Manabu Sugii

Genome-wide Analysis of Chlamydophila pneumoniae Gene Expression at the Late Stage of Infection

Chlamydophila pneumoniae, an obligate intracellular eubacterium, changes its form from a vegetative reticulate body into an infectious elementary body during the late stage of its infection cycle. Comprehension of the molecular events in the morphological change is important to understand the switching mechanism between acute and chronic infection, which is deemed to relate to the pathogenesis of atherosclerosis. Herein, we have attempted to screen genes expressed in the late stage with a genome-wide DNA microarray, resulting in nomination of 17 genes as the late-stage genes. Fourteen of the 17 genes and six other genes predicted as late-stage genes were confirmed to be up-regulated in the late stage with a quantitative reverse transcriptase–polymerase chain reaction. These 20 late-stage genes were classified into two groups by clustering analysis: ‘drastically induced’ and ‘moderately induced’ genes. Out of eight drastically induced genes, four contain σ28 promoter-like sequences and the other four contain an upstream common sequence. It suggests that besides σ28, there are certain up-regulatory mechanisms at the late stage, which may be involved in the chlamydial morphological change and thus pathogenesis.

Machine learning prediction of amino acid patterns in protein n-myristoylation

Protein N-myristoylation is the lipid modification in which the 14-carbon saturated fatty acid binds covalently to N-terminal of virus-based and eukaryotic protein. In this study, we suggest an approach to predict the pattern of N-myristoylation signal using the machine learning system BONSAI. BONSAI finds rules in combination of an alphabet indexings and decision trees. Computational experiments with BONSAI classified amino acid residues depending on effect for N-myristoylation and found rules of the alphabet indexing. In addition, BONSAI suggested new requirements for the position of an amino acid in N-myristoylation signal.

Purification of GVBD-inducing protein from the ciliate Tetrahymena thermophila

Abstract Germinal-vesicle-breakdown (GVBD) was induced if a 132,000-g supernatant of Tetrahymena thermophila homogenates was injected into Xenopus oocytes. Using this induction of GVBD as a bioassay system, a GVBD-inducing substance was purified from the Tetrahymena by ultra-filtration, liquid chromatography, and electroelution from a band on native-PAGE gel. Proteins eluted from the single band on the native-PAGE gel induced GVBD in the absence of oocyte protein synthesis. This band resolved into two bands on SDS-PAGE: 60 and 112 kDa. The 60 kDa protein was the active fraction inducing GVBD. Immunoprecipitation of the 60 kDa protein prevented the GVBD-inducing activity, supporting the conclusion that the 60 kDa protein is the GVBD-inducing substance. An immunoblot with anti-60 kDa monoclonal antibody and PSTAIR antibody showed that p13suc1-beads could remove cdc2 homologues from T. thermophila supernatant but could not remove the GVBD-inducing activity. The 60-kDa protein appeared at the same time as micronuclear division and disappeared at the beginning of the macronuclear division during synchronous cell division. The cyclic appearance of the 60-kDa protein in the T. thermophila cell cycle suggests that this protein has a cell cycle function.

Purification of germinal-vesicle-breakdown-inducing proteins from Tetrahymena pyriformis

Summary Germinal-vesicle-breakdown (GVBD) of Xenopus laevis oocytes was induced by injecting 132,000 g supernatant of Tetrahymena pyriformis homogenates into oocytes. Using induction of GVBD as a bioassay system, we succeeded in purifying two GVBD-inducing proteins from the 132,000 g supernatant by liquid chromatography with an anion-exchange MonoQ column, ultra-filtration, and electroelution from bands on a native-PAGE gel. A protein of 40 kDa induced GVBD in the absence of protein synthesis of Xenopus oocytes whereas another protein of 56 kDa induced GVBD only in the presence of the oocyte protein synthesis. These proteins did not react with the PSTAIR antibody, which is specific for cdc2 homologues.

Computational Predictions for Functional Proteins Working after Cleaved in Apoptotic Pathway

Protein cleavage by a caspase enzyme is observed in many pathways including apoptosis pathway, which induces protein modifications such as N-myristoylation. By exhaustive search of NCBI GenBank database based on the information in PeptideCutter website, we have identified a protein very likely to control apoptosis after N-myristoylation which is caused by the cleavage by caspase-8. To find more rules for sequences cleaved by caspase enzymes, we conducted computational experiments using the machine learning system BONSAI, and extracted some characteristic sequences involving serine-threonine kinase motif. This suggests the new possibility that the machine learning technique finds the significant sequence such as the recognition site of the enzyme.