Reversal of elastase-induced pulmonary emphysema and promotion of alveolar epithelial cell proliferation by simvastatin in mice

Besides lowering cholesterol, statins exert multiple effects, such as anti-inflammatory activity and improvement of endothelial cell function. We examined whether simvastatin (SS) protects against the development of elastase-induced pulmonary emphysema in mice by using mean linear intercepts of alveoli (Lm) as a morphometric parameter of emphysema. After injection of intratracheal elastase on day 0, C57BL/6 mice were treated daily with SS (SS+ group) or PBS (SS- group) for 2 wk. A 21% decrease in Lm on day 7 was observed in the SS+ group vs. the SS- group. Anti-inflammatory effects of SS were observed as a decrease in percentage of neutrophils up to day 3, and in hydroxyproline concentration on day 3, in bronchoalveolar lavage fluid (BALF). SS also increased the number of proliferating cell nuclear antigen (PCNA)-positive alveolar epithelial cells between days 3 and 14. To confirm the role of statins in promoting proliferation of alveolar cells, mice were treated with SS (SS+) vs. PBS (SS-) for 12 days, starting 3 wk after elastase administration. After SS treatment, Lm decreased by 52% and PCNA-positive alveolar epithelial cells increased compared with the SS- group. Concentrations of vascular endothelial growth factor in BALF and endothelial nitric oxide synthase protein expression in pulmonary vessels tended to be higher in the SS+ group vs. the SS- group in this protocol. In conclusion, SS inhibited the development of elastase-induced pulmonary emphysema in mice. This therapeutic effect was due not only to anti-inflammation but also to the promotion of alveolar epithelial cell regeneration, partly mediated by restoring endothelial cell functions.     

Interaction between <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> oncoprotein 6b and a tobacco nucleolar protein that is homologous to TNP1 encoded by a transposable element of <Emphasis Type="Italic">Antirrhinum majus</Emphasis>

When gene 6b on the T-DNA of Agrobacterium tumefaciens is transferred to plant cells, its expression causes plant hormone-independent division of cells in in vitro culture and abnormal cell growth, which induces various morphological defects in 6b-expressing transgenic Arabidopsis thaliana and Nicotiana tabacum plants. Protein 6b localizes to the nuclei, a requirement for the abnormal cell growth, and binds to a tobacco nuclear protein called NtSIP1 and histone H3. In addition, 6b has histone chaperone-like activity in vitro and affects the expression of various plant genes, including cell division-related genes and meristem-related class 1 KNOX homeobox genes, in transgenic Arabidopsis. Here, we report that 6b binds to a newly identified protein NtSIP2, whose amino acid sequence is predicted to be 30% identical and 51% similar to that of the TNP1 protein encoded by the transposon Tam1 of Antirrhinum majus. Immunolocalization analysis using anti-T7 antibodies showed nucleolar localization of most of the T7 epitope-tagged NtSIP2 proteins. A similar analysis with the T7-tagged 6b protein also showed subnucleolar as well as nuclear localization of the 6b protein. These results suggest the involvement of 6b along with NtSIP2 in certain molecular processes in the nucleolus as well as the nucleoplasm.     

Regulation of Chk2 ubiquitination and signaling through autophosphorylation of serine 379

The Chk2 protein kinase protects genome integrity by promoting cell cycle arrest or apoptosis in response to DNA double-strand breaks, and Chk2 mutations are found in both familial and sporadic cancers. Exposure of cells to ionizing radiation (IR) or radiomimetic drugs induces Chk2 phosphorylation by ATM, followed by Chk2 oligomerization, auto-/transphosphorylation, and activation. Here we demonstrate that Chk2 is ubiquitinated upon activation and that this requires Chk2 kinase activity. Serine 379 (S379) was identified as a novel IR-inducible autophosphorylation site required for ubiquitination of Chk2 by a Cullin 1-containing E3 ligase complex. Importantly, S379 was required for Chk2 to induce apoptosis in cells with DNA double-strand breaks. Thus, auto-/transphosphorylation of S379 is required for Chk2 ubiquitination and effector function.     

Contrasting responses of web-building spiders to deer browsing among habitats and feeding guilds

We examined web-building spider species richness and abundance in forests across a deer density gradient to determine the effects of sika deer browsing on spiders among habitats and feeding guilds. Deer decreased the abundance of web-building spiders in understory vegetation but increased their abundance in the litter layer. Deer seemed to affect web-building spiders in the understory vegetation by reducing the number of sites for webs because vegetation complexity was positively correlated with spider density and negatively correlated with deer density. In contrast, the presence of vegetation just above the litter layer decreased the spider density, and deer exerted a negative effect on this vegetation, possibly resulting in an indirect positive effect on spider density. The vegetation just above the litter layer may be unsuitable as a scaffold for building webs if it is too flexible to serve as a reliable web support, and may even hinder spiders from building webs on litter. Alternatively, the negative effect of this vegetation on spiders in the litter may be as a result of reduced local prey availability under the leaves because of the reduced accessibility of aerial insects. The response to deer browsing on web-building spiders that inhabit the understory vegetation varied with feeding guild. Deer tended to affect web-invading spiders, which inhabit the webs of other spiders and steal prey, more heavily than other web-building spiders, probably because of the accumulated effects of habitat fragmentation through the trophic levels. Thus, the treatment of a particular higher-order taxon as a homogeneous group could result in misleading conclusions about the effects of mammalian herbivores.     

