Neuronal roles of the integrin-associated protein (IAP/CD47) in developing cortical neurons

Little is known about the role of the integrin-associated protein (IAP, or CD47) in neuronal development and its function in the central nervous system. We investigated neuronal responses in IAP-overexpressing cortical neurons using a virus-gene transfer system. We found that dendritic outgrowth was significantly enhanced in IAP (form 4)-transfected neurons. Furthermore, synaptic proteins including synaptotagmin, syntaxin, synapsin I, and SNAP25 (25-kDa synaptosomal associated protein) were up-regulated. In accordance with this finding, the release of the excitatory transmitter glutamate and the frequencies of Ca2+ oscillations (glutamate-mediated synaptic transmission) were increased. Interestingly, the overexpression of IAP activated mitogen-activated protein kinase (MAPK), and this activation was required for the IAP-dependent biological effects. After down-regulation of the endogenous IAP by small interfering RNA, MAPK activity, synaptic protein levels, and glutamate release decreased. These observations suggest that the IAP plays important roles in dendritic outgrowth and synaptic transmission in developing cortical neurons through the activation of MAPK.     

Langerhans cells exhibit low responsiveness to double-stranded RNA

Double-stranded RNA (dsRNA) is a viral product recognized by Toll-like receptor 3 (TLR3), and it is a potent activator of dendritic cells (DC). We compared Langerhans cells (LC) and splenic CD11c(+) DC and investigated the responsiveness to dsRNA. We prepared highly purified LC (> 95%) using the panning method. TLR3 mRNA was expressed in LC, splenic DC, and keratinocytes (KC). The expression of IFN-beta mRNA was enhanced in LC and splenic DC by Poly(I:C) stimulation. However, cytokine/chemokine production in response to Poly(I:C) by LC was much lower than that by splenic DC. In addition, Poly(I:C) induced further maturation in splenic DC, but not in LC. Finally, we found that the mouse KC cell line, PAM212, produced a great amount of IL-1alpha by Poly(I:C) stimulation, and that IL-1alpha promoted the maturation of LC. These data altogether indicate that LC exhibit low responsiveness to dsRNA. It is possible that KC may primarily trigger anti-viral immune responses in the skin via cytokine production such as IL-1alpha.     

Contribution of bone-marrow-derived cells to choroidal neovascularization

We investigated the involvement of bone-marrow derived cells to experimental choroidal neovascularization (CNV) in mice, whose bone marrow was reconstituted by either unfractionated bone-marrow cells or Lin-c(-)Kit(+)Sca-1+ enriched presumable hematopoietic stem cells from the green fluorescent protein (GFP) transgeneic mice. Immunohistochemical analysis demonstrated the presence of GFP-positive cells in the CNV lesion after unfractionated bone-marrow transplantation, as well as Lin-c(-)Kit(+)Sca-1+ cell transplantation. Some of the GFP-expressing cells also expressed CD-31 and PanEC antigen, markers of vascular endothelial cells. Our results suggest that bone-marrow derived cells may contribute endothelial cells in CNV.     

Phosphatidylinositol 3-kinase is involved in alpha2(I) collagen gene expression in normal and scleroderma fibroblasts

TGF-beta is implicated in the pathogenesis of fibrotic disorders. It has been shown that Smad3 promotes the human alpha2(I) collagen (COL1A2) gene expression by TGF-beta1 in human dermal fibroblasts. Here, we investigated the role of phosphatidylinositol 3-kinase (PI3K) in the COL1A2 gene expression in normal and scleroderma fibroblasts. In normal fibroblasts, the PI3K inhibitor, LY294002, significantly decreased the basal and the TGF-beta1-induced increased stability of COL1A2 mRNA. The TGF-beta1-induced COL1A2 promoter activity, but not the basal activity, was significantly attenuated by LY294002 or the dominant negative mutant of p85 subunit of PI3K, while the constitutive active mutant of p110 subunit of PI3K did not affect the basal or the TGF-beta1-induced COL1A2 promoter activity. LY294002 significantly decreased the phosphorylation of Smad3 induced by TGF-beta1. Furthermore, the transient overexpression of 2xFYVE, which induces the mislocalization of FYVE domain proteins, decreased the TGF-beta1-induced Smad3 phosphorylation to a similar extent to LY294002. In scleroderma fibroblasts, the blockade of PI3K significantly decreased the mRNA stability and the promoter activity of the COL1A2 gene. Furthermore, LY294002 and the transient overexpression of 2xFYVE completely diminished the constitutive phosphorylation of Smad3. These results indicate that 1) the basal activity of PI3K is necessary for the COL1A2 mRNA stabilization in normal and scleroderma fibroblasts, 2) there is an unidentified FYVE domain protein specifically interacting with Smad3, and 3) the basal activity of PI3K and the FYVE domain protein are indispensable for the efficient TGF-beta/Smad3 signaling in normal fibroblasts and for the establishment of the constitutive activation of TGF-beta/Smad3 signaling in scleroderma fibroblasts.     

