Reduced sensitivity of Niemann-Pick C1-deficient cells to θ-toxin (perfringolysin O): sequestration of toxin to raft-enriched membrane vesicles

-Toxin (perfringolysin O) binds to cell surface cholesterol and forms oligomeric pores that cause membrane damage. Both in cytotoxicity and cell survival assays, a mutant Chinese hamster ovary cell line NPC1(–) that lacked Niemann-Pick C1 showed reduced sensitivity to -toxin, compared with wild-type (wt) cells. BC is a derivative of -toxin that retains cholesterol-binding activity but lacks cytotoxicity. Confocal and electron microscopy revealed the presence of multiple vesicles which bound BC, both on the cell surface and in the extracellular space of these cells. BC binding to raft microdomains was verified by its resistance to 1% Triton X-100 at 4°C and recovery of bound BC in floating low-density fractions on sucrose density gradient fractionation. BC-labeled vesicles were abolished when NPC1(–) cells were depleted of lipoproteins and also when treated with a Rho-associated kinase inhibitor Y-27632. In addition, similar vesicles were observed in wt cells treated with progesterone. In parallel with these results, -toxin sensitivity of NPC1(–) cells was increased when cells were depleted of lipoproteins or treated with Y-27632, whereas that of wt cells was decreased by progesterone. Our findings suggest that sequestration of toxin to raft-enriched cell surface vesicles may underlie reduced sensitivity of NPC1-deficient cells to -toxin.     

Generation of a purely clonal defective interfering influenza virus

Defective interfering (DI) influenza viruses carry a large deletion in a gene segment that interferes with the replication of infectious virus; thus, such viruses have potential for antiviral therapy. However, because DI viruses cannot replicate autonomously without the aid of an infectious helper virus, clonal DI virus stocks that are not contaminated with helper virus have not yet been generated. To overcome this problem, we used reverse genetics to generate a clonal DI virus with a PB2 DI gene, amplified the clonal DI virus using a cell line stably expressing the PB2 protein, and confirmed its ability to interfere with infectious virus replication in vitro. Thus, our approach is suitable for obtaining purely clonal DI viruses, will contribute to the understanding of DI virus interference mechanisms and can be used to develop DI virus‐based antivirals.     

Interaction of human and bacterial AlkB proteins with DNA as probed through chemical cross-linking studies

The Escherichia coli AlkB protein was recently found to repair cytotoxic DNA lesions 1-methyladenine and 3-methylcytosine by using a novel iron-catalyzed oxidative demethylation mechanism. Three human homologs, ABH1, ABH2 and ABH3, have been identified, and two of them, ABH2 and ABH3, were shown to have similar repair activities to E.coli AlkB. However, ABH1 did not show any repair activity. It was suggested that ABH3 prefers single-stranded DNA and RNA substrates, whereas AlkB and ABH2 can repair damage in both single- and double-stranded DNA. We employed a chemical cross-linking approach to probe the structure and substrate preferences of AlkB and its three human homologs. The putative active site iron ligands in these proteins were mutated to cysteine residues. These mutant proteins were used to cross-link to different DNA probes bearing thiol-tethered bases. Disulfide-linked protein–DNA complexes can be trapped and analyzed by SDS–PAGE. Our results show that ABH2 and ABH3 have structural and functional similarities to E.coli AlkB. ABH3 shows preference for the single-stranded DNA probe. ABH1 failed to cross-link to the probes tested. This protein, unlike other AlkB proteins, does not seem to interact with DNA in its E.coli expressed form.     

Temporal and spatial variation of forest biomass in relation to stand dynamics in a mature,lowland tropical rainforest,Malaysia

To clarify consistency in the size of carbon pool of a lowland tropical rainforest, we calculated changes in above-ground biomass in the Pasoh Forest Reserve, Peninsular Malaysia. We estimated the total above-ground biomass of a mature stand using tree census data obtained in a 6-ha plot every 2years from 1994 to 1998. The total above-ground biomass decreased consistently from 1994 (431Mgha^–1) to 1998 (403Mgha^–1) (1Mg=10³ kg). These are much lower than that in 1973 for a 0.2ha portion of the same area, suggesting that the the total above-ground biomass reduction might have been consistent in recent decades. This trend contrasted with a major trend for neotropical forests. During 1994–1998, the forest gained 23.0 and 0.88Mgha^–1 of the total above-ground biomass by tree growth and recruitment, respectively, and lost 51.9Mgha^–1 by mortality. Overall, the biomass decreased by 28.4Mgha^–1 (i.e. 7.10Mgha^–1·year^–1), which is almost equivalent to losing a 76-cm-diameter living tree per hectare per year. Analysis of positive and negative components of biomass change revealed that deaths of large trees dominated the total above-ground biomass decrease. The forest biomass also varied spatially, with the total above-ground biomass density ranging 212–655Mgha^–1 on a 0.2-ha basis (n= 30 subplots, 1998) and 365–440Mgha^–1 on a 1ha basis. A large decrease of the total above-ground biomass density (>50Mg per ha per 2years) in several 0.2-ha subplots contributed to the overall decrease in the 6-ha total above-ground biomass. In the present study, we discuss the association between forest dynamics and biomass fluctuation, and the implication for carbon cycling in mature forests with emphasis on forest monitoring and assessments of soil and decomposition systems.     

