The protein-tyrosine phosphatase PTPMEG interacts with glutamate receptor delta 2 and epsilon subunits

Glutamate receptor (GluR) delta2 is selectively expressed in cerebellar Purkinje cells and plays a crucial role in cerebellum-dependent motor learning. Although GluRdelta2 belongs to an ionotropic GluR family, little is known about its pharmacological features and downstream signaling cascade. To study molecular mechanisms underlying GluRdelta2-dependent motor learning, we employed yeast two-hybrid screening to isolate GluRdelta2-interacting molecules and identified protein-tyrosine phosphatase PTPMEG. PTPMEG is a family member of band 4.1 domain-containing protein-tyrosine phosphatases and is expressed prominently in brain. Here, we showed by in situ hybridization analysis that the PTPMEG mRNA was enriched in mouse thalamus and Purkinje cells. We also showed that PTPMEG interacted with GluRdelta2 as well as with N-methyl-d-aspartate receptor GluRepsilon1 in cultured cells and in brain. PTPMEG bound to the putative C-terminal PDZ target sequence of GluRdelta2 and GluRepsilon1 via its PDZ domain. Examination of the effect of PTPMEG on tyrosine phosphorylation of GluRepsilon1 unexpectedly revealed that PTPMEG enhanced Fyn-mediated tyrosine phosphorylation of GluRepsilon1 in its PTPase activity-dependent manner. Thus, we conclude that PTPMEG associates directly with GluRdelta2 and GluRepsilon1. Moreover, our data suggest that PTPMEG plays a role in signaling downstream of the GluRs and/or in regulation of their activities through tyrosine dephosphorylation.     

Cytotoxic cycloartane-type triterpenes from Combretum quadrangulare

Seven novel cycloartane-type triterpenes were isolated from Combretum quadrangulare, and their structures were elucidated on the basis of spectral analysis. All these compounds were tested for their cytotoxicity against murine colon 26-L5 carcinoma cells. Methyl quadrangularate B (2) and methyl quadrangularate D (4) exhibited potent cytotoxicity having ED₅₀ values 9.54 and 5.42 μM, respectively.     

Acetylcholine promotes the emergence and elongation of lateral roots of Raphanus sativus

Radish (Raphanus sativus L.) was grown on four layers of paper towel moistened with distilled water with and without acetylcholine (ACh) for five days in the dark after sowing. ACh at 1 nM promoted the growth (emergence and elongation) of lateral roots of radish plants, but had no effect on the stems and main roots. Moreover, ACh enhanced the dry weight of roots [main (primary) + lateral roots]. Neostigmine, an inhibitor of acetylcholinesterase (AChE) also promoted the emergence and elongation of lateral roots, and atropine, a competitive inhibitor of ACh receptor, suppressed the emergence and elongation. ACh promoted the activities of glyceraldehyde-3-phosephate dehydrogenase (G-3-PD), nicotinamide adenine dinucleotide-specific isocitrate dehydrogenase (NAD-ICDH), succinate dehydrogenase (SDH) and cytochrome-c oxidase (Cyt-c OD) in seedlings. Moreover, ACh suppressed the activity of AChE and increased the amount of proteins and pyridine nucleotides (NAD and NADH) in the roots of the seedlings. It also increased the activities of NAD-forming enzymes [NAD synthetase and ATP-nicotinamide mononucleotide (ATP-NMN) adenyltransferase], and enhanced the amount of DNA in the roots of the seedlings. The relationship between ACh and the emergence and growth of lateral roots was discussed from a biochemical viewpoint.Key words: acetylcholine, acetylcholine esterase, DNA, lateral root, NAD, NAD-forming enzyme, protein, Raphanus sativus     

Pseudo-Response Regulator (PRR) Homologues of the Moss Physcomitrella patens: Insights into the Evolution of the PRR Family in Land Plants

