Effects of remnant primary forests on ant and dung beetle species diversity in a secondary forest in Sarawak, Malaysia

Tropical landscape structures have been transformed into mosaic structures consisting of small patches of primary and secondary forests, and areas of other land use. Diversity of insect assemblages is often higher in primary forests than in surrounding secondary forests. However, little is known about how the primary forests affect diversity in surrounding secondary forests in a landscape. In Sarawak, Malaysia, the typical landscape in areas from which lowland tropical rainforests had originally spread consists mainly of primary and secondary forests, with small areas of cultivation. In this study, we examined how the proportion of remnant primary forests in a landscape affects species diversity and species composition of ants and dung beetles in Macaranga-dominated secondary forests. The proportions were quantified based on remote-sensing data at various spatial scales, ranging from 100- to 5,000-m radius from each of the target forests. We found that the proportions of remnant primary forests within a 100-m radius had a significant positive effect on ant species diversity, and those within 100-, 300-, and 500-m radii significantly affected species compositions. However, the proportions of remnant primary forests had no significant relationship with dung beetle diversity, while those within 100- and 1,000-m radii had significant effects on species composition. The different responses to the remnant primary forests are likely to be related to differences in the movement and dispersal traits between the two taxa.     

Loss of aPKCλ in Differentiated Neurons Disrupts the Polarity Complex but Does Not Induce Obvious Neuronal Loss or Disorientation in Mouse Brains

Cell polarity plays a critical role in neuronal differentiation during development of the central nervous system (CNS). Recent studies have established the significance of atypical protein kinase C (aPKC) and its interacting partners, which include PAR-3, PAR-6 and Lgl, in regulating cell polarization during neuronal differentiation. However, their roles in neuronal maintenance after CNS development remain unclear. Here we performed conditional deletion of aPKCλ, a major aPKC isoform in the brain, in differentiated neurons of mice by camk2a-cre or synapsinI-cre mediated gene targeting. We found significant reduction of aPKCλ and total aPKCs in the adult mouse brains. The aPKCλ deletion also reduced PAR-6β, possibly by its destabilization, whereas expression of other related proteins such as PAR-3 and Lgl-1 was unaffected. Biochemical analyses suggested that a significant fraction of aPKCλ formed a protein complex with PAR-6β and Lgl-1 in the brain lysates, which was disrupted by the aPKCλ deletion. Notably, the aPKCλ deletion mice did not show apparent cell loss/degeneration in the brain. In addition, neuronal orientation/distribution seemed to be unaffected. Thus, despite the polarity complex disruption, neuronal deletion of aPKCλ does not induce obvious cell loss or disorientation in mouse brains after cell differentiation.     

IL1RAPL1 Associated with Mental Retardation and Autism Regulates the Formation and Stabilization of Glutamatergic Synapses of Cortical Neurons through RhoA Signaling Pathway

Interleukin-1 receptor accessory protein-like 1 (IL1RAPL1) is associated with X-linked mental retardation and autism spectrum disorder. We found that IL1RAPL1 regulates synapse formation of cortical neurons. To investigate how IL1RAPL1 controls synapse formation, we here screened IL1RAPL1-interacting proteins by affinity chromatography and mass spectroscopy. IL1RAPL1 interacted with Mcf2-like (Mcf2l), a Rho guanine nucleotide exchange factor, through the cytoplasmic Toll/IL-1 receptor domain. Knockdown of endogenous Mcf2l and treatment with an inhibitor of Rho-associated protein kinase (ROCK), the downstream kinase of RhoA, suppressed IL1RAPL1-induced excitatory synapse formation of cortical neurons. Furthermore, we found that the expression of IL1RAPL1 affected the turnover of AMPA receptor subunits. Insertion of GluA1-containing AMPA receptors to the cell surface was decreased, whereas that of AMPA receptors composed of GluA2/3 was enhanced. Mcf2l knockdown and ROCK inhibitor treatment diminished the IL1RAPL1-induced changes of AMPA receptor subunit insertions. Our results suggest that Mcf2l-RhoA-ROCK signaling pathway mediates IL1RAPL1-dependent formation and stabilization of glutamatergic synapses of cortical neurons.     

