Isolation of a new potent cytotoxic pigment along with indigotin from the pathogenic basidiomycetous fungus Schizophyllum commune

An indole derivative, schizocommunin, was isolated along with indigotin (indigo), indirubin, isatin, and tryptanthrin, from the liquid culture medium in which a culture of Schizophyllum commune, isolated from the bronchus of a human patient with allergic bronchopulmonary mycosis, had been grown. The structure of schizocommunin was established by spectroscopic investigation. Schizocommunin showed the strong cytotoxicity against murine lymphoma cells. The assignments of the ¹H- and ¹³C-NMR signals of indigotin were also listed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Genetic polymorphisms and antiviral activity in the bovine MX1 gene

Bovine MX1 cDNAs consisting of 2280 bp from 11 animals of five breeds and from a cultured cell line were sequenced and compared with previously reported data. Ten nucleotide substitutions were synonymous mutations, and a single nucleotide substitution at 458 resulted in an amino acid exchange of Ile (ATT) and Met (ATG). A 13-bp deletion-insertion mutation was also found in the 3'-UTR. Based on the nucleotide substitutions found in this study, bovine MX1 cDNA was classified into 11 genotypes. A phylogenetic tree of the 11 genotypes suggested that the genotypes observed in Brahman were a great genetic distance from other genotypes. An 18-bp deletion-insertion variation at position 171 was found to be the result of alternative splicing. The 18-bp deletion-insertion is located at the boundary between exon 3 and intron 3. Permanently transfected 3T3 cell lines expressing bovine MX1 mRNA were established to analyse the antiviral potential against VSVDeltaG*-G infection. Transfected cell clones expressing bovine MX1 mRNA showed a significantly smaller number of cells infected with VSVDeltaG*-G compared with the control cells. These results indicate that the bovine MX1 protein has potent antiviral activity.     

Identification of the mouse H-ficolin gene as a pseudogene and orthology between mouse ficolins A/B and human L-/M-ficolins

Ficolin is a collagenous lectin which plays a crucial role in innate immunity. Three and two ficolins have been identified in human and mice, respectively. To identify the mouse homologue of human H-ficolin and to elucidate the orthology between mouse ficolins A/B and human L-/M-ficolins, the gene structures were explored. The mouse homologue of the H-ficolin gene was identified as a pseudogene on chromosome 4. The mouse ficolin A gene was located far from the ficolin B gene on chromosome 2, whereas the human L-ficolin and M-ficolin genes were close in the region homologous to the ficolin B locus. Together with the exon-intron structures and the phylogenetic tree, these results suggest that ficolin B is the mouse orthologue of M-ficolin and that the genes encoding serum-type ficolins, ficolin A and L-ficolin, were generated independently from the ficolin B/M-ficolin lineage each in mice and primates.     

Molecular cloning and characterization of a cDNA encoding ent-cassa-12,15-diene synthase, a putative diterpenoid phytoalexin biosynthetic enzyme, from suspension-cultured rice cells treated with a chitin elicitor

We have isolated and characterized a cDNA encoding a novel diterpene cyclase, OsDTC1, from suspension-cultured rice cells treated with a chitin elicitor. OsDTC1 functions as ent-cassa-12,15-diene synthase, which is considered to play a key role in the biosynthesis of (-)-phytocassanes recently isolated as rice diterpenoid phytoalexins. The expression of OsDTC1 mRNA was also confirmed in ultraviolet (UV)-irradiated rice leaves. In addition, we identified ent-cassa-12,15-diene, a putative diterpene hydrocarbon precursor of (-)-phytocassanes, as an endogenous compound in the chitin-elicited suspension-cultured rice cells and the UV-irradiated rice leaves. The OsDTC1 cDNA isolated here will be a useful tool to investigate the regulatory mechanisms of the biosynthesis of (-)-phytocassanes in rice.     

The participation of nitric oxide in peritoneal exudate cell cytotoxicity of mice by Fusobacterium nucleatum

Previously we reported that mice infected recurrently with live Fusobacterium nucleatum(Fn) synthesize a significant amount of NO between 12 hr and 24 hr after Fn injection. Fn is a gram-negative rod periodontal pathogen. NO could not be induced by heat-killed Fn or in untreated mice. This NO, derived from the iNOS after infection of live Fn, was not involved in the Fn reduction because Fn clearance occurs within 6 hr. We investigated in this study whether this NO was involved in cytotoxicity in peritoneal exudate cells (PEC) in vivo. The mice were divided into two groups: those treated with live Fn (immune) and those left untreated (normal). PEC number, NO production, detection of apoptosis or death cells, and lactate dehydrogenase (LDH) release activity after injection of live Fn were compared in these groups. In the immune group, the increase of the total cell numbers caused by an increase in neutrophils, a significant NO production only after injection of live Fn at 24 hr and identification of iNOS positive macrophages were confirmed. The apoptotic rate was very low and did not increase at 24 hr in vivo. Therefore, apoptosis was seldom relevant to the NO. In the immune group, LDH activity was remarkable high at 24 hr, and dead cells and macrophages phagocytizing cell fragments increased at the same time. Pretreatment of L NMMA, an inhibitor of iNOS, suppressed LDH activity and cell death. Therefore, the NO derived from the iNOS is involved in the cytotoxicity. These results suggest that NO may contribute to the inflammatory response during Fn infection in periodontitis.