The pseudo-response regulators (PRRs) are the circadian clock component proteins in the model dicot Arabidopsis thaliana. They contain a receiver-like domain (RLD) similar to the receiver domains of the RRs in the His–Asp phosphorelay system, but the RLDs lack the phosphoacceptor aspartic acid residue invariably conserved in the receiver domains. To study the evolution of PRR genes in plants, here we characterize their homologue genes, PpPRR1, PpPRR2, PpPRR3 and PpPRR4, from the moss Physcomitrella patens. In the phylogenetic analysis, PpPRRs cluster together, sister to an angiosperm PRR gene subfamily, illustrating their close relationships with the angiosperm PRRs. However, distinct from the angiosperm sequences, the RLDs of PpPRR2/3/4 exhibit a potential phosphoacceptor aspartic acid–aspartic acid–lysine (DDK) motif. Consistently, the PpPRR2 RLD had phosphotransfer ability in vitro, suggesting that PpPRR2 functions as an RR. The PpPRR1 RLD, on the other hand, shows a partially diverged DDK motif, and it did not show phosphotransfer ability. All PpPRRs were expressed in a circadian and light-dependent manner, with differential regulation between PpPRR2/4 and PpPRR1/3. Altogether, our results illustrate that PRRs originated from an RR(s) and that there are intraspecific divergences among PpPRRs. Finally, we offer scenarios for the evolution of the PRR family in land plants.     

The crystal structure of polyhydroxybutyrate depolymerase from Penicillium funiculosum provides insights into the recognition and degradation of biopolyesters

Polyhydroxybutyrate is a microbial polyester that can be produced from renewable resources, and is degraded by the enzyme polyhydroxybutyrate depolymerase. The crystal structures of polyhydroxybutyrate depolymerase from Penicillium funiculosum and its S39 A mutant complexed with the methyl ester of a trimer substrate of (R)-3-hydroxybutyrate have been determined at resolutions of 1.71 A and 1.66 A, respectively. The enzyme is comprised of a single domain, which represents a circularly permuted variant of the alpha/beta hydrolase fold. The catalytic residues Ser39, Asp121, and His155 are located at topologically conserved positions. The main chain amide groups of Ser40 and Cys250 form an oxyanion hole. A crevice is formed on the surface of the enzyme, to which a single polymer chain can be bound by predominantly hydrophobic interactions with several hydrophobic residues. The structure of the S39A mutant-trimeric substrate complex reveals that Trp307 is responsible for the recognition of the ester group adjacent to the scissile group. It is also revealed that the substrate-binding site includes at least three, and possibly four, subsites for binding monomer units of polyester substrates. Thirteen hydrophobic residues, which are exposed to solvent, are aligned around the mouth of the crevice, forming a putative adsorption site for the polymer surface. These residues may contribute to the sufficient binding affinity of the enzyme for PHB granules without a distinct substrate-binding domain.     

Hypoglycemic effects of the wood of Taxus yunnanensis on streptozotocin-induced diabetic rats and its active components

Hypoglycemic effects of the H(2)O and MeOH extracts of the wood of Taxus yunnanensis were examined in streptozotocin (STZ)-induced diabetic rats. The H(2)O extract significantly lowered the fasting blood glucose level by 33.7% at a 100mg/kg dose on intraperitoneal administration. From the active H(2)O extract of the wood, three lignans, i.e., isotaxiresinol (1), secoisolariciresinol (2) and taxiresinol (3), were isolated as major components. These lignans were further tested for their hypoglycemic effects on the same experimental model. At a dose of 100mg/kg (i.p.), isotaxiresinol (1) reduced the fasting blood glucose level of diabetic rats by 34.5%, while secoisolariciresinol (2) and taxiresinol (3) reduced by 33.4% and 20.9%, respectively. The blood glucose lowering effects of 1 and 2 were stronger than the mixture of tolbutamide (200mg/kg) and buformin (1mg/kg) used as a positive control, which lowered fasting blood glucose level by 24.0%.     