Extracts of Adipose Derived Stem Cells Slows Progression in the R6/2 Model of Huntington's Disease

Stem cell therapy is a promising treatment for incurable disorders including Huntington''s disease (HD). Adipose-derived stem cell (ASC) is an easily available source of stem cells. Since ASCs can be differentiated into nervous stem cells, it has clinically feasible potential for neurodegenerative disease. In addition, ASCs secrete various anti-apoptotic growth factors, which improve the symptoms of disease from transplanted ASCs. Thus, cell-free extracts of ASCs (ASCs-E) could be a potential candidate for treatment of HD. Here, we investigated effects of ASCs-E on R6/2 HD mouse model and neuronal cells. In R6/2 HD model, injection of ASCs-E improved the performance in Rotarod test. ASCs-E also ameliorated striatal atrophy and mutant huntingtin aggregation in the striatum. In Western blot increased expressions of p-Akt, p-CREB and PGC1α were noted by injection of ASCs-E, when comparing to the R6/2 HD model. Neuro2A neuroblastoma cells treated with ASCs-E showed increased expression of p-CREB and PGC1α. In conclusion, ASCs-E delayed disease progression in animal model of HD by restoring of CREB-PGC1α pathway and could be a potential resource for treatment of HD.     

The Flavonoid Apigenin Downregulates CDK1 by Directly Targeting Ribosomal Protein S9

Flavonoids have been reported to inhibit tumor growth by causing cell cycle arrest. However, little is known about the direct targets of flavonoids in tumor growth inhibition. In the present study, we developed a novel method using magnetic FG beads to purify flavonoid-binding proteins, and identified ribosomal protein S9 (RPS9) as a binding partner of the flavonoid apigenin. Similar to treatment with apigenin, knockdown of RPS9 inhibited the growth of human colon cancer cells at the G2/M phase by downregulating cyclin-dependent kinase 1 (CDK1) expression at the promoter level. Furthermore, knockdown of RPS9 suppressed G2/M arrest caused by apigenin. These results suggest that apigenin induces G2/M arrest at least partially by directly binding and inhibiting RPS9 which enhances CDK1 expression. We therefore raise the possibility that identification of the direct targets of flavonoids may contribute to the discovery of novel molecular mechanisms governing tumor growth.     

Homostachydrine is a Xenobiotic Substrate of OCTN1/SLC22A4 and Potentially Sensitizes Pentylenetetrazole-Induced Seizures in Mice

Neurochemical Research - Understanding of the underlying mechanism of epilepsy is desired since some patients fail to control their seizures. The carnitine/organic cation transporter OCTN1/SLC22A4...     

Fibroblasts-dependent invasion of podoplanin-positive cancer stem cells in squamous cell carcinoma

To clear whether podoplanin-positive cancer stem cells in squamous cell carcinoma have higher invasion activity during a fibroblasts-dependent invasion. A collagen gel invasion assay was performed using fluorescent ubiquitination-based cell cycle indicator-labeled A431 cells. The total number and number of invading cells in S/G2/M phase were counted using time-lapse imaging cocultured with fibroblasts. There was no significant difference between the number of invading podoplanin-positive and negative A431 cells when fibroblasts did not exist. On the contrary, the number of invading podoplanin-positive cells was significantly higher when fibroblasts existed. The frequency of cells in S/G2/M phase among invasion was no difference. Knockdown of podoplanin decreased the number of invaded A431 cells significantly when fibroblasts existed. Podoplanin-positive A431 cells display higher invasion activity when fibroblasts exist, suggesting that some biological functions of cancer stem cells might become evident only within the fibrous tumor microenvironment.     

Structure and Function of Nucleoside Hydrolases from Physcomitrella patens and Maize Catalyzing the Hydrolysis of Purine,Pyrimidine, and Cytokinin Ribosides

