Pharmacological assessment of methamphetamine-induced behavioral hyperactivity mediated by dopaminergic transmission in planarian Dugesia japonica

The freshwater planarian Dugesia japonica has a simple central nervous system (CNS) and can regenerate complete organs, even a functional brain. Recent studies demonstrated that there is a great variety of neuronal-related genes, specifically expressed in several domains of the planarian brain. We identified a planarian dat gene, named it D. japonica dopamine transporter (Djdat), and analyzed its expression and function. Both in situ hybridization and immunofluorescence revealed that localization of Djdat mRNA and protein was the same as that of D. japonica tyrosine hydroxylase (DjTH). Although, dopamine (DA) content in Djdat(RNAi) planarians was not altered, Djdat(RNAi) planarians showed increased spontaneous locomotion. The hyperactivity in the Djdat(RNAi) planarians was significantly suppressed by SCH23390 or sulpiride pretreatment, which are D₁ or D₂ receptor antagonists, respectively. These results suggest that planarians have a Djdat ortholog and the ability to regulate dopaminergic neurotransmission and association with spontaneous locomotion.     

BRAF Drives Synovial Fibroblast Transformation in Rheumatoid Arthritis

Synovial fibroblasts destroy articular cartilage and bone in rheumatoid arthritis, but the mechanism of fibroblast transformation remains elusive. Because gain-of-function mutations of BRAF can transform fibroblasts, we examined BRAF in rheumatoid synovial fibroblasts. The strong gain-of-function mutation, V600R, of BRAF found in melanomas and other cancers was identified in first passage synovial fibroblasts from two of nine rheumatoid arthritis patients and confirmed by restriction site mapping. BRAF-specific siRNA inhibited proliferation of synovial fibroblasts with V600R mutations. A BRAF aberrant splice variant with an intact kinase domain and partial loss of the N-terminal autoinhibitory domain was identified in fibroblasts from an additional patient, and fibroblast proliferation was inhibited by BRAF-specific siRNA. Our finding is the first to establish mechanisms for fibroblast transformation responsible for destruction of articular cartilage and bone in rheumatoid arthritis and establishes a new target for therapeutic intervention.     

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.     

Tripeptidic BACE1 inhibitors devised by in-silico conformational structure-based design

Previously reported pentapeptidic BACE1 inhibitors, designed using a substrate-based approach, were used as lead compounds for the further design of non-peptidic BACE1 inhibitors. Although these peptidic and non-peptidic inhibitors, with a hydroxymethylcarbonyl isostere as a substrate transition-state mimic, exhibited potent BACE1 inhibitory activities, their molecular-sizes appeared a little too big (molecular weight of >600daltons) for developing practical anti-Alzheimer's disease drugs. To develop lower weight BACE1 inhibitors, a series of tripeptidic BACE1 inhibitors were devised using a design approach based on the conformation of a virtual inhibitor bound to the BACE1 active site, also called 'in-silico conformational structure-based design'. Although these tripeptidic BACE1 inhibitors contained some natural amino acid residues, they are expected to be useful as lead compounds for developing the next generation BACE1 inhibitors, due to their low molecular size and unique structural features compared with previously reported inhibitors.     

Glycoform-dependent conformational alteration of the Fc region of human immunoglobulin G1 as revealed by NMR spectroscopy

