SmpB triggers GTP hydrolysis of elongation factor Tu on ribosomes by compensating for the lack of codon-anticodon interaction during trans-translation initiation

Bacterial tmRNA rescues ribosomes that stall because of defective mRNAs via the trans-translation process. Although entry of the charged transfer messenger RNA (tmRNA) into the ribosome proceeded in the absence of elongation factor (EF-Tu) and in the presence of EF-Tu and the antibiotic kirromycin, evidence was found for the involvement of EF-Tu in trans-translation initiation. The polyalanine synthesis system attained by using a tmRNA variant consisting of only the tRNA-like domain revealed that it was completely dependent on the presence of SmpB and greatly enhanced by EF-Tu and EF-G. Actually, ribosome-dependent GTPase activity of EF-Tu was stimulated by the addition of SmpB and tmRNA but independently of template mRNA, demonstrating that SmpB compensates for the lack of codon-anticodon interaction during the first step of the trans-translation initiation. Based on these results, we suggest that SmpB structurally mimics the anticodon arm of tRNA and elicits GTP hydrolysis of EF-Tu upon tmRNA accommodation in the A site of the ribosome.     

Comparative effects of alendronate and alfacalcidol on cancellous and cortical bone mass and bone mechanical properties in ovariectomized rats.

The purpose of the present study was to compare the effects of alendronate and alfacalcidol on cancellous and cortical bone mass and bone mechanical properties in ovariectomized rats. Twenty-six female Sprague-Dawley rats, 7 months of age, were randomized by the stratified weight method into four groups: the sham-operated control (Sham) group and the three ovariectomy (OVX) groups, namely, OVX + vehicle, OVX + alendronate (2.5 mg/kg, p.o., daily), and OVX + alfacalcidol (0.5 mug/kg, p.o., daily). At the end of the 8-week experimental period, bone histomorphometric analyses of cancellous bone at the proximal tibial metaphysis and cortical bone at the tibial diaphysis were performed, and the mechanical properties of the femoral distal metaphysis and femoral diaphysis were evaluated. OVX decreased cancellous bone volume per total tissue volume (BV/TV), and the maximum load of the femoral distal metaphysis, as a result of increases in serum osteocalcin (OC) levels, and also the number of osteoclasts (N.Oc), osteoclast surface (OcS) and bone formation rate (BFR) per bone surface (BS), and BFR/BV, without any effect on cortical area (Ct Ar), or maximum load of the femoral diaphysis. Alendronate prevented this decrease in cancellous BV/TV by suppressing increases in N.Oc/BS, OcS/BS, BFR/BS, and BFR/BV, without any apparent effect on Ct Ar, or maximum load of the femoral distal metaphysis and femoral diaphysis. On the other hand, alfacalcidol increased cancellous BV/TV, Ct Ar, and the maximum load of the femoral distal metaphysis and femoral diaphysis, by mildly decreasing trabecular BFR/BV, maintaining trabecular mineral apposition rate and osteoblast surface per BS, increasing periosteal and endocortical BFR/BS, and preventing an increase in endocortical eroded surface per BS. The present study clearly showed the differential skeletal effects of alendronate and alfacalcidol in ovariectomized rats. Alendronate prevented OVX-induced cancellous bone loss by suppressing bone turnover, while alfacalcidol improved cancellous and cortical bone mass and bone strength by suppressing bone resorption and maintaining or even increasing bone formation.     

Pure and Mixed Genetic Lines of Saccharomyces bayanus and Saccharomyces pastorianus and Their Contribution to the Lager Brewing Strain Genome

The yeast species Saccharomyces bayanus and Saccharomyces pastorianus are of industrial importance since they are involved in the production process of common beverages such as wine and lager beer; however, they contain strains whose variability has been neither fully investigated nor exploited in genetic improvement programs. We evaluated this variability by using PCR-restriction fragment length polymorphism analysis of 48 genes and partial sequences of 16. Within these two species, we identified “pure” strains containing a single type of genome and “hybrid” strains that contained portions of the genomes from the “pure” lines, as well as alleles termed “Lager” that represent a third genome commonly associated with lager brewing strains. The two pure lines represent S. uvarum and S. bayanus, the latter a novel group of strains that may be of use in strain improvement programs. Hybrid lines identified include (i) S. cerevisiae/S. bayanus/Lager, (ii) S. bayanus/S. uvarum/Lager, and (iii) S. cerevisiae/S. bayanus/S. uvarum/Lager. The genome of the lager strains may have resulted from chromosomal loss, replacement, or rearrangement within the hybrid genetic lines. This study identifies brewing strains that could be used as novel genetic sources in strain improvement programs and provides data that can be used to generate a model of how naturally occurring and industrial hybrid strains may have evolved.     