Effect of dietary calcium: Phosphorus ratio on bone mineralization and intestinal calcium absorption in ovariectomized rats

We investigated the effect of dietary calcium:phosphorus (Ca:P) ratio on bone mineralization and intestinal Ca absorption in ovariectomized (OVX) rat models of osteoporosis and sham-operated rats. Thirty 12-wk-old female Wistar rats were divided into three groups of OVX rats and three groups of sham rats. Thirty days after the adaptation period, OVX rats and sham rats were fed a diet formulated Ca:P, 1:0.5, 1:1 or 1:2 (each diet containing 0.5% Ca), respectively for 42 d. In both sham and OVX rats, serum osteocalcin, a marker of bone turnover, was increased by decreasing Ca:P ratio (1:2). In contrast, rats fed the Ca:P = 1:0.5 diet (dietary P restriction) suppressed the increased serum parathyroid hormone, osteocalcin and urinary deoxypyridinoline, and increased Ca absorption in both sham and OVX rats compared to the Ca:P = 1:1 and 1:2 diets. Especially, in OVX rats, the decreased bone mineral density of the fifth lumbar was also suppressed when rats were fed the Ca:P = 1:0.5 diet. These results indicated that the elevation of dietary Ca:P ratio may inhibit bone loss and increase intestinal Ca absorption in OVX rats.     

Effect of high phosphorus diet on phosphorus metabolism in parathyroidectomized rats

To determine the parathyroid hormone (PTH) action on kidney and bone by high phosphorus (P) diet, this study investigated PTH/PTH-related peptide (PTHrP) receptor mRNA expression in 6-week-old parathyroidectomized (PTX) rats received constant amount of PTH. To maintain serum PTH levels equally to sham operated rats, PTX rats were constantly exposed to rPTH (1-34) and fed a control diet (0.3% P) and a high P diet (1.2% P) for 7 days, respectively. There were no significant differences in serum PTH (1-34) concentration in rats fed the control diet. In sham groups, serum PTH concentrations, both (1-84) and (1-34) fragments, were increased in rats fed the high P diet than in rats fed the control diet. Urinary excretions of P and C-terminal telopeptides of type I collagen were significantly increased in both PTX and sham rats by the high P diet. PTH/PTHrP receptor mRNA expression in kidney and femur was not changed in both PTX and sham rats by the high P diet. In conclusion, high P diet did not change PTH action in PTX rats and increased urinary excretion of P and bone resorption regardless of PTH action.     

Sex differences in kidney mineral concentrations and urinary albumin excretion in rats given high-phosphorus feed

We examined sex differences in kidney mineral concentrations and urinary albumin excretion in rats given feed containing various phosphorus (P) levels. With feed that was 0.6%, 0.9%, 1.2%, and 1.5% P, kidney calcium and P concentrations were higher in female rats than in male rats. With 1.2% or 1.5% P, urinary albumin excretion was higher in the female rats. The sex of the animal affected the kidney mineral concentrations and urinary albumin excretion in rats with a high P intake.     

Recombinant bovine zona pellucida glycoproteins ZP3 and ZP4 coexpressed in Sf9 cells form a sperm-binding active hetero-complex

The zona pellucida (ZP) is a transparent envelope that surrounds the mammalian oocyte and mediates species-selective sperm-egg interactions. Porcine and bovine ZPs are composed of the glycoproteins ZP2, ZP3, and ZP4. We previously established an expression system for porcine ZP glycoproteins (ZPGs) using baculovirus in insect Sf9 cells. Here we established a similar method for expression of bovine ZPGs. The recombinant ZPGs were secreted into the medium and purified by metal-chelating column chromatography. A mixture of bovine recombinant ZP3 (rZP3) and rZP4 coexpressed in Sf9 cells exhibited inhibitory activity for bovine sperm-ZP binding similar to that of a native bovine ZPG mixture, whereas neither bovine rZP3 nor rZP4 inhibited binding. An immunoprecipitation assay revealed that the coexpressed rZP3/rZP4 formed a hetero-complex. We examined the functional domain structure of bovine rZP4 by constructing ZP4 mutants lacking the N-terminal domain or lacking both the N-terminal and trefoil domains. When either of these mutant proteins was coexpressed with bovine rZP3, the resulting mixtures exhibited inhibitory activity comparable to that of the bovine rZP3/rZP4 complex. Hetero-complexes of bovine rZP3 and porcine rZP4, or porcine rZP3 and bovine rZP4, also inhibited bovine sperm-ZP binding. Our results demonstrate that the N-terminal and trefoil domains of bovine rZP4 are dispensable for formation of the sperm-binding active bovine rZP3/rZP4 complex and, furthermore, that the molecular interactions between rZP3 and rZP4 are conserved in the bovine and porcine systems.