Effective handling of induced-fit motion in flexible docking

For structure-based drug design, where various ligand structures need to be docked to a target protein structure, a docking method that can handle conformational flexibility of not only the ligand, but also the protein, is indispensable. We have developed a simple and effective approach for dealing with the local induced-fit motion of the target protein, and implemented it in our docking tool, ADAM. Our approach efficiently combines the following two strategies: a vdW-offset grid in which the protein cavity is enlarged uniformly, and structure optimization allowing the motion of ligand and protein atoms. To examine the effectiveness of our approach, we performed docking validation studies, including redocking in 18 test cases and foreign-docking, in which various ligands from foreign crystal structures of complexes are docked into a target protein structure, in 22 cases (on five target proteins). With the original ADAM, the correct docking modes (RMSD < 2.0 A) were not present among the top 20 models in one case of redocking and four cases of foreign-docking. When the handling of induced-fit motion was implemented, the correct solutions were acquired in all 40 test cases. In foreign-docking on thymidine kinase, the correct docking modes were obtained as the top-ranked solutions for all 10 test ligands by our combinatorial approach, and this appears to be the best result ever reported with any docking tool. The results of docking validation have thus confirmed the effectiveness of our approach, which can provide reliable docking models even in the case of foreign-docking, where conformational change of the target protein cannot be ignored. We expect that this approach will contribute substantially to actual drug design, including virtual screening.     

Molecular phylogenetic analyses reveal a close relationship between powdery mildew fungi on some tropical trees and Erysiphe alphitoides, an oak powdery mildew

To investigate the phylogenetic relationships among the powdery mildew fungi of some economically important tropical trees belonging to Oidium subgenus Pseudoidium, we conducted molecular phylogenetic analyses using 30 DNA sequences of the rDNA internal transcribed spacer (ITS) regions and 26 sequences of the domains D1 and D2 of the 28S rDNA obtained from the powdery mildews on Hevea brasiliensis (para rubber tree), Anacardium occidentale (cashew), Bixa orellana, Citrus spp., Mangifera indica (mango), and Acacia spp. The results indicate that the powdery mildew fungi isolated from these tropical trees are closely related to one another. These powdery mildews are also closely related to E. alphitoides (including Erysiphe sp. on Quercus phillyraeoides). Because of the obligate biotrophic nature of the powdery mildew fungi, the relationship between powdery mildews and their host plants is conservative. However, the present study suggests that a particular powdery mildew species has expanded its host ranges on a wide range of the tropical trees. This article also suggests that a powdery mildew fungus distributed in temperate regions of the Northern Hemisphere expanded its host ranges onto tropical plants and may be a good example of how geographical and host range expansion has occurred in the Erysiphales.     

Assembly of the CotSA coat protein into spores requires CotS in Bacillus subtilis

The CotSA protein, encoded by cotSA (ytxN) of Bacillus subtilis, was detected from the cells at 5 h after the onset of sporulation (T5) and in the spore coat of wild-type cells, but not in cotE, cotS, gerE, or cotSA mutant spores. CotSA was also detected in the sporangium at T5 to T7 but not in the sporangium at T18 of cotS mutant cells, while the incorporation of CotS into the coat was not dependent upon CotSA. These results suggested that CotSA was synthesized simultaneously with CotS during T5 to T7 of sporulation and assembled into the coat dependent upon CotS.     

Use of virtual slide system for quick frozen intra-operative telepathology diagnosis in Kyoto,Japan

We started to use virtual slide (VS) and virtual microscopy (VM) systems for quick frozen intra-operative telepathology diagnosis in Kyoto, Japan. In the system we used a digital slide scanner, VASSALO by CLARO Inc., and a broadband optic fibre provided by NTT West Japan Inc. with the best effort capacity of 100 Mbps. The client is the pathology laboratory of Yamashiro Public Hospital, one of the local centre hospitals located in the south of Kyoto Prefecture, where a full-time pathologist is not present. The client is connected by VPN to the telepathology centre of our institute located in central Kyoto. As a result of the recent 15 test cases of VS telepathology diagnosis, including cases judging negative or positive surgical margins, we could estimate the usefulness of VS in intra-operative remote diagnosis. The time required for the frozen section VS file making was found to be around 10 min when we use x10 objective and if the maximal dimension of the frozen sample is less than 20 mm. Good correct focus of VS images was attained in all cases and all the fields of each tissue specimen. Up to now the capacity of best effort B-band appears to be sufficient to attain diagnosis on time in intra-operation. Telepathology diagnosis was achieved within 5 minutes in most cases using VS viewer provided by CLARO Inc. The VS telepathology system was found to be superior to the conventional still image telepathology system using a robotic microscope since in the former we can observe much greater image information than in the latter in a certain limited time of intra-operation and in the much more efficient ways. In the near future VS telepathology will replace conventional still image telepathology with a robotic microscope even in quick frozen intra-operative diagnosis.     

