GDNF promotes neurite outgrowth and upregulates galectin-1 through the RET/PI3K signaling in cultured adult rat dorsal root ganglion neurons

Galectin-1 (GAL-1), a member of a family of β-galactoside binding animal lectins, is predominantly expressed in isolectin B4 (IB4)-binding small non-peptidergic (glial cell line-derived neurotrophic factor (GDNF)-responsive) sensory neurons in the sections of adult rat dorsal root ganglia (DRG), but its functional role and the regulatory mechanisms of its expression in the peripheral nervous system remain unclear. In the present study, both recombinant nerve growth factor (NGF) and GDNF (50 ng/ml) promoted neurite outgrowth from cultured adult rat DRG neurons, whereas GDNF, but not NGF, significantly increased the number of IB4-binding neurons and the relative protein expression of GAL-1 in the neuron-enriched culture of DRG. The GAL-1 expression in immortalized adult rat Schwann cells IFRS1 and DRG neuron-IFRS1 cocultures was unaltered by treatment with GDNF, which suggests that GDNF/GAL-1 signaling axis is more related to neurite outgrowth, rather than neuron-Schwann cell interactions. The GDNF-induced neurite outgrowth and GAL-1 upregulation were attenuated by anti-GDNF family receptor (RET) antibody and phosphatidyl inositol-3′-phosphate-kinase (PI3K) inhibitor LY294002, suggesting that the neurite-outgrowth promoting activity of GDNF may be attributable, at least partially, to the upregulation of GAL-1 through RET-PI3K pathway. On the contrary, no significant differences were observed between GAL-1 knockout and wild-type mice in DRG neurite outgrowth in the presence or absence of GDNF. Considerable immunohistochemical colocalization of GAL-3 with GAL-1 in DRG sections and GDNF-induced upregulation of GAL-3 in cultured DRG neurons imply the functional redundancy between these galectins.     

Mechanism of Intramembrane Cleavage of Alcadeins by γ-Secretase

Background

Alcadein proteins (Alcs; Alcα, Alcβand Alcγ) are predominantly expressed in neurons, as is Alzheimer''s β-amyloid (Aβ) precursor protein (APP). Both Alcs and APP are cleaved by primary α- or β-secretase to generate membrane-associated C-terminal fragments (CTFs). Alc CTFs are further cleaved by γ-secretase to secrete p3-Alc peptide along with the release of intracellular domain fragment (Alc ICD) from the membrane. In the case of APP, APP CTFβ is initially cleaved at the ε-site to release the intracellular domain fragment (AICD) and consequently the γ-site is determined, by which Aβ generates. The initial ε-site is thought to define the final γ-site position, which determines whether Aβ40/43 or Aβ42 is generated. However, initial intracellular ε-cleavage sites of Alc CTF to generate Alc ICD and the molecular mechanism that final γ-site position is determined remains unclear in Alcs.

Methodology

Using HEK293 cells expressing Alcs plus presenilin 1 (PS1, a catalytic unit of γ-secretase) and the membrane fractions of these cells, the generation of p3-Alc possessing C-terminal γ-cleavage site and Alc ICD possessing N-terminal ε-cleavage site were analysed with MALDI-TOF/MS. We determined the initial ε-site position of all Alcα, Alcβ and Alcγ, and analyzed the relationship between the initially determined ε-site position and the final γ-cleavage position.

Conclusions

The initial ε-site position does not always determine the final γ-cleavage position in Alcs, which differed from APP. No additional γ-cleavage sites are generated from artificial/non-physiological positions of ε-cleavage for Alcs, while the artificial ε-cleavage positions can influence in selection of physiological γ-site positions. Because alteration of γ-secretase activity is thought to be a pathogenesis of sporadic Alzheimer''s disease, Alcs are useful and sensitive substrate to detect the altered cleavage of substrates by γ-secretase, which may be induced by malfunction of γ-secretase itself or changes of membrane environment for enzymatic reaction.     