Pantothenate Kinase from the Thermoacidophilic Archaeon Picrophilus torridus

Pantothenate kinase (CoaA) catalyzes the first step of the coenzyme A (CoA) biosynthetic pathway and controls the intracellular concentrations of CoA through feedback inhibition in bacteria. An alternative enzyme found in archaea, pantoate kinase, is missing in the order Thermoplasmatales. The PTO0232 gene from Picrophilus torridus, a thermoacidophilic euryarchaeon, is shown to be a distant homologue of the prokaryotic type I CoaA. The cloned gene clearly complements the poor growth of the temperature-sensitive Escherichia coli CoaA mutant strain ts9, and the recombinant protein expressed in E. coli cells transfers phosphate to pantothenate at pH 5 and 55°C. In contrast to E. coli CoaA, the P. torridus enzyme is refractory to feedback regulation by CoA, indicating that in P. torridus cells the CoA levels are not regulated by the CoaA step. These data suggest the existence of two subtypes within the class of prokaryotic type I CoaAs.Coenzyme A (CoA) is an essential cofactor synthesized from pantothenate (vitamin B₅), cysteine, and ATP (1, 20, 30). The thiol group derived from the cysteine moiety in a CoA molecule forms a thioester bond, which is a high-energy bond, with carboxylates including fatty acids. The resulting compounds are called acyl-CoAs (CoA thioesters) and function as the major acyl group carriers in numerous metabolic and energy-yielding pathways. Since it is thought that the pantetheine moiety in CoA existed when life first came about on Earth (25) and at present, a CoA, acyl-CoA, or 4′-phosphopantethein moiety that is common to CoA and acyl carrier proteins is utilized by about 4% of all enzymes as a substrate (6), these compounds are thought to play a crucial role in the earliest metabolic system.Bacteria, fungi, and plants can produce pantothenate, which is the starting material of CoA biosynthesis, although animals must take it from their diet (41). The canonical CoA biosynthetic pathway consists of five enzymatic steps: i.e., pantothenate kinase (CoaA in prokaryotes and PanK in eukaryotes; EC 2.7.1.33), phosphopantothenoylcysteine synthetase (CoaB; EC 6.3.2.5), phosphopantothenoylcysteine decarboxylase (CoaC: EC 4.1.1.36), phosphopantetheine adenylyltransferase (CoaD; EC 2.7.7.3), and dephospho-CoA kinase (CoaE; EC 2.7.1.24). The organisms belonging to the domains Bacteria and Eukarya have this pathway (20, 30). CoaB, CoaC, CoaD, and CoaE are detectable in the complete genome sequences as orthologs of the counterparts from E. coli and humans (15, 16, 32). However, there is diversity among the CoaAs and PanKs, depending on their primary structures, and to date, three types of CoaA in bacteria and one type of PanK in eukaryotes have been identified. CoaAs and PanK catalyze the phosphorylation of pantothenate to produce 4′-phosphopantothenate at the first step of the pathway. First, the Escherichia coli CoaA (CoaA_Ec) was cloned as a prokaryotic type I CoaA after characterization of the properties enzymatically (42-44, 48). Thereafter, the eukaryotic PanK isoforms were isolated from Aspergillus nidulans (AnPanK), mice (mPanK), and humans (hPanK) (10, 17, 28, 29, 33, 34, 54-56). These enzyme activities were clearly regulated by end products of the biosynthetic pathway such as CoA, acetyl-CoA, and malonyl-CoA, and the pantothenate kinases governed the intracellular concentrations of CoA and acyl-CoAs (10, 17, 28, 29, 33, 34, 43, 44, 48, 54, 55). However, CoaAs insensitive to CoA and acyl-CoAs were recently identified from Staphylococcus aureus (CoaA_Sa), Pseudomonas aeruginosa (CoaA_Pa), and Helicobacter pylori (CoaA_Hp) as prokaryotic type II and III CoaAs (9, 11, 18, 27). The structural and functional diversity among pantothenate kinases suggests that they are key indicators of the regulation of the CoA biosynthesis. In archaea neither CoaA nor pantothenate synthetase (PanC; EC 6.3.2.1), which catalyzes the condensation of pantoate and β-alanine to produce pantothenate, had been identified biochemically until very recently. COG1829 and COG1701 were assigned as the respective candidates based on comparative genomic analysis (15). COG1701 was reported to be PanC (36), and later the enzyme was revised to phosphopantothenate synthetase, which catalyzed the condensation of phosphopantoate and β-alanine (52). Together with the identification of COG1701, COG1829 was found to be pantoate kinase, responsible for the phosphorylation of pantoate (52). Homologues of pantoate kinase and phosphopantothenate synthetase are found in most archaeal genomes, thus establishing a noncanonical CoA biosynthetic pathway involving the two novel enzymes. However, homologues of the two novel enzymes are missing in the order Thermoplasmatales.Hence, we proceeded with a search for the kinase genes of the remaining archaea to elucidate the regulatory mechanism(s) underlying archaeal CoA biosynthesis. The PTO0232 gene in the complete genome sequence of Picrophilus torridus was identified as encoding a distant homologue of CoaA_Ec by a BLAST search. The recombinant protein phosphorylated pantothenate, but the activity was not inhibited at all by CoA or CoA thioesters despite its classification as prokaryotic type I CoaA. This functional difference between P. torridus CoaA (CoaA_Pt) and CoaA_Ec can be accounted for by an amino acid substitution at position 247 which possibly interacts with CoA. Here we describe the existence of a second subtype in the class of prokaryotic type I CoaAs.     

