The human-specific action of intermedilysin, a homolog of streptolysin O, is dictated by domain 4 of the protein

Intermedilysin is a pore-forming cytolysin belonging to the streptolysin O gene family known as the 'Cholesterol-binding/dependent cytolysins' and is unique within the family in that it is highly humanspecific. This specificity suggests interaction with a component of human cells other than cholesterol, the proposed receptor for the other toxins of the gene family. Indeed, intermedilysin showed no significant degree of affinity to free or liposome-embedded cholesterol. Characterization of intermedilysin undecapeptide mutants revealed that this lack of affinity to cholesterol was a result of the substitutions of intermedilysin in this region. Absorption assays with erythrocyte membranes from various animals, competitive inhibition with domain 4 of intermedilysin and liposome-binding assays of streptolysin O and intermedilysin indicated that cell membrane binding is the human-specific step of intermedilysin action, that the host cell membrane-binding site is located within domain 4 in common with other members of the family and that the receptor for this toxin is not cholesterol. The species specificity of undecapeptide mutants of intermedilysin and streptolysin O and chimeric mutants between intermedilysin and streptolysin O, and intermedilysin and pneumolysin indicated that domain 4 of intermedilysin determines the human-specific action step and the cell-binding site of domain 4 lies within the 56 amino acids of the C-terminal, excluding the undecapeptide region.     

Simple RNAi vectors for stable and transient suppression of gene function in rice

Since the recent sequencing of the rice genome, the functional identification of rice genes has become increasingly important. Various tagged lines have been generated; however, the number of tagged genes available is not sufficient for extensive study of gene function. To help identify the functions of genes in rice, we developed a Gateway vector, pANDA, for RNA interference of rice genes. This vector can be used for Agrobacterium transformation of rice and allows easy and fast construction of efficient RNAi vectors. In the construct, hairpin RNA derived from a given gene is transcribed from a strong maize ubiquitin promoter, and an intron is placed 5' upstream of inverted repeats to enhance RNA expression. Analysis of rice genes using this vector showed that suppression of mRNA expression was observed in more than 90% of transgenic plants examined, and short interfering RNA indicative of RNA silencing was detected in each silenced plant. A similar vector, pANDA-mini, was also developed for direct transfer into leaf cells or protoplasts. This vector can be used for transient suppression of gene function in rice. These vectors should help identify the functions of rice genes whose tagged mutants are not available at present and complement existing methods for functional genomics of rice.     

C17,20-lyase inhibitors I. Structure-based de novo design and SAR study of C17,20-lyase inhibitors

Novel nonsteroidal C(17,20)-lyase inhibitors were synthesized using de novo design based on its substrate, 17 alpha-hydroxypregnenolone, and several compounds exhibited potent C(17,20)-lyase inhibition. However, in vivo activities were found to be short-lasting, and in order to improve the duration of action, a series of benzothiophene derivatives were evaluated. As a result, compounds 9h, (S)-9i, and 9k with nanomolar enzyme inhibition (IC(50)=4-9 nM) and 9e (IC(50)=27 nM) were identified to have powerful in vivo efficacy with extended duration of action. The key structural determinants for the in vivo efficacy were demonstrated to be the 5-fluoro group on the benzothiophene ring and the 4-imidazolyl moiety. Superimposition of 9k and 17 alpha-hydroxypregnenolone demonstrated their structural similarity and enabled rationalization of the pharmacological results. In addition, selected compounds were also identified to be potent inhibitors of human enzyme with IC(50) values of 20-30 nM.     