Involvement of PRIP, phospholipase C-related, but catalytically inactive protein, in bone formation

PRIP (phospholipase C-related, but catalytically inactive protein) is a novel protein isolated in this laboratory. PRIP-deficient mice showed increased serum gonadotropins, but decreased gonadal steroid hormones. This imbalance was similar to that for the cause of bone disease, such as osteoporosis. In the present study, therefore, we analyzed mutant mice with special reference to the bone property. We first performed three-dimensional analysis of the femur of female mice. The bone mineral density and trabecular bone volume were higher in mutant mice. We further performed histomorphometrical assay of bone formation parameters: bone formation rate, mineral apposition rate, osteoid thickness, and osteoblast number were up-regulated in the mutant, indicating that increased bone mass is caused by the enhancement of bone formation ability. We then cultured primary cells isolated from calvaria prepared from both genotypes. In mutant mice, osteoblast differentiation, as assessed by alkaline phosphatase activity and the expression of osteoblast differentiation marker genes, was enhanced. Moreover, we analyzed the phosphorylation of Smad1/5/8 in response to bone morphogenetic protein, with longer phosphorylation in the mutant. These results indicate that PRIP is implicated in the negative regulation of bone formation.     

Nup358, a nucleoporin, functions as a key determinant of the nuclear pore complex structure remodeling during skeletal myogenesis

The nuclear pore complex (NPC) is the only gateway for molecular trafficking across the nuclear envelope. The NPC is not merely a static nuclear-cytoplasmic transport gate; the functional analysis of nucleoporins has revealed dynamic features of the NPC in various cellular functions, such as mitotic spindle formation and protein modification. However, it is not known whether the NPC undergoes dynamic changes during biological processes such as cell differentiation. In the present study, we evaluate changes in the expression levels of several nucleoporins and show that the amount of Nup358/RanBP2 within individual NPCs increases during muscle differentiation in C2C12 cells. Using atomic force microscopy, we demonstrate structural differences between the cytoplasmic surfaces of myoblast and myotube NPCs and a correlation between the copy number of Nup358 and the NPC structure. Furthermore, small interfering RNA-mediated depletion of Nup358 in myoblasts suppresses myotube formation without affecting cell viability, suggesting that NUP358 plays a role in myogenesis. These findings indicate that the NPC undergoes dynamic remodeling during muscle cell differentiation and that Nup358 is prominently involved in the remodeling process.     

Transforming activity of the lymphotoxin-beta receptor revealed by expression screening

Pancreatic ductal carcinoma (PDC) remains one of the most intractable human malignancies. To obtain insight into the molecular pathogenesis of PDC, we constructed a retroviral cDNA expression library with total RNA isolated from the PDC cell line MiaPaCa-2. Screening of this library with the use of a focus formation assay with NIH 3T3 mouse fibroblasts resulted in the identification of 13 independent genes with transforming activity. One of the cDNAs thus identified encodes an NH(2)-terminally truncated form of the lymphotoxin-beta receptor (LTBR). The transforming activity of this short-type LTBR in 3T3 cells was confirmed by both an in vitro assay of cell growth in soft agar and an in vivo assay of tumorigenicity in nude mice. The full-length (wild-type) LTBR protein was also found to manifest similar transforming activity. These observations suggest that LTBR, which belongs to the tumor necrosis factor receptor superfamily of proteins, may contribute to human carcinogenesis.     