We present a comprehensive characterization of the nucleoside N-ribohydrolase (NRH) family in two model plants, Physcomitrella patens (PpNRH) and maize (Zea mays; ZmNRH), using in vitro and in planta approaches. We identified two NRH subclasses in the plant kingdom; one preferentially targets the purine ribosides inosine and xanthosine, while the other is more active toward uridine and xanthosine. Both subclasses can hydrolyze plant hormones such as cytokinin ribosides. We also solved the crystal structures of two purine NRHs, PpNRH1 and ZmNRH3. Structural analyses, site-directed mutagenesis experiments, and phylogenetic studies were conducted to identify the residues responsible for the observed differences in substrate specificity between the NRH isoforms. The presence of a tyrosine at position 249 (PpNRH1 numbering) confers high hydrolase activity for purine ribosides, while an aspartate residue in this position confers high activity for uridine. Bud formation is delayed by knocking out single NRH genes in P. patens, and under conditions of nitrogen shortage, PpNRH1-deficient plants cannot salvage adenosine-bound nitrogen. All PpNRH knockout plants display elevated levels of certain purine and pyrimidine ribosides and cytokinins that reflect the substrate preferences of the knocked out enzymes. NRH enzymes thus have functions in cytokinin conversion and activation as well as in purine and pyrimidine metabolism.Nucleoside hydrolases or nucleoside N-ribohydrolases (NRHs; EC 3.2.2.-) are glycosidases that catalyze the cleavage of the N-glycosidic bond in nucleosides to enable the recycling of the nucleobases and Rib (Fig. 1A). The process by which nucleosides and nucleobases are recycled is also known as salvaging and is a way of conserving energy, which would otherwise be needed for the de novo synthesis of purine- and pyrimidine-containing compounds. During the salvage, bases and nucleosides can be converted into nucleoside monophosphates by the action of phosphoribosyltransferases and nucleoside kinases, respectively, and further phosphorylated into nucleoside diphosphates and triphosphates (Moffatt et al., 2002; Zrenner et al., 2006; Fig. 1B). Uridine kinase and uracil phosphoribosyl transferase are key enzymes in the pyrimidine-salvaging pathway in plants (Mainguet et al., 2009; Chen and Thelen, 2011). Adenine phosphoribosyltransferase and adenosine kinase (ADK) are important in purine salvaging (Moffatt and Somerville, 1988; Moffatt et al., 2002), and their mutants cause reductions in fertility or sterility, changes in transmethylation, and the formation of abnormal cell walls. In addition, both enzymes were also reported to play roles in cytokinin metabolism (Moffatt et al., 1991, 2000; von Schwartzenberg et al., 1998; Schoor et al., 2011). Cytokinins (N⁶-substituted adenine derivatives) are plant hormones that regulate cell division and numerous developmental events (Mok and Mok, 2001; Sakakibara, 2006). Cytokinin ribosides are considered to be transport forms and have little or no activity.Open in a separate windowFigure 1.A, Scheme of the reactions catalyzed by plant NRHs when using purine (inosine), pyrimidine (uridine), and cytokinin (iPR) ribosides as the substrates. B, Simplified schematic overview of cytokinin, purine, and pyrimidine metabolism in plants. The diagram is adapted from the work of Stasolla et al. (2003) and Zrenner et al. (2006) with modifications. The metabolic components shown are as follows: 1, cytokinin nucleotide phosphoribohydrolase; 2, adenine phosphoribosyltransferase; 3, adenosine kinase; 4, 5′-nucleotidase; 5, adenosine phosphorylase; 6, purine/pyrimidine nucleoside ribohydrolase; 7, cytokinin oxidase/dehydrogenase; 8, AMP deaminase; 9, hypoxanthine phosphoribosyltransferase; 10, inosine kinase; 11, inosine-guanosine phosphorylase; 12, IMP dehydrogenase; 13, xanthine dehydrogenase; 14, 5′-nucleotidase; 15, GMP synthase; 16, hypoxanthine-guanine phosphoribosyltransferase; 17, guanosine deaminase; 18, guanine deaminase; 19, guanosine kinase; 20, uracil phosphoribosyltransferase; 21, uridine cytidine kinase; 22, pyrimidine 5′-nucleotidase; 23, cytidine deaminase; 24, adenosine/adenine deaminase. CK, Cytokinin; CKR, cytokinin riboside; CKRMP, cytokinin riboside monophosphate.NRHs are metalloproteins first identified and characterized in parasitic protozoa such as Trypanosoma, Crithidia, and Leishmania