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.     

Loss of calcineurin homologous protein-1 in chicken B lymphoma DT40 cells destabilizes Na+/H+ exchanger isoform-1 protein

NHE1/SLC9A1 is a ubiquitous isoform of vertebrate Na⁺/H⁺ exchangers (NHEs) functioning in maintaining intracellular concentrations of Na⁺ and H⁺ ions. Calcineurin homologous protein-1 (CHP1) binds to the hydrophilic region of NHE1 and regulates NHE1 activity but reportedly does not play a role in translocating NHE1 from the endoplasmic reticulum to the plasma membrane. However, an antiport function of NHE1 requiring CHP1 remains to be clarified. Here we established CHP1-deficient chicken B lymphoma DT40 cells by gene targeting to address CHP1 function. CHP1-deficient cells showed extensive decreases in Na⁺/H⁺ activities in intact cells. Although NHE1 mRNA levels were not affected, NHE1 protein levels were significantly reduced not only in the plasma membrane but in whole cells. The expression of a CHP1 transgene in CHP1-deficient cells rescued NHE1 protein expression. Expression of mutant forms of CHP1 defective in Ca²⁺ binding or myristoylation also partially decreased NHE1 protein levels. Knockdown of CHP1 also caused a moderate decrease in NHE1 protein in HeLa cells. These data indicate that CHP1 primarily plays an essential role in stabilization of NHE1 for reaching of NHE1 to the plasma membrane and its exchange activity. membrane protein; transporter; antiporter; quality control; degradation     

Repellent from traditional Chinese medicine, Periploca sepium Bunge

By using a new bioassay-guided method, 2-hydroxy-4-methoxybenzaldehyde isolated from the root bark of Periploca sepium, a traditional Chinese medicine, showed repellent activity against the olive weevil (Dyscerus perforatus) at 62.5, 125, 250 and 500 microg/disc, respectively. In addition, it also exhibited antinematodal activity against Bursaphelenchus xylophilus at a minimum effective dose of 200 microg/ball. The three related compounds obtained were also evaluated for the above-mentioned bioactivities.     

N-haloacetylimino neonicotinoids: potency and molecular recognition at the insect nicotinic receptor

This structure-activity relationship study for neonicotinoids with an N-haloacetylimino pharmacophore identifies several candidate compounds showing outstanding insecticidal potency and consequently leads to establishing their molecular recognition at an insect nicotinic receptor structural model, wherein the neonicotinoid halogen atoms (fluorine, chlorine, bromine, and iodine) variously interact with the receptor loops C-D interfacial niche via H-bonding and/or hydrophobic interactions.     

Expression, purification and characterization of isoforms of peripheral stalk subunits of human V-ATPase

The vacuolar-type H+-ATPase (V-ATPase) is a multi-subunit proton pump that is involved in both intra- and extracellular acidification processes throughout human body. Subunits constituting the peripheral stalk of the V-ATPase are known to have several isoforms responsible for tissue/cell specific different physiological roles. To study the different interaction of these isoforms, we expressed and purified the isoforms of human V-ATPase peripheral stalk subunits using Escherichia coli cell-free protein synthesis system: E1, E2, G1, G2, G3, C1, C2, H and N-terminal soluble part of a1 and a2 isoforms. The purification conditions were different depending on the isoforms, maybe reflecting the isoform specific biochemical characteristics. The purified proteins are expected to facilitate further experiments to study about the cell specific interaction and regulation and thus provide insight into physiological meaning of the existence of several isoforms of each subunit in V-ATPase.     

Identification of Rab GTPase-activating protein-like protein (RabGAPLP) as a novel Alix/AIP1-interacting protein

Alix/AIP1 is a multifunctional adaptor protein involved in endocytosis, cell adhesion, and cell death. By yeast two-hybrid screening we identified a novel Alix/AIP1-interacting protein named Rab GTPase-activating protein-like protein (RabGAPLP). Interaction between Alix and RabGAPLP was confirmed by pull-down assays using fusion proteins of either glutathione-S-transferase (GST) or chitin-binding domain (CBD) and lysates of cultured mammalian cells expressing the respective proteins. Partial colocalization of FLAG-tagged RabGAPLP and green fluorescent protein (GFP)-fused Alix was observed at cell edges and filopodia-like structures by fluorescence confocal laser scanning microscopic analysis. The identity of RabGAPLP to merlin-associated protein (MAP), one of the interacting partners of neurofibromatosis type 2 (NF2) tumor suppressor gene product (merlin), implies cross-talk of membrane traffic and cell adhesion.     

Dysplastic development of seminiferous tubules and interstitial tissue in rat hypogonadic (hgn/hgn) testes

The hypogonadic rat is characterized by male sterility, reduced female fertility, and renal hypoplasia controlled by a single recessive allele (hgn) on chromosome 10. Plasma testosterone is low and levels of gonadotropins are high in adult male hgn/hgn rats, indicating that the cause of hypogonadism lies within the testis itself. We found that the postnatal growth of the seminiferous tubules was severely affected. Here we describe the details of postnatal testicular pathogenesis of the hgn/ hgn rats. In these rats, gonadal sex determination and initial differentiation of each type of testicular cell occur, but proliferation, differentiation, and maturation of these cells during postnatal testicular development is severely affected. Postnatal pathological changes include reduced proliferation and apoptotic cell death of Sertoli cells, abnormal mitosis and cell death of gonocytes, reduced deposition of extracellular matrix proteins into the basal lamina, lack of the formation of an outer basal lamina, formation of multiple layers of undifferentiated peritubular cells, and the delayed appearance and islet conformation of adult-type Leydig cells. Apoptotic cell death of Sertoli cells and disappearance of FSH receptor mRNA expression indicate that this mutant rat is a useful model for Sertoli cell dysfunction. The abnormalities listed above might be caused by defective interactions between Sertoli cells and other types of testicular cells. Because the results presented here strongly indicate that a normal allele for hgn encodes a factor playing a critical role in testicular development, the determination of the gene responsible for hgn and the analysis of early alterations of gene expression caused by mutations in this gene would provide important information on the mechanisms of testicular development.     

Monocarboxylate transporters in subsarcolemmal and intermyofibrillar mitochondria

Whether subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria contain monocarboxylate transporters (MCTs) is controversial. We have examined the presence of MCT1, 2, and 4 in highly purified SS and IMF mitochondria. These mitochondria were not contaminated with plasma membrane, sarcoplasmic reticulum or endosomal compartments, as the marker proteins for these sub-cellular compartments (Na+-K+-ATPase, Ca2+-ATPase, and the transferrin receptor) were not present in SS or IMF mitochondria. MCT1, MCT2, and MCT4 were all present at the plasma membrane. However, MCT1 and MCT4 were associated with SS mitochondria. In contrast, the IMF mitochondria were completely devoid of MCT1 and MCT4. However, MCT2 was associated with both SS and IMF mitochondria. These observations suggest that SS and IMF mitochondria have different capacities for metabolizing monocarboxylates. Thus, the controversy as to whether mitochondria can take up and oxidize lactate will need to take account of the different distribution of MCTs between SS and IMF mitochondria.