Alfacalcidol prevents age-related bone loss and causes an atypical pattern of bone formation in aged male rats

The current study was designed to investigate the skeletal effects of alfacalcidol in aged rats. Eighteen-month-old male rats were treated with 0, 0.1, or 0.2 microg/kg/d of alfacalcidol by daily oral gavage, 5 days/week for 12 weeks. At the beginning of the treatments, one group of rats was euthanized to serve as a baseline control. At the end of the study, the second lumbar vertebrae and the right tibial diaphysess were processed for bone histomorphometric analysis. The fourth lumbar vertebrae were subjected to strength testing. The control group of rats at 21 months of age had decreased serum testosterone levels and decreased cancellous bone mass associated with increased bone turnover on the trabecular surface. The older rats had increased bone turnover on the endocortical surface and decreased bone formation on the periosteal surface compared with the 18-month group. In contrast, alfacalcidol treatment increased cancellous and cortical bone mass in aged male rats. Trabecular bone resorption was decreased whereas bone formation was maintained or increased in the rats treated with alfacalcidol. In addition, endocortical bone formation was decreased whereas periosteal bone formation was increased in the rats treated with alfacalcidol compared with vehicle-treated rats. Marrow trabecular bone area was increased by alfacalcidol treatment in tibial diaphyses. Furthermore, bone strength of the lumbar vertebral body was increased after alfacalcidol treatment. An atypical pattern of bone formation on endosteal bone surfaces was seen in the rats treated with alfacalcidol. The atypical bone formation is characterized by small, focal packets of newly formed bone on trabecular and endocortical bone surfaces. This gave the appearance of the formation of "bone buds" emanating from trabecular surfaces. These bony outgrowths were mineralized and demonstrated significant fluorochrome label indicating recent mineralization. Also, lamellae of the bony buds did not run parallel to those of the trabecular plate to which they are attached. Arrest lines presented in most of the "bone buds". In summary, alfacalcidol treatment increased cancellous and cortical bone mass and improved bone strength, resulting in the prevention of age-related bone loss in aged male rats. An atypical pattern of bone formation observed in this study may be a result of minimodeling based bone formation stimulated by alfacalcidol treatment.     

Inducible nitric-oxide synthase is an important contributor to prolonged protective effects of ischemic preconditioning in the mouse kidney

Ischemic preconditioning renders the mouse kidney resistant to subsequent ischemia. Understanding the mechanisms responsible for ischemic preconditioning is important for formulating therapeutic strategies aimed at mimicking protective mechanisms. We report that the resistance afforded by 30 min of bilateral kidney ischemia persists for 12 weeks after preconditioning. The protection is reflected by improved postischemic renal function, reduced leukocyte infiltration, reduced postischemic disruption of the actin cytoskeleton, and reduced postischemic expression of kidney injury molecule-1 (Kim-1). The protection is observed in both BALB/c and C57BL/6J strains of mice. Thirty minutes of prior ischemia increases the expression of inducible nitric-oxide synthase (iNOS) and endothelial NOS (eNOS) and the expression of heat shock protein (HSP)-25 and is associated with increased interstitial expression of alpha-smooth muscle actin (alpha-SMA), an indication of long term postischemic sequelae. Treatment with Nomega-nitro-l-arginine (l-NNA), an inhibitor of NO synthesis, increases kidney susceptibility to ischemia. Gene deletion of iNOS increases kidney susceptibility to ischemia, whereas gene deletion of eNOS has no effect. Pharmacological inhibition of NOS by l-NNA or l-N6-(1-iminoethyl) lysine (l-NIL, a specific inhibitor of iNOS) mitigates the kidney protection afforded by 30 min of ischemic preconditioning. Fifteen minutes of prior ischemic preconditioning, which does not result in the disruption of the actin cytoskeleton, impairment of renal function, increased interstitial alpha-SMA, or increased iNOS or eNOS expression, but does increase HSP-25 expression, partially protects the kidney from ischemia on day 8 via a mechanism that is not abolished by l-NIL treatment. Thus, iNOS is responsible for a significant component of the long term protection afforded the kidney by ischemic preconditioning, which results in persistent renal interstitial disease, but does not explain the preconditioning seen with shorter periods of ischemia.     