Sulfatide and its synthetic analogues recognition by Moraxella catarrhalis

Moraxella catarrhalis is one of the major pathogens of respiratory and middle ear infections. Attachment of this bacterium to the surface of human pharyngeal epithelial cells is the first step in the pathogenesis of infections. This study revealed that sulfatide might act as a binding molecule for the attachment of M. catarrhalis to human pharyngeal epithelial cells. Furthermore, six different synthetic sulfatides were found to inhibit the attachment of M. catarrhalis significantly at an optimum concentration of 10 microg/ml. Synthetic sulfatides may have the potential to be used as a therapy to prevent M. catarrhalis infections.     

T-type Ca2+ Channels Promote Oxygenation-induced Closure of the Rat Ductus Arteriosus Not Only by Vasoconstriction but Also by Neointima Formation

The ductus arteriosus (DA), an essential vascular shunt for fetal circulation, begins to close immediately after birth. Although Ca²⁺ influx through several membrane Ca²⁺ channels is known to regulate vasoconstriction of the DA, the role of the T-type voltage-dependent Ca²⁺ channel (VDCC) in DA closure remains unclear. Here we found that the expression of α1G, a T-type isoform that is known to exhibit a tissue-restricted expression pattern in the rat neonatal DA, was significantly up-regulated in oxygenated rat DA tissues and smooth muscle cells (SMCs). Immunohistological analysis revealed that α1G was localized predominantly in the central core of neonatal DA at birth. DA SMC migration was significantly increased by α1G overexpression. Moreover, it was decreased by adding α1G-specific small interfering RNAs or using R(−)-efonidipine, a highly selective T-type VDCC blocker. Furthermore, an oxygenation-mediated increase in an intracellular Ca²⁺ concentration of DA SMCs was significantly decreased by adding α1G-specific siRNAs or using R(−)-efonidipine. Although a prostaglandin E receptor EP4 agonist potently promoted intimal thickening of the DA explants, R(−)-efonidipine (10⁻⁶ m) significantly inhibited EP4-promoted intimal thickening by 40% using DA tissues at preterm in organ culture. Moreover, R(−)-efonidipine (10⁻⁶ m) significantly attenuated oxygenation-induced vasoconstriction by ∼27% using a vascular ring of fetal DA at term. Finally, R(−)-efonidipine significantly delayed the closure of in vivo DA in neonatal rats. These results indicate that T-type VDCC, especially α1G, which is predominantly expressed in neonatal DA, plays a unique role in DA closure, implying that T-type VDCC is an alternative therapeutic target to regulate the patency of DA.The ductus arteriosus (DA)² is an essential vascular shunt between the aortic arch and the pulmonary trunk during a fetal period (1). After birth, the DA closes immediately in accordance with its smooth muscle contraction and vascular remodeling, whereas the connecting vessels such as the aorta and pulmonary arteries remain open. When the DA fails to close after birth, the condition is known as patent DA, which is a common form of congenital heart defect. Patent DA is also a frequent problem with significant morbidity and mortality in premature infants. Investigating the molecular mechanism of DA closure is important not only for vascular biology but also for clinical problems in pediatrics.Voltage-dependent Ca²⁺ channels (VDCCs) consist of multiple subtypes, named L-, N-, P/Q-, R-, and T-type. L-type VDCCs are known to play a primary role in regulating Ca²⁺ influx and thus vascular tone in the development of arterial smooth muscle including the DA (2–4). Our previous study demonstrated that all T-type VDCCs were expressed in the rat DA (5). α1G subunit, especially, was the most dominant isoform among T-type VDCCs. The abundant expression of α1G subunit suggests that it plays a role in the vasoconstriction and vascular remodeling of the DA. In this regard, Nakanishi et al. (6) demonstrated that 0.5 mm nickel, which blocks T-type VDCC, inhibited oxygen-induced vasoconstriction of the rabbit DA. On the other hand, Tristani-Firouzi et al. (7) demonstrated that T-type VDCCs exhibited little effect on oxygen-sensitive vasoconstriction of the rabbit DA. Thus, the role of T-type VDCCs in DA vasoconstriction has remained controversial.In addition to their role in determining the contractile state, a growing body of evidence has demonstrated that T-type VDCCs play an important role in regulating differentiation (8, 9), proliferation (10–12), migration (13, 14), and gene expression (15) in vascular smooth muscle cells (SMCs). Hollenbeck et al. (16) and Patel et al. (17) demonstrated that nickel inhibited platelet-derived growth factor-BB-induced SMC migration. Rodman et al. (18) demonstrated that α1G promoted SMC proliferation in the pulmonary artery. The DA dramatically changes its morphology during development. Intimal cushion formation, a characteristic feature of vascular remodeling of the DA (19–21), involves many cellular processes: an increase in SMC migration and proliferation, production of hyaluronic acid under the endothelial layer, impaired elastin fiber assembly, and so on (1, 19, 21–23). Although our previous study demonstrated that T-type VDCCs are involved in smooth muscle cell proliferation in the DA (5), the role of T-type VDCCs in vascular remodeling of the DA has remained poorly understood.In the present study, we hypothesized that T-type VDCCs, especially α1G subunit, associate with vascular remodeling and vasoconstriction in the DA. To test our hypothesis, we took full advantage of recent molecular and pharmacological developments. We chose the recently developed, highly selective T-type VDCC blocker R(−)-efonidipine instead of low dose nickel for our study. Selective inhibition or activation of α1G subunit was also obtained using small interfering RNA (siRNA) technology or by overexpression of the α1G subunit gene, respectively. We found that Ca²⁺ influx through T-type VDCCs promoted oxygenation-induced DA closure through SMC migration and vasoconstriction.     

