Degradation of nuclear DNA by DNase II-like acid DNase in cortical fiber cells of mouse eye lens

The eye lens is composed of fiber cells that differentiate from epithelial cells on its anterior surface. In concert with this differentiation, a set of proteins essential for lens function is synthesized, and the cellular organelles are degraded. DNase II-like acid DNase, also called DNase IIbeta, is specifically expressed in the lens, and degrades the DNA in the lens fiber cells. Here we report that DNase II-like acid DNase is synthesized as a precursor with a signal sequence, and is localized to lysosomes. DNase II-like acid DNase mRNA was found in cortical fiber cells but not epithelial cells, indicating that its expression is induced during the differentiation of epithelial cells into fiber cells. Immunohistochemical and immunocytochemical analyses indicated that DNase II-like acid DNase was colocalized with Lamp-1 in the lysosomes of fiber cells in a relatively narrow region bordering the organelle-free zone, and was often found in degenerating nuclei. A comparison by microarray analysis of the gene expression profiles between epithelial and cortical fiber cells of young mouse lens indicated that some genes for lysosomal enzymes (cathepsins and lipases) were strongly expressed in the fiber cells. These results suggest that the lysosomal system plays a role in the degradation of cellular organelles during lens cell differentiation.     

Comparative morphological examination of vertebral bodies of teleost fish using high-resolution micro-CT scans

Vertebral bodies of teleost fish are formed by the sclerotomal bone covering the chordacentrum. The internal part of the sclerotomal bone is composed of an amphicoelous hourglass shaped autocentrum, which is common in most fish species. In contrast, the external shape of the sclerotomal bone varies extensively among species. There are multiple hypotheses regarding the composition and formation of the external structure. However, as they are based on studies of few extant or extinct species, their applicability to other species remains to be clarified. To understand the morphology, formation, and composition of vertebral bodies in teleosts, we performed a comparative analysis using micro-CT scans of 32 species from 10 orders of Teleostei and investigated the detailed morphology of the sclerotomal bone, especially its plate-like ridge and trabeculae. We discovered two structural characteristics that are shared among most of the examined species. One was the sheet-like trabeculae that extend radially from the center of the vertebral body with a constant thickness. The other was the presence of hollow spaces on the internal parts of the lateral ridge and trabeculae. The combination of different arrangements of sheet-like trabeculae and internal hollow spaces formed different shapes of the lateral structure of the vertebral body. The properties of these two characteristics suggest that the external part of the sclerotomal bone grows outward by deposition at the bone tip, and that, concurrently, bone absorption occurs in the internal part of the sclerotomal bone. The vertebral arches were also formed by the sheet-like trabeculae, indicating that both, the vertebral body and the arches, are formed by the same component. The micro-CT scanning data were uploaded to a public database so they can be used for future studies on fish vertebrae.     

Effect of angiotensin II type 2 receptor on tyrosine kinase Pyk2 and c-Jun NH2-terminal kinase via SHP-1 tyrosine phosphatase activity: evidence from vascular-targeted transgenic mice of AT2 receptor

Angiotensin II (Ang II) has two major receptor isoforms, AT1 and AT2. AT1 transphosphorylates Ca(2+)-sensitive tyrosine kinase Pyk2 to activate c-Jun NH2-terminal kinase (JNK). Although AT2 inactivates extracellular signal-regulated kinase (ERK) via tyrosine phosphatases (PTP), the action of AT2 on Pyk2 and JNK remains undefined. Using AT2-overexpressing vascular smooth muscle cells (AT2-VSMC) from AT2-transgenic mice, we studied these undefined actions of AT2. AT1-mediated JNK activity was increased 2.2-fold by AT2 inhibition, which was abolished by orthovanadate. AT2 did not affect AT1-mediated Pyk2 phosphorylation, but attenuated c-Jun mRNA accumulation by 32%. The activity of src-homology 2 domain-containing PTP (SHP-1) was significantly upregulated 1 min after AT2 stimulation. Stable overexpression of SHP-1 dominant negative mutant in AT2-VSMC completely abolished AT2-mediated inhibition of JNK activation and c-Jun expression. These findings suggest that AT2 inhibits JNK activity by affecting the downstream signal of Pyk2 in a SHP-1-dependent manner, leading to a decrease in c-Jun expression.     

