neuropeptides derived locally in the hippocampus, whereas expression of these four neuropeptide receptors accounts for 50% of G protein-coupled receptors for neuropeptides. We propose that CA1, CA3, and DG have different modalities based on the ligand-receptor system, particularly the "neuropeptidergic system." Our quantitative gene-expression analysis provides fundamental data to support functional differences between the three hippocampal subregions regarding ligand-receptor interactions.

Gene expression profiling of neuropeptides in mouse cerebellum, hippocampus, and retina.

OBJECTIVE: We examined gene expression profiling in single neuron types and small regions of the nervous system. METHODS: The RNAs were extracted from mouse cerebellar Purkinje cells, granule cell layer, hippocampal CA1 and CA3 pyramidal cell layers, and three layers of the retina (outer nuclear layer, inner nuclear layer, and ganglion cell layer) were dissected by laser capture microdissection. The gene expression profiling of each sample was examined by Affymetrix GeneChip and real-time reverse transcription polymerase chain reaction. We studied the gene expression of 62 neuropeptide and hormone genes and 387 G-protein-coupled receptor (GPCR) genes. RESULTS: Among them, cholecystokinin and neuropeptide Y genes were the most widely expressed. The gene expression of cholecystokinin was very high in the hippocampus, suggesting that cholecystokinin transcripts might have unknown roles in the hippocampus. More than 10 neuropeptide genes were expressed in the ganglion cell layer of the retina, whereas the outer nuclear layer of the retina did not express a considerable amount of neuropeptide mRNAs. In total 12 GPCR genes were found in all tissues examined, and half were orphans (6 of 12). CONCLUSION: The high ratio of orphan GPCR genes suggests our limited knowledge of the ligand-receptor system in the nervous system. These results provide basic information for studying the function of neuropeptides.

Actin -related protein 3 (Arp3) is mutated in proteinuric BUF/Mna rats.

The BUF/Mna strain of rat is a model of focal and segmental glomerulosclerosis (FSGS) in which a quantitative trait locus (QTL) for proteinuria, Pur1, has been identified. The aim of the present study was to identify candidates for the Pur1 gene. To narrow the Pur1 QTL, we performed fine QTL mapping and single nucleotide polymorphism (SNP) genotyping. To identify candidate genes, sequencing and gene-expression analyses of all genes contained in the narrowed locus were conducted. The narrowed Pur1 region contained 25 genes. Among these genes, only the Arp3 gene was mutated in the BUF/Mna strain; it contained a missense mutation that caused an (L)111(F) substitution. This leucine is conserved across species. Gene-expression analysis failed to identify any other candidate genes for Pur1. Arp3-mediated actin assembly abnormalities were visible in immunohistochemical and electron microscopic examinations of podocytes in old BUF/Mna rats. Taken together, these data suggest that Arp3 is a candidate for the Pur1 gene. This observation is consistent with our growing recognition that abnormal signaling-induced assembly of actin in podocytes leads to the development of FSGS.

Disruption of the murine alpha1-antitrypsin/PI2 gene.

Alpha-1-antitrypsin (alpha1-AT) is a member of the serine protease inhibitor family regulating numerous proteolytic processes. The genetic disorder, alpha1-AT deficiency, is well known as a cause of hereditary pulmonary emphysema and liver cirrhosis. To create an animal model of human alpha1-AT deficiency, we disrupted the major murine isoform PI2, which is similar to human alpha1-AT and is one of 7 alpha1-AT isoforms found in the mouse. The ability of the serum to inhibit the activities of human leukocyte elastase (HLE) and human chymotrypsin (CYT) was significantly lower in heterozygous mice (alpha1-AT/PI2 -/+) than wild-type (alpha1-AT/PI2 +/+) mice (73.2% vs. 100% for HLE and 67.8% vs.100% for CYT, respectively; P<0.05). The distribution of genotypes among F(2) progeny was not in accordance with Mendelian distribution (P<0.01), as the percentages of wild-type, heterozygotes and homozygotes were 47.8%, 37.3% and 14.9%, respectively. Thus, it is likely that impairment of the protease inhibitor had a critical effect on fetus development. The alpha1-AT/PI2 deficient mouse will be a useful animal model for elucidating the function of alpha1-AT in fetal development, studying the mechanisms of chronic inflammatory disease and evaluating therapeutic candidates for the treatment of inflammatory disease.

MUC20 suppresses the hepatocyte growth factor-induced Grb2-Ras pathway by binding to a multifunctional docking site of met.

A cDNA encoding a novel mucin protein, MUC20, was isolated as a gene that is up-regulated in the renal tissues of patients with immunoglobulin A nephropathy. We demonstrate here that the C terminus of MUC20 associates with the multifunctional docking site of Met without ligand activation, preventing Grb2 recruitment to Met and thus attenuating hepatocyte growth factor (HGF)-induced transient extracellular signal-regulated kinase-1 and -2 activation. Production of MUC20 reduced HGF-induced matrix metalloproteinase expression and proliferation, which require the Grb2-Ras pathway, whereas cell scattering, branching morphogenesis, and survival via the Gab1/phosphatidylinositol 3-kinase (PI3K) pathways was not affected. Thus, MUC20 reduces HGF-induced activation of the Grb2-Ras pathway but not the Gab1/PI3K pathways. We further demonstrate that the cytoplasmic domain of MUC20 has the ability to oligomerize and that the oligomerization augments its affinity for Met. Taken together, these results suggest that MUC20 is a novel regulator of the Met signaling cascade which has a role in suppression of the Grb2-Ras pathway.