Association between Striatal Subregions and Extrastriatal Regions in Dopamine D1 Receptor Expression: A Positron Emission Tomography Study

The mesencephalic dopamine (DA) system is the main DA system related to affective and cognitive functions. The system consists of two different cell groups, A9 and A10, which originate from different regions of the midbrain. The striatum is the main input from the midbrain, and is functionally organized into associative, sensorimotor and limbic subdivisions. At present, there have been few studies investigating the associations of DA functions between striatal subdivisions and extrastriatal regions. The aim of this study was to investigate the relationship of DA D₁ receptor (D₁R) expression between striatal subdivisions and extrastriatal regions in humans using positron emission tomography (PET) with voxel-by-voxel whole brain analysis. The PET study was performed on 30 healthy subjects using [¹¹C]{"type":"entrez-protein","attrs":{"text":"SCH23390","term_id":"1052733334","term_text":"SCH23390"}}SCH23390 to measure D₁R expression. Parametric images of binding potentials (BP _ND) were created using the simplified reference tissue model. Regions of interest were defined for striatal subdivisions. Multiple regression analysis was undertaken to determine extrastriatal regions that were associated with each striatal subdivision in BP _ND using statistical parametric mapping 5. The BP _ND values of associative, sensorimotor and limbic subdivisions were similarly correlated with those of multiple brain regions. Regarding the interrelationships among striatal subdivisions, mutual correlations were found among associative, sensorimotor and limbic subdivisions in BP _ND as well. The relationships in BP _ND between striatal subdivisions and extra-striatal regions suggest that differential striatal subdivisions and extrastriatal regions have a similar biological basis of D₁R expression. Different DA projections from the midbrain did not explain the associations between striatal subdivisions and extrastriatal regions in D₁R expression, and the DA-related neural networks among the midbrain, striatum and the other regions would contribute to a similar D₁R expression pattern throughout the whole brain.     

Biochemical characterization of a novel cycloisomaltooligosaccharide glucanotransferase from Paenibacillus sp. 598K

Cycloisomaltooligosaccharide glucanotransferase (CITase; EC 2.4.1.248), a member of the glycoside hydrolase family 66 (GH66), catalyzes the intramolecular transglucosylation of dextran to produce cycloisomaltooligosaccharides (CIs; cyclodextrans) of varying lengths. Eight CI-producing bacteria have been found; however, CITase from Bacillus circulans T-3040 (CITase-T3040) is the only CI-producing enzyme that has been characterized to date. In this study, we report the gene cloning, enzyme characterization, and analysis of essential Asp and Glu residues of a novel CITase from Paenibacillus sp. 598K (CITase-598K). The cit genes from T-3040 and 598K strains were expressed recombinantly, and the properties of Escherichia coli recombinant enzymes were compared. The two CITases exhibited high primary amino acid sequence identity (67%). The major product of CITase-598K was cycloisomaltoheptaose (CI-7), whereas that of CITase-T3040 was cycloisomaltooctaose (CI-8). Some of the properties of CITase-598K are more favorable for practical use compared with CITase-T3040, i.e., the thermal stability for CITase-598K (≤ 50 °C) was 10 °C higher than that for CITase-T3040 (≤ 40 °C); the k_cat/K_M value of CITase-598K was approximately two times higher (32.2 s^− 1 mM^− 1) than that of CITase-T3040 (17.8 s^− 1 mM^− 1). Isomaltotetraose was the smallest substrate for both CITases. When isomaltoheptaose or smaller substrates were used, a lag time was observed before the intramolecular transglucosylation reaction began. As substrate length increased, the lag time shortened. Catalytically important residues of CITase-598K were predicted to be Asp144, Asp269, and Glu341. These findings will serve as a basis for understanding the reaction mechanism and substrate recognition of GH66 enzymes.     

Cleavage mechanism and anti-tumor activity of 3,6-epidioxy-1,10-bisaboladiene isolated from edible wild plants

A bisabolane sesquiterpene endoperoxide compound, 3,6-epidioxy-1,10-bisaboladiene (EDBD), was isolated from edible wild plants grown in the northern area of Japan, Cacalia delphiniifolia and Cacalia hastata, using a mutant yeast (cdc2-1 rad9Δ). It showed cytotoxicity at IC(50) = 3.4 μM and induced apoptosis against the human promyelocytic leukemia cell line HL60 through a new stable rearrangement product (1) when in the presence of FeSO(4). This conversion mechanism is different from another sesquiterpene endoperoxide lactone compound, dihydroartemisinin (DHA), which is an anti-malarial drug. The cytotoxicity of EDBD decreased in the presence of the ferrous ion chelating drug deferoxamine mesylate (DFOM), and this suggested that the structural change of the drug caused by Fe(2+) may be responsible for its biological activities. EDBD induced apoptosis via phosphorylation of p38 mitogen-activated protein kinase (MAPK) in HL60 cells, and was detected by Western blot. EDBD resulted in an immediate increase in DCF fluorescence intensity in HL60 cells using DCFH-DA (2',7'-dichlorofluorescin diacetate) assay. The in vitro reaction of EDBD with FeSO(4) also increased DCF fluorescence intensity in a dose dependent manner. These results showed that the biological activity of EDBD involves an unstable carbon-centered radical intermediate. Furthermore, there was no similarity between the JFCR39 fingerprints of EDBD and DHA (correlation coefficient on COMPARE Analysis γ = 0.158). EDBD showed anti-tumor effects against a xenograft of Lox-IMVI cells in vivo.     

