Mincle (also called as Clec4e and Clecsf9) is a C-type lectin receptor expressed in activated phagocytes. Recently, we have demonstrated that Mincle is an FcR␥-associated activating receptor that senses damaged cells. To search an exogenous ligand(s), we screened pathogenic fungi using cell line expressing Mincle, FcR␥, and NFAT-GFP reporter. We found that Mincle specifically recognizes the Malassezia species among 50 different fungal species tested. Malassezia is a pathogenic fungus that causes skin diseases, such as tinea versicolor and atopic dermatitis, and fatal sepsis. However, the specific receptor on host cells has not been identified. Mutation of the putative mannose-binding motif within C-type lectin domain of Mincle abrogated Malassezia recognition. Analyses of glycoconjugate microarray revealed that Mincle selectively binds to ␣-mannose but not mannan. Thus, Mincle may recognize specific geometry of ␣-mannosyl residues on Malassezia species and use this to distinguish them from other fungi. Malassezia activated macrophages to produce inflammatory cytokines/ chemokines. To elucidate the physiological function of Mincle, Mincle-deficient mice were established. Malassezia-induced cytokine/chemokine production by macrophages from Mincle ؊/؊ mice was significantly impaired. In vivo inflammatory responses against Malassezia was also impaired in Mincle ؊/؊ mice. These results indicate that Mincle is the first specific receptor for Malassezia species to be reported and plays a crucial role in immune responses to this fungus.ITAM ͉ macrophages ͉ signal transduction

Hydrogen sulfide (H(2)S) has recently been identified as a biological response modifier. Here, we report the design and synthesis of a novel fluorescence probe for H(2)S, HSip-1, utilizing azamacrocyclic copper(II) ion complex chemistry to control the fluorescence. HSip-1 showed high selectivity and high sensitivity for H(2)S, and its potential for biological applications was confirmed by employing it for fluorescence imaging of H(2)S in live cells.

Until recently, Histoplasma capsulatum was believed to harbour three varieties, var. capsulatum (chiefly a New World human pathogen), var. duboisii (an African human pathogen) and var. farciminosum (an Old World horse pathogen), which varied in clinical manifestations and geographical distribution. We analysed the phylogenetic relationships of 137 individuals representing the three varieties from six continents using DNA sequence variation in four independent protein-coding genes. At least eight clades were idengified: (i) North American class 1 clade; (ii) North American class 2 clade; (iii) Latin American group A clade; (iv) Latin American group B clade; (v) Australian clade; (vi) Netherlands (Indonesian?) clade; (vii) Eurasian clade and (viii) African clade. Seven of eight clades represented genetically isolated groups that may be recognized as phylogenetic species. The sole exception was the Eurasian clade which originated from within the Latin American group A clade. The phylogenetic relationships among the clades made a star phylogeny. Histoplasma capsulatum var. capsulatum individuals were found in all eight clades. The African clade included all of the H. capsulatum var. duboisii individuals as well as individuals of the other two varieties. The 13 individuals of var. farciminosum were distributed among three phylogenetic species. These findings suggest that the three varieties of Histoplasma are phylogenetically meaningless. Instead we have to recognize the existence of genetically distinct geographical populations or phylogenetic species. Combining DNA substitution rates of protein-coding genes with the phylogeny suggests that the radiation of Histoplasma started between 3 and 13 million years ago in Latin America.

Hydrogen sulfide (H(2)S) has been recognized as a smooth muscle relaxant. Cystathionine gamma-lyase, which is localized to smooth muscle, is thought to be the major H(2)S-producing enzyme in the thoracic aorta. Here we show that 3-mercaptopyruvate sulfurtransferase (3MST) and cysteine aminotransferase (CAT) are localized to vascular endothelium in the thoracic aorta and produce H(2)S. Both 3MST and CAT were localized to endothelium. Lysates of vascular endothelial cells produced H(2)S from cysteine and alpha-ketoglutarate. The present study provides a new insight into the production and release of H(2)S as a smooth muscle relaxant from vascular endothelium.

Accumulating evidence shows that hydrogen sulfide (H2S) has a variety of physiological functions. H2S is produced from cysteine by 3 sulfurtransferases. H2S, in turn, generates polysulfides, the functions of which are not well understood. H2S induces Ca(2+) influx in astrocytes, a type of glia. However, the receptor that mediates the response has not been identified. Here, we have shown that polysulfides induce Ca(2+) influx by activating transient receptor potential (TRP)A1 channels in rat astrocytes (EC50 91 nM, Hill coefficient value 1.77±0.26) and that the maximum response was induced at 0.5 μM, which is 1/320 of the concentration of H2S required to achieve a response of similar magnitude (160 μM, EC50 116 μM). TRPA1-selective agonists, allyl isothiocyanate and cinnamaldehyde, induced Ca(2+) influx, and responses to polysulfides were suppressed by TRPA1-selective inhibitors, HC-030031 and AP-18, as well as by siRNAs selective to TRPA1. The present study suggests that polysulfides are possible H2S-derived signaling molecules that stimulate TRP channels in the brain.