Phylogenetic and Structural Diversity in the Feline Leukemia Virus Env Gene

Feline leukemia virus (FeLV) belongs to the genus Gammaretrovirus, and causes a variety of neoplastic and non-neoplastic diseases in cats. Alteration of viral env sequences is thought to be associated with disease specificity, but the way in which genetic diversity of FeLV contributes to the generation of such variants in nature is poorly understood. We isolated FeLV env genes from naturally infected cats in Japan and analyzed the evolutionary dynamics of these genes. Phylogenetic reconstructions separated our FeLV samples into three distinct genetic clusters, termed Genotypes I, II, and III. Genotype I is a major genetic cluster and can be further classified into Clades 1–7 in Japan. Genotypes were correlated with geographical distribution; Genotypes I and II were distributed within Japan, whilst FeLV samples from outside Japan belonged to Genotype III. These results may be due to geographical isolation of FeLVs in Japan. The observed structural diversity of the FeLV env gene appears to be caused primarily by mutation, deletion, insertion and recombination, and these variants may be generated de novo in individual cats. FeLV interference assay revealed that FeLV genotypes did not correlate with known FeLV receptor subgroups. We have identified the genotypes which we consider to be reliable for evaluating phylogenetic relationships of FeLV, which embrace the high structural diversity observed in our sample. Overall, these findings extend our understanding of Gammaretrovirus evolutionary patterns in the field, and may provide a useful basis for assessing the emergence of novel strains and understanding the molecular mechanisms of FeLV transmission in cats.     

Effects of Transport Stress on Serum Alkaline Phosphatase Activity in Beagle Dogs

Here, to determine the effects of transport stress on blood parameters in dogs, we investigated the changes in hematologic and serum chemical parameters in healthy beagle dogs transported from Beijing, China, to Osaka, Japan, to obtain the background data. Only the activity of serum alkaline phosphatase increased clearly upon arrival, a change attributed to transport stress, but the activity gradually reduced afterward. No marked changes in levels of other blood parameters were noted. Our findings here suggest that alkaline phosphatase is a useful tool for studying transport stress.     

Properties of Oligonucleotide with Phenyl-Substituted Carbocyclic Nucleoside Analogs for the Formation of Duplex and Triplex DNA

(1S,3S,4R)-1-Phenyl-1-thymidyl-3-hydroxy-4-hydroxymethylcyclopentane (10) and their analogs were synthesized, incorporated into the oligodeoxynucleotides, and their properties were evaluated for the formation of duplex and triplex DNA. The known chiral cyclopentanone derivative was converted into the corresponding ketimine sulfonamide derivative, which was subjected to a stereoselective PhLi addition. The formed sulfonamide was hydrolyzed to afford the primary amino group, on which the thymine moiety was built. The benzyl protecting groups were removed to form the nucleoside analog having a phenyl group and the thymine unit at the 1′ position of a carbocyclic skeleton (10). In the estimation of the oligodeoxynucleotides incorporating 10 for duplex and triplex formation, the carbocyclic nucleoside analog 10 did not show the stabilizing effect for duplex formation; on the other hand, it stabilized the triplex. Therefore, the skeleton of the phenyl-substituted carbocyclic nucleoside analog 10 may be a platform for the formation of stable triplex DNA.     

An evolving tail of centromere histone variant CENP-A

We have recently shown that replication forks pause near origins in normal human fibroblasts (NHF1-hTERT) but not glioblastoma T98G cells. This observation led us to question whether other differences in the replication program may exist between these cell types that may relate to their genetic integrity. To identify differences, we detected immunoflourescently the sequential incorporation of the nucleotide analogs IdU and CldU into replicating DNA at the start of every hour of a synchronized S phase. We then characterized the patterns of labeled replicating DNA tracks and quantified the percentages and lengths of the tracks found at these hourly intervals. From the directionality of labeling in single extended replicating DNA fibers, tracks were categorized as single bidirectional origins, unidirectional elongations, clusters of origins firing in tandem, or merging forks (terminations). Our analysis showed that the start of S phase is enriched in single bidirectional origins in NHF1-hTERT cells, followed by an increase in clustering during mid S phase and an increase in merging forks during late S phase. Early S phase in T98G cells also largely consisted of single bidirectional origin initiations; however, an increase in clustering was delayed until an hour later, and clusters were shorter in mid/late S phase than in NHF1-hTERT cells. The spike in merging forks also did not occur until an hour later in T98G cells. Our observations suggest models to explain the temporal replication of single and clustered origins, and suggest differences in the replication program in a normal and cancer cell line.     