Solution structure of the ribosome recycling factor from Aquifex aeolicus

The solution structure of ribosome recycling factor (RRF) from hyperthermophilic bacterium, Aquifex aeolicus, was determined by heteronuclear multidimensional NMR spectroscopy. Fifteen structures were calculated using restraints derived from NOE, J-coupling, and T1/T2 anisotropies. The resulting structure has an overall L-shaped conformation with two domains and is similar to that of a tRNA molecule. The domain I (corresponding to the anticodon stem of tRNA) is a rigid three alpha-helix bundle. Being slightly different from usual coiled-coil arrangements, each helix of domain I is not twisted but straight and parallel to the main axis. The domain II (corresponding to the portion with the CCA end of tRNA) is an alpha/beta domain with an alpha-helix and two beta-sheets, that has some flexible regions. The backbone atomic root-mean-square deviation (rmsd) values of both domains were 0.7 A when calculated separately, which is smaller than that of the molecule as a whole (1.4 A). Measurement of 15N-[1H] NOE values show that the residues in the corner of the L-shaped molecule are undergoing fast internal motion. These results indicate that the joint region between two domains contributes to the fluctuation in the orientation of two domains. Thus, it was shown that RRF remains the tRNA mimicry in solution where it functions.     

Ceramide generation in nitric oxide-induced apoptosis. Activation of magnesium-dependent neutral sphingomyelinase via caspase-3

Sodium nitroprusside (SNP), a NO donor, has been recognized as an inducer of apoptosis in various cell lines. Here, we demonstrated the intracellular formation of ceramide, a lipid signal mediator, in SNP-induced apoptosis in human leukemia HL-60 cells and investigated the mechanisms of ceramide generation. The levels of intracellular ceramide increased to, at most, 160% of the control level in a time- and dose-dependent manner when the cells were treated with 1 mM SNP. SNP also decreased the sphingomyelin level to approximately 70% of the control level and increased magnesium-dependent neutral sphingomyelinase (N-SMase) activity to 160% of the control activity 2 h after treatment. Neither acid SMase nor magnesium-independent N-SMase was affected by SNP. Caspases are thought to be key enzymes in apoptotic cell death. Acetyl-Asp-Glu-Val-Asp-aldehyde, a synthetic tetrapeptide inhibitor of caspases, inhibited magnesiumdependent N-SMase, ceramide generation, and apoptosis. Moreover, recombinant purified caspase-3 increased magnesium-dependent N-SMase in a cell-free system. These results suggest that the findings that SNP increased ceramide generation and magnesium-dependent N-SMase activity via caspase-3 are interesting to future study to determine the relation between caspases and sphingolipid metabolites in NO-mediated signaling.     

High efficient expression of the functional ligand binding site of the inositol 1,4,5-triphosphate receptor in Escherichia coli

Type 1 inositol 1,4,5-trisphosphate receptor (IP3R1), an inositol 1, 4,5-trisphosphate (IP3)-gated Ca2+ release channel, binds IP3 within the N-terminal ligand-binding region. Here we report an improved Escherichia coli expression system in which large amounts of the IP3 binding sites could be efficiently produced as soluble active proteins. We have found that the structures of IP3 binding constructs expressed in E. coli significantly affect their production as soluble protein. Residues 1-604 (T604), which contain the putative protein folding units, yielded about 4.6% of the total soluble fraction. As a result, soluble active T604 would be 19 mg per liter of culture. The affinity for IP3 of T604 (Kd = 45 nM) is comparable to that of the native IP3R1, whereas that of an R441Q mutant is much higher (8.1 nM). This system should provide an invaluable and powerful means to unveil the molecular recognition of IP3R1 for IP3.     

Sequence analysis of the gene encoding a spotted fever group-specific intracytoplasmic protein PS120 of Rickettsia japonica

The 3,438-nucleotide (nt) sequence containing a 3,054-nt open reading frame of the gene (rps120) encoding an antigenic, intracytoplasmic, spotted fever group-specific and heat-stable 120-kilodalton protein (PS120) of Rickettsia japonica was determined. The nt and deduced 1,018 amino-acid (aa) sequences were compared to those of R. conorii since only those of this species had been determined among SFG rickettsiae. The homologies of these sequences between R. japonica and R. conorii were considerably high at 97 and 95%, respectively. These high homologies were comparable to those of beta-peptides encoded by the ompB genes among SFG rickettsiae. It was also found that the genome of R. prowazekii contained a nt sequence with 68% homology to that of the rps120 gene of R. japonica.