Transducible form of p47phox and p67phox compensate for defective NADPH oxidase activity in neutrophils of patients with chronic granulomatous disease

Protein delivery to primary cells by protein transduction domain (PTD) serves as a novel measure for manipulation of the cells for biological study and for the treatment of various human conditions. Although the method has been employed to modulate cellular function in vitro, only limited reports are available on its application in the replacement of deficient signaling molecules into primary cells. We examined the potential of recombinant proteins to compensate for defective cytosolic components of the NADPH oxidase complex in chronic granulomatous disease (CGD) neutrophils in both p47(phox) and p67(phox) deficiency. The p47(phox) or p67(phox) protein linked to Hph-1 PTD was effectively expressed in soluble form and transduced into human neutrophils efficiently without eliciting unwanted signal transduction or apoptosis. The delivered protein was stable for more than 24h, expressed in the cytoplasm, translocated to the membrane fraction upon activation, and, most importantly able to restored reactive oxygen species (ROS) production. Although research on human primary neutrophils using the protein delivery system is still limited, our data show that the protein transduction approach for neutrophils may be applicable to the control of local infections in CGD patients by direct delivery of the protein product.     

Definition of metabolism-dependent xenobiotic toxicity with co-cultures of human hepatocytes and mouse 3T3 fibroblasts in the novel integrated discrete multiple organ co-culture (IdMOC) experimental system: results with model toxicants aflatoxin B1, cyclophosphamide and tamoxifen

The integrated discrete multiple organ co-culture system (IdMOC) allows the co-culturing of multiple cell types as physically separated cells interconnected by a common overlying medium. We report here the application of IdMOC with two cell types: the metabolically competent primary human hepatocytes, and a metabolically incompetent cell line, mouse 3T3 fibroblasts, in the definition of the role of hepatic metabolism on the cytotoxicity of three model toxicants: cyclophosphamide (CPA), aflatoxin B1 (AFB) and tamoxifen (TMX). The presence of hepatic metabolism in IdMOC with human hepatocytes was demonstrated by the metabolism of the P450 isoform 3A4 substrate, luciferin-IPA. The three model toxicants showed three distinct patterns of cytotoxic profile: TMX was cytotoxic to 3T3 cells in the absence of hepatocytes, with slightly lower cytotoxicity towards both 3T3 cells and hepatocytes in the IdMOC. AFB was selective toxic towards the human hepatocytes and relatively noncytotoxic towards 3T3 cells both in the presence and absence of the hepatocytes. CPA cytotoxicity to the 3T3 cells was found to be significantly enhanced by the presence of the hepatocytes, with the cytotoxicity dependent of the number of hepatocytes, and with the cytotoxicity attenuated by the presence of a non-specific P450 inhibitor, 1-aminobenzotriazole. We propose here the following classification of toxicants based on the role of hepatic metabolism as defined by the human hepatocyte-3T3 cell IdMOC assay: type I: direct-acting cytotoxicants represented by TMX as indicated by cytotoxicity in 3T3 cells in the absence of hepatocytes; type II: metabolism-dependent cytotoxicity represented by AFB1 with effects localized within the site of metabolic activation (i. e. hepatocytes); and type III: metabolism-dependent cytotoxicity with metabolites that can diffuse out of the hepatocytes to cause toxicity in cells distal from the site of metabolism, as exemplified by CPA.     

Synthesis of oligomethylene-strapped chlorophyll derivatives and optical properties of their stereoisomers in a solution

Methyl pheophorbide-a/a′ derivatives covalently linked with oligomethylene chains at the 3-CH₂OCO– and 13²-COO– moieties in a molecule were prepared by modifying chlorophyll-a through intramolecular ring-closing metathesis of vinyl groups. At least, a C10-length between the 3³- and 13⁴-positions was necessary for the cyclization and connection of a C12-strap was the most suitable to achieve the highest closure yield. The oligomethylene chain in 13² R-epimers derived from methyl pheophorbide-a covered the α-face of the chlorin π-plane and the strap in the corresponding 13² S-epimers protected the β-face. Synthetic 13² R-epimer with a dodecamethylene chain gave a flat chlorin π-plane, while the decamethylene chain in the 13² R-epimer distorted the π-system due to its shorter linkage. The distortion by strapping in the 13² R-epimer induced a slight blue-shift of Qy peak in dichloromethane. CD spectra of the 13² R-epimers were similarly dependent on the chain length, i.e., the distortion of π-plane. Visible absorption and CD spectra of all the strapped 13² S-epimers were almost identical and only slightly different from those of the unstrapped. The strapping in the 13² S-epimers shifted the Qy peak bathochromically.     

Nation-wide litter fall data from 21 forests of the Monitoring Sites 1000 Project in Japan

This data paper reports litter fall data collected in a network of 21 forest sites in Japan. This is the largest litter fall data set freely available in Japan to date. The network is a part of the Monitoring Sites 1000 Project launched by the Ministry of the Environment, Japan. It covers subarctic to subtropical climate zones and the four major forest types in Japan. Twenty-three permanent plots in which usually 25 litter traps were installed were established in old-growth or secondary natural forests. Litter falls were collected monthly from 2004, and sorted into leaves, branches, reproductive structures and miscellaneous. The data provide seasonal patterns and inter-annual dynamics of litter falls, and their geographical patterns, and offer good opportunities for meta-analyses and comparative studies among forests.     

Discovery and structure-activity relationship of 2,6-disubstituted pyrazines, potent and selective inhibitors of protein kinase CK2

We report the discovery and structure-activity relationship of 2,6-disubstituted pyrazines, which are potent and selective CK2 inhibitors. Lead compound 1 was identified, and derivatives were prepared to develop potent inhibitory activity. As a result, we obtained compound 7, which was the smallest unit that retained potency. Then, introducing an aminoalkyl group at the 6-position of the indazole ring resulted in improved efficacy in both enzymatic and cell-based CK2 inhibition assays. Moreover, compound 13 showed selectivity against other kinases and in vivo efficacy in a rat nephritis model. These results show that 2,6-disubstituted pyrazines have potential as therapeutic agents for nephritis.     

Identification of the actinorhodin monomer and its related compound from a deletion mutant of the actVA-ORF4 gene of Streptomyces coelicolor A3(2)

An oxygenated derivative of dihydrokalafungin (DHK) was isolated from a deletion mutant of the actVA-ORF4 gene involved in the biosynthesis of a dimeric benzoisochromanequinone (BIQ) antibiotic, actinorhodin (ACT), in Streptomyces coelicolor A3(2). Spectroscopic analysis elucidated its structure as 8-hydroxy-DHK, corresponding to the monomeric unit of ACT. Further metabolite analysis identified its related compound, clearly derived from the reduction of 8-hydroxy-DHK. The structures of these metabolites indicate the essential role of ActVA-ORF4 in ACT biosynthesis, specifically in dimerization of a BIQ intermediate via C-C bond formation.