Involvement of 14-3-3 proteins in the second epidermal growth factor-induced wave of Rac1 activation in the process of cell migration

Immense previous efforts have elucidated the core machinery in cell migration, actin remodeling regulated by Rho family small GTPases including RhoA, Cdc42, and Rac1; however, the spatiotemporal regulation of these molecules remains largely unknown. Here, we report that EGF induces biphasic Rac1 activation in the process of cell migration, and UTKO1, a cell migration inhibitor, inhibits the second EGF-induced wave of Rac1 activation but not the first wave. To address the regulation mechanism and role of the second wave of Rac1 activation, we identified 14-3-3ζ as a target protein of UTKO1 and also showed that UTKO1 abrogated the binding of 14-3-3ζ to Tiam1 that was responsible for the second wave of Rac1 activation, suggesting that the interaction of 14-3-3ζ with Tiam1 is involved in this event. To our knowledge, this is the first report to use a chemical genetic approach to demonstrate the mechanism of temporal activation of Rac1.     

Substrate tRNA recognition mechanism of a multisite-specific tRNA methyltransferase, Aquifex aeolicus Trm1, based on the X-ray crystal structure

Archaeal and eukaryotic tRNA (N(2),N(2)-guanine)-dimethyltransferase (Trm1) produces N(2),N(2)-dimethylguanine at position 26 in tRNA. In contrast, Trm1 from Aquifex aeolicus, a hyper-thermophilic eubacterium, modifies G27 as well as G26. Here, a gel mobility shift assay revealed that the T-arm in tRNA is the binding site of A. aeolicus Trm1. To address the multisite specificity, we performed an x-ray crystal structure study. The overall structure of A. aeolicus Trm1 is similar to that of archaeal Trm1, although there is a zinc-cysteine cluster in the C-terminal domain of A. aeolicus Trm1. The N-terminal domain is a typical catalytic domain of S-adenosyl-l-methionine-dependent methyltransferases. On the basis of the crystal structure and amino acid sequence alignment, we prepared 30 mutant Trm1 proteins. These mutant proteins clarified residues important for S-adenosyl-l-methionine binding and enabled us to propose a hypothetical reaction mechanism. Furthermore, the tRNA-binding site was also elucidated by methyl transfer assay and gel mobility shift assay. The electrostatic potential surface models of A. aeolicus and archaeal Trm1 proteins demonstrated that the distribution of positive charges differs between the two proteins. We constructed a tRNA-docking model, in which the T-arm structure was placed onto the large area of positive charge, which is the expected tRNA-binding site, of A. aeolicus Trm1. In this model, the target G26 base can be placed near the catalytic pocket; however, the nucleotide at position 27 gains closer access to the pocket. Thus, this docking model introduces a rational explanation of the multisite specificity of A. aeolicus Trm1.     

Regulation of anoxic death in Caenorhabditis elegans by mammalian apoptosis signal-regulating kinase (ASK) family proteins

Cells and organisms face anoxia in a wide variety of contexts, including ischemia and hibernation. Cells respond to anoxic conditions through multiple signaling pathways. We report that NSY-1, the Caenorhabditis elegans ortholog of mammalian apoptosis signal-regulating kinase (ASK) family of MAP kinase (MAPK) kinase kinases (MAP3Ks), regulates viability of animals in anoxia. Loss-of-function mutations of nsy-1 increased survival under anoxic conditions, and increased survival was also observed in animals with mutations in tir-1 and the MAPK kinase (MAP2K) sek-1, which are upstream and downstream factors of NSY-1, respectively. Consistent with these findings, anoxia was found to activate the p38 MAPK ortholog PMK-1, and this was suppressed in nsy-1 and tir-1 mutant animals. Furthermore, double-mutant analysis showed that the insulin-signaling pathway, which also regulates viability in anoxia, functioned in parallel to NSY-1. These results suggest that the TIR-1-NSY-1-SEK-1-PMK-1 pathway plays important roles in the reponse to anoxia in C. elegans.