Changes in gene expression during hairy root formation by<Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis> infection in ginseng

Researchers have widely adopted the hairy root culture system as a means for producing secondary metabolites, including ginsenosides from ginseng. Although bacterial genes are involved, the aspects of plant gene expression are unclear. Using a cDNA microarray approach, we identified genes that are differentially expressed in ginseng hairy roots afterAgrobacterium rhizogenes infection. Our goal was to gain an initial understanding of the correlation between hairy root morphology and ginsenoside production. Among the 250 genes analyzed here, 63 (including 14 that are unclassified) were differentially expressed in a hairy root line containing a high level of ginsenosides. Of the genes that had been functionally categorized, 29% and 17% were active in metabolism and stress responses, respectively. Most were primarily associated with ribosomal proteins, thereby functioning in protein synthesis and destination. Their expression was down-regulated in hairy roots having less lateral branching. This phenotype may have resulted from the manipulation of metabolic activities by the translational machinery.     

Structure of the UBA domain of Dsk2p in complex with ubiquitin molecular determinants for ubiquitin recognition

The ubiquitin-associated (UBA) domain is one of the most frequently occurring motifs that recognize ubiquitin tags. Dsk2p, a UBA-containing protein from Saccharomyces cerevisiae, is involved in the ubiquitin-proteasome proteolytic pathway and has been implicated in spindle pole duplication. Here we present the solution structure of the UBA domain of Dsk2p (Dsk2(UBA)) in complex with ubiquitin. The structure reveals that the UBA domain uses a mode of ubiquitin recognition that is similar to that of the CUE domain, another ubiquitin binding motif that shares low sequence homology but high structural similarity with UBA domains. These two domains, as well as the structurally unrelated ubiquitin binding motif UIM, provide a common, crucial recognition site for ubiquitin, comprising a hydrogen-bonding acceptor for the amide group of Gly-47, and a methyl group that packs against the hydrophobic pocket of ubiquitin formed by Leu-8, Ile-44, His-68, and Val-70.     

Calcineurin regulates enteric muscle contraction through EXP-1, excitatory GABA-gated channel, in C. elegans

The enteric muscle contraction (EMC) is the last step of the defecation behavior which occurs every 50 s in Caenorhabditis elegans. This EMC is regulated by intestinal and anal depressor muscles, which are innervated by GABA motor neurons. Our data show that calcineurin (tax-6) is expressed in intestinal muscle and anal depressor muscle, and the gain-of-function mutant of calcineurin, tax-6(jh107), shows defects in enteric muscle contractions. In addition, the intracellular region of EXP-1, an excitatory GABA receptor, specifically binds to calcineurin A. This interaction between TAX-6 and EXP-1 appears to be independent of both calcium and CNB, which is the calcium-binding regulatory subunit. Genetic evidence of epistasis between cnb-1(jh103) and exp-1(sa6) suggests that calcineurin functions as a negative regulator of excitatory GABA receptor in GABA signaling in C.elegans.     

Conservation and Diversification of <Emphasis Type="Italic">SCARECROW</Emphasis> in Maize

The SCARECROW (SCR) gene in Arabidopsis is required for asymmetric cell divisions responsible for ground tissue formation in the root and shoot. Previously, we reported that Zea mays SCARECROW (ZmSCR) is the likely maize ortholog of SCR. Here we describe conserved and divergent aspects of ZmSCR. Its ability to complement the Arabidopsis scr mutant phenotype suggests conservation of function, yet its expression pattern during embryogenesis and in the shoot system indicates divergence. ZmSCR expression was detected early during embryogenesis and localized to the endodermal lineage in the root, showing a gradual regionalization of expression. Expression of ZmSCR appeared to be analogous to that of SCR during leaf formation. However, its absence from the maize shoot meristem and its early expression pattern during embryogenesis suggest a diversification of ZmSCR in the patterning processes in maize. To further investigate the evolutionary relationship of SCR and ZmSCR, we performed a phylogenetic analysis using Arabidopsis, rice and maize SCARECROW-LIKE genes (SCLs). We found SCL23 to be the most closely related to SCR in both eudicots and monocots, suggesting that a gene duplication resulting in SCR and SCL23 predates the divergence of dicots and monocots. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.