In vivo anti-osteoporotic activity of isotaxiresinol, a lignan from wood of Taxus yunnanensis

Isotaxiresinol, the main lignan isolated from the water extract of wood of Taxus yunnanensis, was investigated for its effect on bone loss, on serum biochemical markers for bone remodeling and on uterine tissue, using ovariectomized (OVX) rats as the model of postmenopausal osteoporosis. After oral administration of isotaxiresinol (50 and 100mg/kg/d) for 6 weeks, bone mineral content (BMC) and bone mineral density (BMD) in total and cortical bones were increased as compared to those of OVX control rats, and decreases of three bone strength indexes induced by OVX surgery were prevented. Serum biochemical markers for bone remodeling revealed that isotaxiresinol slightly increased bone formation and significantly inhibited bone resorption without side effect on uterine tissue. These results suggest that isotaxiresinol may be useful for treatment of postmenopausal osteoporosis, especially for prevention of bone fracture induced by estrogen deficiency.     

Molecular cloning and gene expression of endoplasmic reticulum stress proteins in Japanese monkey, Macaca fuscata

BACKGROUND: Immunoglobulin heavy-chain binding protein (BiP), calreticulin (Crt), and protein disulfide isomerase (PDI), are major resident endoplasmic reticulum (ER) stress proteins which are involved in diverse roles relating to successful folding, assembly, intracellular localization, and degradation of other proteins. METHODS: In this study, we molecular cloned cDNAs for BiP, Crt, and PDI from Japanese monkey (Macaca fuscata), and analyzed tissue-specific expression of respective genes. RESULTS AND CONCLUSIONS: The lengths of protein-coding regions of these cDNAs for BiP, Crt and PDI are 1965, 1254, and 1533 bp, respectively. Each protein has a signal peptide and a KDEL motif in N- and C-terminal parts respectively, showing its intracellular localization to be the lumen of the ER. These stress proteins are highly conserved, showing that their similarities among mammals are more than 90% in the level of amino acid. The expression of the genes for stress proteins differed among the monkey tissues examined. BiP and PDI gene expression was predominant in secretory tissues such as liver and kidney, and brain tissues. But Crt gene expressed rather ubiquitously in a variety of tissues.     

Expression and function of inducible co-stimulator in patients with systemic lupus erythematosus: possible involvement in excessive interferon-γ and anti-double-stranded DNA antibody production

Inducible co-stimulator (ICOS) is the third member of the CD28/cytotoxic T-lymphocyte associated antigen-4 family and is involved in the proliferation and activation of T cells. A detailed functional analysis of ICOS on peripheral blood T cells from patients with systemic lupus erythematosus (SLE) has not yet been reported. In the present study we developed a fully human anti-human ICOS mAb (JTA009) with high avidity and investigated the immunopathological roles of ICOS in SLE. JTA009 exhibited higher avidity for ICOS than a previously reported mAb, namely SA12. Using JTA009, ICOS was detected in a substantial proportion of unstimulated peripheral blood T cells from both normal control individuals and patients with SLE. In CD4⁺CD45RO⁺ T cells from peripheral blood, the percentage of ICOS⁺ cells and mean fluorescence intensity with JTA009 were significantly higher in active SLE than in inactive SLE or in normal control individuals. JTA009 co-stimulated peripheral blood T cells in the presence of suboptimal concentrations of anti-CD3 mAb. Median values of [³H]thymidine incorporation were higher in SLE T cells with ICOS co-stimulation than in normal T cells, and the difference between inactive SLE patients and normal control individuals achieved statistical significance. ICOS co-stimulation significantly increased the production of IFN-γ, IL-4 and IL-10 in both SLE and normal T cells. IFN-γ in the culture supernatants of both active and inactive SLE T cells with ICOS co-stimulation was significantly higher than in normal control T cells. Finally, SLE T cells with ICOS co-stimulation selectively and significantly enhanced the production of IgG anti-double-stranded DNA antibodies by autologous B cells. These findings suggest that ICOS is involved in abnormal T cell activation in SLE, and that blockade of the interaction between ICOS and its receptor may have therapeutic value in the treatment of this intractable disease.