species that rely on the import and salvage of nucleotide derivatives. They have since been characterized in other organisms such as bacteria, yeast, and insects (Versées and Steyaert, 2003) but never in mammals (Parkin et al., 1991). They have been divided into four classes based on their substrate specificity: nonspecific NRHs, which hydrolyze inosine and uridine (IU-NRHs; Parkin et al., 1991; Shi et al., 1999); purine-specific inosine/adenosine/guanosine NRHs (Parkin, 1996); the 6-oxopurine-specific guanosine/inosine NRHs (Estupiñán and Schramm, 1994); and the pyrimidine nucleoside-specific cytidine/uridine NRHs (CU-NRHs; Giabbai and Degano, 2004). All NRHs exhibit a stringent specificity for the Rib moiety and differ in their preferences regarding the nature of the nucleobase. Crystal structures are available for empty NRH or in complex with inhibitors from Crithidia fasciculata (CfNRH; Degano et al., 1998), Leishmania major (LmNRH; Shi et al., 1999), and Trypanosoma vivax (TvNRH; Versées et al., 2001, 2002). The structures of two CU-NRHs from Escherichia coli, namely YeiK (Iovane et al., 2008) and YbeK (rihA; Muzzolini et al., 2006; Garau et al., 2010), are also available. NRHs are believed to catalyze N-glycosidic bond cleavage by a direct displacement mechanism. An Asp from a conserved motif acts as a general base and abstracts a proton from a catalytic water molecule, which then attacks the C1′ atom of the Rib moiety of the nucleoside. Kinetic isotope-effect studies on CfNRH (Horenstein et al., 1991) showed that the substrate’s hydrolysis proceeds via an oxocarbenium ion-like transition state and is preceded by protonation at the N7 atom of the purine ring, which lowers the electron density on the purine ring and destabilizes the N-glycosidic bond. A conserved active-site His is a likely candidate for this role in IU-NRHs and CU-NRHs. In the transition state, the C1′-N9 glycosidic bond is almost 2 Å long, with the C1′ atom being sp² hybridized while the C3′ atom adopts an exo-conformation, and the whole ribosyl moiety carries a substantial positive charge (Horenstein et al., 1991).Several NRH enzymes have been identified in plants, including a uridine-specific NRH from mung bean (Phaseolus radiatus; Achar and Vaidyanathan, 1967), an inosine-specific NRH (EC 3.2.2.2) and a guanosine-inosine-specific NRH, both from yellow lupine (Lupinus luteus; Guranowski, 1982; Szuwart et al., 2006), and an adenosine-specific NRH (EC 3.2.2.7) from coffee (Coffea arabica), barley (Hordeum vulgare), and wheat (Triticum aestivum; Guranowski and Schneider, 1977; Chen and Kristopeit, 1981; Campos et al., 2005). However, their amino acid sequences have not been reported so far. A detailed study of the NRH gene family from Arabidopsis (Arabidopsis thaliana) has recently been reported (Jung et al., 2009, 2011). The AtNRH1 enzyme exhibits highest hydrolase activity toward uridine and xanthosine. It can also hydrolyze the cytokinin riboside N⁶-(2-isopentenyl)adenosine (iPR), which suggests that it may also play a role in cytokinin homeostasis. However, Riegler et al. (2011) analyzed the phenotypes of homozygous nrh1 and nrh2 single mutants along with the homozygous double mutants and concluded that AtNRHs are probably unimportant in cytokinin metabolism.Here, we identify and characterize plant IU-NRHs from two different model organisms, Physcomitrella patens and maize (Zea mays), combining structural, enzymatic, and in planta functional approaches. The moss P. patens was chosen to represent the bryophytes, which can be regarded as being evolutionarily basal terrestrial plants, and is suitable for use in developmental and metabolic studies (Cove et al., 2006; von Schwartzenberg, 2009), while maize is an important model system for cereal crops. We report the crystal structures of NRH enzymes from the two plant species, PpNRH1 and ZmNRH3. Based on these structures, we performed site-directed mutagenesis experiments and kinetic analyses of point mutants of PpNRH1 in order to identify key residues involved in nucleobase interactions and catalysis. To analyze the physiological role of the PpNRHs, single knockout mutants were generated. NRH deficiency caused significant changes in the levels of purine, pyrimidine, and cytokinin metabolites relative to those seen in the wild type, illustrating the importance of these enzymes in nucleoside and cytokinin metabolism.