Suppression of Adipocyte Differentiation by Aldo-keto Reductase 1B3 Acting as Prostaglandin F2�� Synthase

Prostaglandin (PG) F_2α suppresses adipocyte differentiation by inhibiting the function of peroxisome proliferator-activated receptor γ. However, PGF_2α synthase (PGFS) in adipocytes remains to be identified. Here, we studied the expression of members of the aldo-keto reductase (AKR) 1B family acting as PGFS during adipogenesis of mouse 3T3-L1 cells. AKR1B3 mRNA was expressed in preadipocytes, and its level increased about 4-fold at day 1 after initiation of adipocyte differentiation, and then quickly decreased the following day to a level lower than that in the preadipocytes. In contrast, the mRNA levels of Akr1b8 and 1b10 were clearly lower than that level of Akr1b3 in preadipocytes and remained unchanged during adipogenesis. The transient increase in Akr1b3 during adipogenesis was also observed by Western blot analysis. The mRNA for the FP receptor, which is selective for PGF_2α, was also expressed in preadipocytes. Its level increased about 2-fold within 1 h after the initiation of adipocyte differentiation and was maintained at almost the same level throughout adipocyte differentiation. The small interfering RNA for Akr1b3, but not for Akr1b8 or 1b10, suppressed PGF_2α production and enhanced the expression of adipogenic genes such as peroxisome proliferator-activated receptor γ, fatty acid-binding protein 4 (aP2), and stearoyl-CoA desaturase. Moreover, an FP receptor agonist, Fluprostenol, suppressed the expression of those adipogenic genes in 3T3-L1 cells; whereas an FP receptor antagonist, AL-8810, efficiently inhibited the suppression of adipogenesis caused by the endogenous PGF_2α. These results indicate that AKR1B3 acts as the PGFS in adipocytes and that AKR1B3-produced PGF_2α suppressed adipocyte differentiation by acting through FP receptors.     

Composition of the bacterial flora in tonsilloliths

Tonsilloliths are a potential cause of oral malodor. In this study, microbial profiles and composition of tonsilloliths were determined using culture-independent molecular methods and scanning electron microscopy. 16S ribosomal RNA bacterial genes (16S rDNAs) isolated from tonsilloliths of 6 individuals were amplified by PCR and cloned into Escherichia coli. Partial 16S rDNA sequences of approximately 600 bases of cloned inserts were used to determine species identity by comparison with sequences of known species. Characteristics of bacteria on the surface and inside the tonsillolith were analyzed using scanning electron microscopy. Anaerobic bacteria detected in tonsilloliths belonged to the genera Eubacterium, Fusobacterium, Megasphaera, Porphyromonas, Prevotella, Selenomonas and Tannerella, all of which appear to be associated with production of volatile sulfur compounds. Electron microscopy revealed cocci and rods on the surface and rods predominating inside the tonsilloliths. These results support the tonsillolith as an origin of oral malodor.