Heterologous production of bisucaberin using a biosynthetic gene cluster cloned from a deep sea metagenome

A siderophore biosynthetic gene cluster was cloned from a metagenomic library generated from deep sea sediment. The gene cluster was successfully expressed in Escherichia coli to produce bisucaberin, a siderophore originally reported from the marine bacterium Alteromonas haloplanktis. The cloned bisucaberin biosynthetic gene cluster was moderately similar to that of the known bisucaberin producer Vibrio salmonicida. However, the cloned gene cluster consists of four genes rather than three genes found in the V. salmonicida cluster. The low overall homology of the amino acid and nucleotide sequences with those of other species suggests that the cloned genes were derived from one of the unsequenced bacteria including uncultured species.     

Structure of an archaeal homolog of the human protein complex Rpp21-Rpp29 that is a key core component for the assembly of active ribonuclease P

Ribonuclease P (RNase P) is a ribonucleoprotein complex involved in the processing of the 5′-leader sequence of precursor tRNA. Human RNase P protein subunits Rpp21 and Rpp29, which bind to each other, with catalytic RNA (H1 RNA) are sufficient for activating endonucleolytic cleavage of precursor tRNA. Here we have determined the crystal structure of the complex between the Pyrococcus horikoshii RNase P proteins PhoRpp21 and PhoRpp29, the archaeal homologs of Rpp21 and Rpp29, respectively. PhoRpp21 and PhoRpp29 form a heterodimeric structure where the two N-terminal helices (α1 and α2) in PhoRpp21 predominantly interact with the N-terminal extended structure, the β-strand (β2), and the C-terminal helix (α3) in PhoRpp29. The interface is dominated by hydrogen bonds and several salt bridges, rather than hydrophobic interactions. The electrostatic potential on the surface of the heterodimer shows a positively charged cluster on one face, suggesting a possible RNA-binding surface of the PhoRpp21-PhoRpp29 complex. The present structure, along with the result of a mutational analysis, suggests that heterodimerization between PhoRpp21 and PhoRpp29 plays an important role in the function of P. horikoshii RNase P.     

Depolarization-induced differentiation of PC12 cells is mediated by phospholipase D2 through the transcription factor CREB pathway

The present study examined the role of phospholipase D2 (PLD2) in the regulation of depolarization-induced neurite outgrowth and the expression of growth-associated protein-43 (GAP-43) and synapsin I in rat pheochromocytoma (PC12) cells. Depolarization of PC12 cells with 50 mmol/L KCl increased neurite outgrowth and elevated mRNA and protein expression of GAP-43 and synapsin I. These increases were suppressed by inhibition of Ca²⁺-calmodulin-dependent protein kinase II (CaMKII), PLD, or mitogen-activated protein kinase kinase (MEK). Knockdown of PLD2 by small interfering RNA (siRNA) suppressed the depolarization-induced neurite outgrowth, and the increase in GAP-43 and synapsin I expression. Depolarization evoked a Ca²⁺ rise that activated various signaling enzymes and the cAMP response element-binding protein (CREB). Silencing CaMKIIδ by siRNA blocked KCl-induced phosphorylation of proline-rich protein tyrosine kinase 2 (Pyk2), Src kinase, and extracellular signal-regulated kinase (ERK). Inhibition of Src or MEK abolished phosphorylation of ERK and CREB. Furthermore, phosphorylation of Pyk2, ERK, and CREB was suppressed by the PLD inhibitor, 1-butanol and transfection of PLD2 siRNA, whereas it was enhanced by over-expression of wild-type PLD2. Depolarization-induced PLD2 activation was suppressed by CaMKII and Src inhibitors, but not by MEK or protein kinase A inhibitors. These results suggest that the signaling pathway of depolarization-induced PLD2 activation was downstream of CaMKIIδ and Src, and upstream of Pyk2(Y881) and ERK/CREB, but independent of the protein kinase A. This is the first demonstration that PLD2 activation is involved in GAP-43 and synapsin I expression during depolarization-induced neuronal differentiation in PC12 cells.     

Observations on Cryptosporidium life cycle stages during excystation

Cryptosporidium parvum (HNJ-1 strain, genotype 2) merozoites were released from oocysts directly during an incubation and excystation procedure without bleach treatment. They were polymorphic, mostly spindle-shaped; others were bean shaped, actively motile, and underwent division. Merozoites survived for short time-period in an in vitro culture system, but could not be established in a subsequent cultivation effort in RPMI medium.