H2S (hydrogen sulfide) has recently been recognized as a signalling molecule as well as a cytoprotectant. We recently demonstrated that 3MST (3-mercaptopyruvate sulfurtransferase) produces H2S from 3MP (3-mercaptopyruvate). Although a reducing substance is required for an intermediate persulfide at the active site of 3MST to release H2S, the substance has not been identified. In the present study we show that Trx (thioredoxin) and DHLA (dihydrolipoic acid) associate with 3MST to release H2S. Other reducing substances, such as NADPH, NADH, GSH, cysteine and CoA, did not have any effect on the reaction. We also show that 3MST produces H2S from thiosulfate. The present study provides a new insight into a mechanism for the production of H2S by 3MST.

We determined the genomic sequence of Nocardia farcinica IFM 10152, a clinical isolate, and revealed the molecular basis of its versatility. The genome consists of a single circular chromosome of 6,021,225 bp with an average G؉C content of 70.8% and two plasmids of 184,027 (pNF1) and 87,093 (pNF2) bp with average G؉C contents of 67.2% and 68.4%, respectively. The chromosome encoded 5,674 putative protein-coding sequences, including many candidate genes for virulence and multidrug resistance as well as secondary metabolism. Analyses of paralogous protein families suggest that gene duplications have resulted in a bacterium that can survive not only in soil environments but also in animal tissues, resulting in disease.N ocardia are filamentous-growing Gram-positive soil saprophytes that belong to the family Actinomycetales, which also includes clinically and industrially important genera such as Mycobacterium, Streptomyces, Corynebacterium, and Rhodococcus. Many species of Nocardia cause the disease nocardiosis in humans and animals on lung, central nervous system, brain, and cutaneous tissues (1), and a species Nocardia asteroides is suspected to be an etiological agent of Parkinson's disease (2). Nocardiosis is on the rise, with an estimated 109-136 new cases occurring annually in Japan (3) and 500-1,000 in the U.S. (www.cdc.gov͞ncidod͞dbmd͞diseaseinfo͞nocardiosis t.htm). However, there are only a few studies on the mechanisms of nocardial virulence. Nocardia species are resistant to many front-line antibiotics. Because treatment for nocardiosis relies heavily on chemotherapy, their intrinsic multiple drug resistance is a serious problem.Another feature of the nocardia is that many strains, even clinical isolates, also have the capability to produce bioactive molecules such as antibiotics (4, 5) and enzymes that are industrially important (6). A monobactam antibiotic, nocardicin, was isolated from Nocardia sp. (7), and a terpenoid brasilicardin with immunosuppressive activity was isolated from a clinical isolate (4).Nocardia farcinica IFM 10152 was isolated from the bronchus of a 68-year-old male Japanese patient. Despite the complicated taxonomy of the nocardia, the species N. farcinica was found to be nearly homogeneous (8). Subsequently, to elucidate the molecular basis of the versatility of the nocardia, we analyzed the genomic sequence of N. farcinica IFM 10152. MethodsGenome Sequencing and Assembly. The nucleotide sequence of the N. farcinica IFM 10152 genome was determined by a wholegenome shotgun strategy. We constructed small-insert (2 kb) and large-insert (10 kb) genomic libraries and generated 127,077 sequences (giving 9-fold coverage) from both ends of the genomic clones. Sequence assembly was carried out by using the PHRED͞PHRAP͞CONSED package (9). Remaining gaps were closed by transcriptional sequencing (Nippon Gene, Toyama, Japan) or by primer walking. There were no ambiguous nucleotides in the genomic sequence that we could determine. Consistency of the final assembly was confirmed in terms of res...

Further characterization of the genetic environment of the gene encoding the Escherichia coli extendedspectrum ␤-lactamase, bla VEB-1 , revealed the presence of a plasmid-located class 1 integron, In53, which carried eight functional resistance gene cassettes in addition to bla VEB-1 . While the aadB and the arr-2 gene cassettes were identical to those previously described, the remaining cassettes were novel: (i) a novel nonenzymatic chloramphenicol resistance gene of the cmlA family, (ii) a qac allele encoding a member of the small multidrug resistance family of proteins, (iii) a cassette, aacA1b/orfG, which encodes a novel 6-N-acetyltransferase, and (iv) a fused gene cassette, oxa10/aadA1, which is made of two cassettes previously described as single cassettes. In addition, oxa10 and aadA1 genes were expressed from their own promoter sequence present upstream of the oxa10 cassette. arr-2 coded for a protein that shared 54% amino acid identity with the rifampin ADP-ribosylating transferase encoded by the arr-1 gene from Mycobacterium smegmatis DSM43756. While in M. smegmatis, the main inactivated compound was 23-ribosyl-rifampin, the inactivated antibiotic recovered from E. coli culture was 23-O-ADP-ribosyl-rifampin. The integrase gene of In53 was interrupted by an IS26 insertion sequence, which was also present in the 3 conserved segment. Thus, In53 is a truncated integron located on a composite transposon, named Tn2000, bounded by two IS26 elements in opposite orientations. Target site duplication at both ends of the transposon indicated that the integron likely was inserted into the plasmid through a transpositional process. This is the first description of an integron located on a composite transposon.