Involvement of Sema4A in the progression of experimental autoimmune myocarditis

Dilated cardiomyopathy often results from autoimmunity triggered by microbial infections during myocarditis. However, it remains unclear how immunological disorders are implicated in pathogenesis of autoimmune myocarditis. Here, we demonstrated that Sema4A, a class IV semaphorin, plays key roles in experimental autoimmune myocarditis (EAM). Dendritic cells pulsed with myosin heavy chain-α peptides induced severe myocarditis in wild-type mice, but not in Sema4A-deficient mice. In adoptive transfer experiments, CD4⁺ T-cells from wild-type mice induced severe myocarditis, while CD4⁺ T-cells from Sema4A-deficient mice exhibited considerably attenuated myocarditis. Our results indicated that Sema4A is critically involved in EAM by regulating differentiation of T-cells.     

Significant contribution of the pgdA gene to the virulence of Streptococcus suis

Streptococcus suis is a major swine pathogen and emerging zoonotic agent. In this study we have determined the muropeptide composition of S. suis peptidoglycan (PG) and found, among other modifications, N-deacetylated compounds. Comparison with an isogenic mutant showed that the product of the pgdA gene is responsible for this specific modification which occurred in very low amounts. Low level of PG N-deacetylation correlated with absence of significant lysozyme resistance when wild-type S. suis was grown in vitro. On the other hand, expression of the pgdA gene was increased upon interaction of the bacterium with neutrophils in vitro as well as in vivo in experimentally inoculated mice, suggesting that S. suis may enhance PG N-deacetylation under these conditions. Evaluation of the DeltapgdA mutant in both the CD1 murine and the porcine models of infection revealed a significant contribution of the pgdA gene to the virulence traits of S. suis. Reflecting a severe impairment in its ability to persist in blood and decreased ability to escape immune clearance mechanisms mediated by neutrophils, the DeltapgdA mutant was highly attenuated in both models. The results of this study suggest that modification of PG by N-deacetylation is an important factor in S. suis virulence.     

Molecular phylogeny and evolution of the genus Neoerysiphe (Erysiphaceae, Ascomycota)

The genus Neoerysiphe belongs to the tribe Golovinomyceteae of the Erysiphaceae together with the genera Arthrocladiella and Golovinomyces. This is a relatively small genus, comprising only six species, and having ca 300 species from six plant families as hosts. To investigate the molecular phylogeny and evolution of the genus, we determined the nucleotide sequences of the rDNA ITS regions and the divergent domains D1 and D2 of the 28S rDNA. The 30 ITS sequences from Neoerysiphe are divided into three monophyletic groups that are represented by their host families. Groups 1 and 3 consist of N. galeopsidis from Lamiaceae and N. galii from Rubiaceae, respectively, and the genetic diversity within each group is extremely low. Group 2 is represented by N. cumminsiana from Asteraceae. This group also includes Oidium baccharidis, O. maquii, and Oidium spp. from Galinsoga (Asteraceae) and Aloysia (Verbenaceae), and is further divided into four subgroups. N. galeopsidis is distributed worldwide, but is especially common in western Eurasia from Central Asia to Europe. N. galii is also common in western Eurasia. In contrast, the specimens of group 2 were all collected in the New World, except for one specimen that was collected in Japan; this may indicate a close relationship of group 2 with the New World. Molecular clock calibration demonstrated that Neoerysiphe split from other genera of the Erysiphaceae ca 35–45 M years ago (Mya), and that the three groups of Neoerysiphe diverged between 10 and 15 Mya, in the Miocene. Aloysia citriodora is a new host for the Erysiphaceae and the fungus on this plant is described as O. aloysiae sp. nov.     

A novel Coptis japonica multidrug-resistant protein preferentially expressed in the alkaloid-accumulating rhizome.

A full-length cDNA, Cjmdr1, which belongs to the multidrug-resistant (mdr) gene family, was isolated by nested RT-PCR from alkaloid-producing cultured cells of Coptis japonica. The cDNA is 4192 nucleotides long and has an ORF of 1289 amino acids. Northern analysis of the intact plant showed a clear preference in its expression in the rhizome, where alkaloids are highly accumulated compared to other organs.     

Abnormal biantennary sugar chains are expressed in human chorionic gonadotropin produced in the choriocarcinoma cell line, JEG-3

Over the past two decades, sugar chain structures of human chorionic gonadotropin (hCG) produced in healthy people, three types of trophoblastic disease, and some types of cell lines have been analyzed. The abnormal biantennary structure of hCG is a good marker for the diagnosis of malignant choriocarcinoma. In spite of much research, hCG with an abnormal biantennary structure is only detected in the urine of choriocarcinoma or pregnant diabetic patients. We hypothesized that the formation mechanism of the abnormal biantennary sugar chain structure is mainly caused by high GnT-IV activity. To confirm this, we measured the N-acetylglucosaminyltransferase (GnT)-IV activity and hCG productivity in three choriocarcinoma cell lines, and selected JEG-3 cells. hCG samples were purified from medium conditioned by JEG-3 cells, and their sugar chain structures were analyzed. We detected an abnormal biantennary structure, and the proportions were different from those previously reported in the urine samples of choriocarcinoma patients. These findings proved our hypothesis and suggest the usefulness of JEG-3 cells for further analyses of abnormal biantennary structure formation. Published in 2004.