Arginine carrier peptide bearing Ni(II) chelator to promote cellular uptake of histidine-tagged proteins

Arginine-rich peptide-mediated protein delivery into living cells is a novel technology for controlling cell functions with therapeutic potential. In this report, a novel approach for the intracellular delivery of histidine-tagged proteins was introduced where a Ni(II) chelate of octaarginine peptide bearing nitrilotriacetic acid [R8-NTA-Ni(II)] was used as a membrane-permeable carrier molecule. Significant internalization of histidine-tagged enhanced green fluorescent protein (EGFP) into HeLa cells was observed by confocal microscopic observation in the presence of R8-NTA-Ni(II). Nuclear condensation characteristic in apoptotic cell death was also induced in the cells treated with a histidine-tagged apoptosis-inducing peptide [pro-apoptotic domain peptide (PAD)], indicating that the cargo molecules really went through the membrane to reach the cytosol. The apoptosis-inducing activity of the peptide thus delivered was compared with that of the PAD peptide covalently connected with the octaarginine peptide.     

Gicerin,a cell adhesion molecule,promotes the metastasis of lymphoma cells of the chicken

Gicerin is an immunoglobulin superfamily cell adhesion molecule purified from chicken gizzards. This molecule displays an adhesive interaction with a laminin-like protein as well as with gicerin itself. Gicerin appears in embryonic tissues and plays a role in chick development through its cell adhesive properties. An increase in gicerin expression is found in some sporadic tumors of the chicken. To elucidate the possible role of gicerin in tumor progression in chickens, we introduced gicerin cDNA into an endogenous gicerin negative lymphoma MDCC-MSB1 cell line, and subsequently analyzed them for changes in their metastatic potentials. After intravenous implantation of the gicerin transfectants into chickens, the metastatic potential to the lung, liver and kidney was enhanced compared with parental MDCC-MSB1 cells. Self-aggregation activity was increased in gicerin transfectants. In addition, adhesive and migratory activities of the gicerin transfectants to the gicerin ligands were enhanced in vitro. These findings indicate that gicerin can contribute to the malignancy and metastatic properties of lymphoma.This work was supported in part by a Grant-in-Aid for Scientific Research (No. 13760210), and a grant for Scientific Research on Priority Areas "Cancer" (No. 12215133), from the Ministry of Education, Science, Sports and Culture, Japan, grants from the Uehara Memorial Foundation and Senri Life Science and a Grant-in-Aid for Advanced Scientific Research from Osaka Prefecture University     

Relationship between renal sympathetic nerve activity and arterial pressure during REM sleep in rats

The relationship between renal sympathetic nerve activity (RSNA) and systemic arterial pressure obtained during rapid eye movement (REM) sleep was compared with that obtained in other sleep and awake states. Electrodes for the measurements of RSNA, electrocardiogram, electromyogram, and electroencephalogram and a catheter for the measurement of systemic arterial pressure were implanted while the animals were under aseptic conditions at least 5 days before the experiment. During the transition from non-REM (NREM) to REM sleep, RSNA and heart rate (HR) decreased immediately by 46 +/- 2% (P < 0.05) and 22 +/- 3 beats/min (P < 0.05), respectively, over 3 s after the onset of REM sleep. Meanwhile, systemic arterial pressure increased gradually after the onset of REM sleep, which was apparently independent of the changes in RSNA. During REM sleep, the relationships between RSNA/HR and systemic arterial pressure were dissociated compared with that obtained during the other behavioral states. These data indicate that the interdependency between systemic arterial pressure and RSNA during REM sleep is likely to be modified compared with other behavioral states.     

Crystal structure of nitrile hydratase from a thermophilic Bacillus smithii

The crystal structure of the nitrile hydratase (NHase) from Bacillus smithii SC-J05-1 was determined. Our analysis of the structure shows that some residues that seem to be responsible for substrate recognition are different from those of other NHases. In particular, the Phe52 in the beta subunit of NHase from B. smithii covers the metal center partially like a small lid and narrows the active site cleft. It is well known that the NHase from B. smithii especially prefers aliphatic nitriles for its substrate rather than aromatic ones, and we can now infer that the Phe52 residue may play a key role in the substrate specificity for this enzyme. This finding leads us to suggest that substitution of these residues may alter the substrate specificity of the enzyme.