The Fc portion of immunoglobulin G (IgG) expresses the biantennary complex type oligosaccharides at Asn297 of the C(H)2 domain of each heavy chain with microheterogeneities depending on physiological and pathological states. These N-glycans are known to be essential for promotion of proper effector functions of IgG such as complement activation and Fcgamma receptor (FcgammaR)-mediated activities. To gain a better understanding of the role of Fc glycosylation, we prepared a series of truncated glycoforms of human IgG1-Fc and analyzed their interactions with human soluble FcgammaRIIIa (sFcgammaRIIIa) and with staphylococcal protein A by surface plasmon resonance and nuclear magnetic resonance (NMR) methods. Progressive but less pronounced reductions in the affinity for sFcgammaRIIIa were observed as a result of the galactosidase and subsequent N-acetylhexosaminidase treatments of IgG1-Fc. The following endoglycosidase D treatment, giving rise to a disaccharide structure composed of a fucosylated GlcNAc, abrogated the affinity of IgG1-Fc for sFcgammaRIIIa. On the other hand, those glycosidase treatments did not significantly affect the affinity of IgG1-Fc for protein A. Inspection of stable-isotope-assisted NMR data of a series of Fc glycoforms indicates that the stepwise trimming out of the carbohydrate residues results in concomitant increase in the number of amino acid residues perturbed thereby in the C(H)2 domains. Furthermore, the cleavage at the GlcNAcbeta1-4GlcNAc glycosidic linkage induced the conformational alterations of part of the lower hinge region, which makes no direct contact with the carbohydrate moieties and forms the major FcgammaR-binding site, while the conformation of the C(H)2/C(H)3 interface was barely perturbed that is the protein A-binding site. These results indicate that the carbohydrate moieties are required for maintaining the structural integrity of the FcgammaR-binding site.     

The budget of dissolved trace metals in Lake Biwa,Japan

A comprehensive study on the dynamics of dissolved elements (Mg, Al, Si, P, Ca, V, Cr, Mn, Fe, Ni, Zn, As, Sr, Y, W, and U) in Lake Biwa was carried out using a clean technique. Lake water samples (n = 523) were collected from six stations in the North Basin and three stations in the South Basin. River water samples (n = 178) were collected from 14 major rivers flowing into the North Basin. Rainwater samples (n = 89) were collected at Otsu. The river water was enriched with Mn, Al, Fe, P, and Zn and the rainwater was enriched with Zn, Al, Fe, and Mn compared to North Basin water during winter mixing. The residence times of dissolved species were estimated on the basis of input through the rivers and rain. The residence times for Ca, Mg, and Sr were about 8 years, the same as that for water. Mn, Al, Fe, and Zn showed the shortest residence times (0.05–0.19 year). A budget calculation suggested that more than 60% of the input of dissolved Si, P, V, Cr, Mn, Fe, Ni, and Zn was scavenged and retained in the lake sediments and/or discharged as suspended particles.     

NPH3- and PGP-like genes are exclusively expressed in the apical tip region essential for blue-light perception and lateral auxin transport in maize coleoptiles

Phototropic curvature results from differential growth on two sides of the elongating shoot, which is explained by asymmetrical indole-3-acetic acid (IAA) distribution. Using 2 cm maize coleoptile segments, 1st positive phototropic curvature was confirmed here after 8 s irradiation with unilateral blue light (0.33 μmol m(-2) s(-1)). IAA was redistributed asymmetrically by approximately 20 min after photo-stimulation. This asymmetric distribution was initiated in the top 0-3 mm region and was then transmitted to lower regions. Application of the IAA transport inhibitor, 1-N-naphthylphthalamic acid (NPA), to the top 2 mm region completely inhibited phototropic curvature, even when auxin was simultaneously applied below the NPA-treated zone. Thus, lateral IAA movement occurred only within the top 0-3 mm region after photo-stimulation. Localized irradiation experiments indicated that the photo-stimulus was perceived in the apical 2 mm region. The results suggest that this region harbours key components responsible for photo-sensing and lateral IAA transport. In the present study, it was found that the NPH3- and PGP-like genes were exclusively expressed in the 0-2 mm region of the tip, whereas PHOT1 and ZmPIN1a, b, and c were expressed relatively evenly along the coleoptile, and ZmAUX1, ZMK1, and ZmSAURE2 were strongly expressed in the elongation zone. These results suggest that the NPH3-like and PGP-like gene products have a key role in photo-signal transduction and regulation of the direction of auxin transport after blue light perception by phot1 at the very tip region of maize coleoptiles.