Growth-promoting effect of carbon material upon bacterial cells propagating through a distance

Carbon material such as graphite and activated charcoal, but not diamond, causes the promotion of growth of certain bacteria under ordinarily non-permissive stress conditions over a distance of several centimeters. Bacillus carboniphilus under the stress of a high KCl concentration and high temperature responded to this remote effect of carbon material with enhanced growth, and thermophile bacterium Bacillus stearothermophilus responded similarly yet moderately under the stress of low temperature. The remote effect of carbon was caused by its activation with external energy, probably of electromagnetic nature, as this effect was markedly decreased by sheltering the experimental system with an iron or aluminum barrier. Carbon material probably transforms the external oscillatory pulses or radiation into a signal exerting, far-reaching, growth-promoting effect upon cells. The most plausible candidate of signals emitted from carbon was considered to be (ultra)sonic.     

Analysis of two major intracellular phospholipases A(2) (PLA(2)) in mast cells reveals crucial contribution of cytosolic PLA(2)α, not Ca(2+)-independent PLA(2)β, to lipid mobilization in proximal mast cells and distal fibroblasts

Mast cells release a variety of mediators, including arachidonic acid (AA) metabolites, to regulate allergy, inflammation, and host defense, and their differentiation and maturation within extravascular microenvironments depend on the stromal cytokine stem cell factor. Mouse mast cells express two major intracellular phospholipases A(2) (PLA(2)s), namely group IVA cytosolic PLA(2) (cPLA(2)α) and group VIA Ca(2+)-independent PLA(2) (iPLA(2)β), and the role of cPLA(2)α in eicosanoid synthesis by mast cells has been well documented. Lipidomic analyses of mouse bone marrow-derived mast cells (BMMCs) lacking cPLA(2)α (Pla2g4a(-/-)) or iPLA(2)β (Pla2g6(-/-)) revealed that phospholipids with AA were selectively hydrolyzed by cPLA(2)α, not by iPLA(2)β, during FcεRI-mediated activation and even during fibroblast-dependent maturation. Neither FcεRI-dependent effector functions nor maturation-driven phospholipid remodeling was impaired in Pla2g6(-/-) BMMCs. Although BMMCs did not produce prostaglandin E(2) (PGE(2)), the AA released by cPLA(2)α from BMMCs during maturation was converted to PGE(2) by microsomal PGE synthase-1 (mPGES-1) in cocultured fibroblasts, and accordingly, Pla2g4a(-/-) BMMCs promoted microenvironmental PGE(2) synthesis less efficiently than wild-type BMMCs both in vitro and in vivo. Mice deficient in mPGES-1 (Ptges(-/-)) had an augmented local anaphylactic response. These results suggest that cPLA(2)α in mast cells is functionally coupled, through the AA transfer mechanism, with stromal mPGES-1 to provide anti-anaphylactic PGE(2). Although iPLA(2)β is partially responsible for PGE(2) production by macrophages and dendritic cells, it is dispensable for mast cell maturation and function.