Plant acetyl-CoA carboxylase: structure, biosynthesis, regulation, and gene manipulation for plant breeding

Acetyl-CoA carboxylase (ACCase) catalyzes the first committed step of fatty acid synthesis, the carboxylation of acetyl-CoA to malonyl-CoA. Two physically distinct types of enzymes are found in nature. Heteromeric ACCase composed of four subunits is usually found in prokaryotes, and homomeric ACCase composed of a single large polypeptide is found in eukaryotes. Most plants have both forms, the heteromeric form in plastids, in which de novo fatty acids are synthesized, and the homomeric form in cytosol. This review focuses on the structure and regulation of plant heteromeric ACCase and its manipulation for plant breeding.     

Endothelin-1 regulates the dorsoventral branchial arch patterning in mice

Endothelin-1 (ET-1), a 21-amino acid peptide secreted by the epithelium and core mesenchyme in the branchial arches as well as vascular endothelium, is involved in craniofacial and cardiovascular development through endothelin receptor type-A (EdnrA) expressed in the neural crest-derived ectomesenchyme. Here we show that ET-1(-/-) mutant mice exhibit a homeotic-like transformation of the lower jaw to an upper jaw. Most of the maxillary arch-derived components are duplicated and replaced mandibular arch-derived structures, resulting in a mirror image of the upper and lower jaws in the ET-1(-/-) mutant. As for hyoid arch-derivatives, the ventral structures are severely affected in comparison to the dorsal ones in the ET-1(-/-) mutant. Correspondingly, the expression of Dlx5 and Dlx6, Distalless-related homeobox genes determining the ventral identity of the anterior branchial arches, and of the mandibular marker gene Pitx1 is significantly downregulated in the ET-1(-/-) mutant, whereas the expression of Dlx2 and the maxillary marker gene Prx2 is unaffected or rather upregulated. These findings indicate that the ET-1/EdnrA signaling may contribute to the dorsoventral axis patterning of the branchial arch system as a mediator of the regional intercellular interactions.     

Immunolocalization of vascular endothelial growth factor in rat condylar cartilage during postnatal development

It is well known that angiogenesis is essential for the replacement of cartilage by bone during skeletal growth and regeneration. To address angiogenesis of endochondral ossification in the condyle, we examined the appearance of vascular endothelial growth factor (VEGF) and its receptor Flt-1 in condylar cartilage of the growing rat. The early expression of VEGF at various sites during condylar cartilage development indicates that VEGF plays a role in the regulation of angiogenesis at each site of bone formation. From the findings of Flt-1 immunoreactivity, the VEGF produced by the chondrocytes of the hypertrophic zone should contribute to the promotion of endothelial cell proliferation and to stimulate migration and activation of osteoclasts in condylar cartilage, resulting in the invasion of these cells into the mineralized zone.Junko Aoyama and Eiji Tanaka contributed equally to this work     

Effect of lectins on the transport of food ingredients in Caco-2 cell cultures

We investigated the effect of several lectins, such as soy bean lectin (SBA), concanavalin A (Con A), and wheat germ agglutinin (WGA), on the transport of some food ingredients (isoflavones, quercetin glycosides, carnosine/anserine) across Caco-2 cell monolayers. After incubation of food ingredients (0.03 approximately 2 mmol/L) in the presence or absence of lectins (1 approximately 180 microg/ml) on the apical side, aliquots were taken from the apical and basolateral solution, and were subjected to HPLC analysis. We also examined the effect of lectins on the permeability of the tight junction by measuring the transepithelial electrical resistance (TER) value of the Caco-2 cell monolayer. Isoflavones, which was not transported to the basolateral solution without lectins, could be transported in the presence of lectins, whereas their aglycones were detected at the same levels with or without the lectin treatment. The transport of quercetin glycosides also increased in the presence of lectins, however, that of peptides was not affected by the lectins. Con A and WGA, but SBA, decreased the TER value, indicating that Con A and WGA increased the transport via paracellular pathway, whereas SBA did via a different pathway.