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     

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.     

Molecular cloning and characterization of a cDNA encoding ent-cassa-12,15-diene synthase, a putative diterpenoid phytoalexin biosynthetic enzyme, from suspension-cultured rice cells treated with a chitin elicitor

We have isolated and characterized a cDNA encoding a novel diterpene cyclase, OsDTC1, from suspension-cultured rice cells treated with a chitin elicitor. OsDTC1 functions as ent-cassa-12,15-diene synthase, which is considered to play a key role in the biosynthesis of (-)-phytocassanes recently isolated as rice diterpenoid phytoalexins. The expression of OsDTC1 mRNA was also confirmed in ultraviolet (UV)-irradiated rice leaves. In addition, we identified ent-cassa-12,15-diene, a putative diterpene hydrocarbon precursor of (-)-phytocassanes, as an endogenous compound in the chitin-elicited suspension-cultured rice cells and the UV-irradiated rice leaves. The OsDTC1 cDNA isolated here will be a useful tool to investigate the regulatory mechanisms of the biosynthesis of (-)-phytocassanes in rice.     

Isolation and characterization of genes encoding polycyclic aromatic hydrocarbon dioxygenase from acenaphthene and acenaphthylene degrading Sphingomonas sp. strain A4

Sphingomonas sp. strain A4 is capable of utilizing acenaphthene and acenaphthylene as sole carbon and energy sources, but it is unable to grow on other polycyclic aromatic hydrocarbons (PAHs). The genes encoding terminal oxygenase components of ring-hydroxylating dioxygenase (arhA1 and arhA2) were isolated from this strain by means of the ability to oxidize indole to indigo of the Escherichia coli clone containing electron transport proteins from phenanthrene-degrading Sphingobium sp. strain P2. The translated products of arhA1 and arhA2 exhibited moderate sequence identity (less than 56%) to large and small subunits of dioxygenase of other ring-hydroxylating dioxygenases. Biotransformation with recombinant E. coli clone revealed the broad substrate specificity of this oxygenase toward several PAHs including acenaphthene, acenaphthylene, naphthalene, phenanthrene, anthracene and fluoranthene. Southern hybridization analysis revealed the presence of a putative arhA1 homologue on a locus different from that of the arhA1 gene. Insertion inactivation of the arhA1 gene in strain A4 suggested that the gene but not the putative homologue one was involved in the degradation of acenaphthene and acenaphthylene in this strain.     

Quantitative PCR with 16S rRNA-gene-targeted species-specific primers for analysis of human intestinal bifidobacteria

A highly sensitive quantitative PCR detection method has been developed and applied to the distribution analysis of human intestinal bifidobacteria by combining real-time PCR with Bifidobacterium genus- and species-specific primers. Real-time PCR detection of serially diluted DNA extracted from cultured bifidobacteria was linear for cell counts ranging from 10(6) to 10 cells per PCR assay. It was also found that the method was applicable to the detection of Bifidobacterium in feces when it was present at concentrations of >10(6) cells per g of feces. Concerning the distribution of Bifidobacterium species in intestinal flora, the Bifidobacterium adolescentis group, the Bifidobacterium catenulatum group, and Bifidobacterium longum were found to be the three predominant species by examination of DNA extracted from the feces of 46 healthy adults. We also examined changes in the population and composition of Bifidobacterium species in human intestinal flora of six healthy adults over an 8-month period. The results showed that the composition of bifidobacterial flora was basically stable throughout the test period.     

Exposure to pressure stimulus enhances succinate dehydrogenase activity in L6 myoblasts

Contraction of skeletal muscle generates pressure stimuli to intramuscular tissues. However, the effects of pressure stimuli, other than those created by electricity or nerve impulse, on physiological and biochemical responses in skeletal muscles are unknown. The purpose of this study is to examine the effects of a pure pressure stimulus on metabolic responses in a skeletal muscle cell line. Atmospheric pressure was applied to L6 myoblasts using an original apparatus. Succinate dehydrogenase (SDH) activity was evaluated by colorimetric assay using tetrazolium monosodium salt. The amounts of 2-deoxy-[(3)H]glucose uptake and lactate release were measured. SDH activity was 2.6- to 2.9-fold higher in pressurized L6 cells than in nonpressurized L6 cells (P < 0.01), and 2-deoxy-[(3)H]glucose uptake was 2.2-fold higher (P < 0.001). In addition, the amount of released lactate decreased from 6.8 to 3.7 mumol/dish when pressure was applied (P < 0.001). In contrast, the intracellular lactate contents of the pressurized cells were higher than those of nonpressurized cells (P < 0.01). However, the total amount of released lactate and intracellular lactate was lower in the pressurized cells than in nonpressurized cells. These findings demonstrate that a pure pressure stimulus enhances aerobic metabolism in L6 skeletal muscle cells and raise the possibility that elevated intramuscular pressure during muscle activity may be an important factor in stimulating oxidative metabolic responses in skeletal muscles.     

Structural basis of sugar-recognizing ubiquitin ligase

SCF(Fbs1) is a ubiquitin ligase that functions in the endoplasmic reticulum (ER)-associated degradation pathway. Fbs1/Fbx2, a member of the F-box proteins, recognizes high-mannose oligosaccharides. Efficient binding to an N-glycan requires di-N-acetylchitobiose (chitobiose). Here we report the crystal structures of the sugar-binding domain (SBD) of Fbs1 alone and in complex with chitobiose. The SBD is composed of a ten-stranded antiparallel beta-sandwich. The structure of the SBD-chitobiose complex includes hydrogen bonds between Fbs1 and chitobiose and insertion of the methyl group of chitobiose into a small hydrophobic pocket of Fbs1. Moreover, NMR spectroscopy has demonstrated that the amino acid residues adjoining the chitobiose-binding site interact with the outer branches of the carbohydrate moiety. Considering that the innermost chitobiose moieties in N-glycans are usually involved in intramolecular interactions with the polypeptide moieties, we propose that Fbs1 interacts with the chitobiose in unfolded N-glycoprotein, pointing the protein moiety toward E2 for ubiquitination.     

20S proteasome prevents aggregation of heat-denatured proteins without PA700 regulatory subcomplex like a molecular chaperone

The eukaryotic 20S proteasome is the multifunctional catalytic core of the 26S proteasome, which plays a central role in intracellular protein degradation. Association of the 20S core with a regulatory subcomplex, termed PA700 (also known as the 19S cap), forms the 26S proteasome, which degrades ubiquitinated and nonubiquitinated proteins through an ATP-dependent process. Although proteolytic assistance by this regulatory particle is a general feature of proteasome-dependent turnover, the 20S proteasome itself can degrade some proteins directly, bypassing ubiquitination and PA700, as an alternative mechanism in vitro. The mechanism underlying this pathway is based on the ability of the 20S proteasome to recognize partially unfolded proteins. Here we show that the 20S proteasome recognizes the heat-denatured forms of model proteins such as citrate synthase, malate dehydrogenase. and glyceraldehydes-3-phosphate dehydrogenase, and prevents their aggregation in vitro. This process was not followed by the refolding of these denatured substrates into their native states, whereas PA700 or the 26S proteasome generally promotes their reactivation. These results indicate that the 20S proteasome might play a role in maintaining denatured and misfolded substrates in a soluble state, thereby facilitating their refolding or degradation.