Signaling through Arf6 guanine-nucleotide exchange factor cytohesin-1 regulates migration in Schwann cells

During development of the peripheral nervous system (PNS), Schwann cells migrate along neuronal axons before initiating myelination of the axons. While intercellular signals controlling migration, between Schwann cells and peripheral neurons, are established, how their intracellular transduction of the signals into Schwann cells still remains to be clarified. Here, we show that cytohesin-1, a guanine-nucleotide exchange factor (GEF), and the effector Arf6 are required for migration of primary Schwann cells. Knockdown of cytohesin-1 or Arf6 in Schwann cells, as well as treatment with the chemical cytohesin inhibitor SecinH3 or knockout of cytohesin-1, inhibits peripheral neuronal conditioned medium-mediated migration. Similar effects are also observed following stimulation with each of growth factors contained in a conditioned medium, suggesting that cytohesin-1 plays a role in transducing soluble ligand signals from neurons. Reintroduction of small interfering (si)RNA-resistant cytohesin-1 into Schwann cells reverses blunted migration in the siRNA-transfected Schwann cells, illustrating the importance of cytohesin-1 in migration. On the other hand, introduction of cytohesin-1 that harbors the Tyr-382 mutation, which is an amino acid that is important for its activation, failed to reverse the reduction in primary Schwann cell migration. These results suggest that signaling through cytohesin-1 is required for Schwann cell migration, revealing a novel mechanism for Schwann cell migration.     

Role of Rho-kinase signaling and endothelial dysfunction in modulating blood flow distribution in pulmonary hypertension

Rho-kinase-mediated vasoconstriction and endothelial dysfunction are considered two primary instigators of pulmonary arterial hypertension (PAH). However, their contribution to the adverse changes in pulmonary blood flow distribution associated with PAH has not been addressed. This study utilizes synchrotron radiation microangiography to assess the specific role, and contribution of, Rho-kinase-mediated vasoconstriction and endothelial dysfunction in PAH. Male adult Sprague-Dawley rats were injected with saline (Cont-rats) or monocrotaline (MCT-rats) 3 wk before microangiography was performed on the left lung. We assessed dynamic changes in vessel internal diameter (ID) in response to 1) the Rho-kinase inhibitor fasudil (10 mg/kg iv); or 2) ACh (3 μg · kg?1 · min?1), sodium nitroprusside (SNP, 5 μg · kg?1 · min?1), and N(ω)-nitro-l-arginine methyl ester (l-NAME, 50 mg/kg iv). We observed that MCT-rats had fewer vessels of the microcirculation compared with Cont-rats. The fundamental result of this study is that fasudil improved pulmonary blood flow distribution and reduced pulmonary pressure in PAH rats, not only by dilating already-perfused vessels (ID > 100 μm), but also by restoring blood flow to vessels that had previously been constricted closed (ID < 100 μm). Endothelium-dependent vasodilation was impaired in MCT-rats primarily in vessels with an ID < 200 μm. Moreover the vasoconstrictor response to l-NAME was accentuated in MCT-rats, but only in the 200- to 300-μm vessels. These results highlight the importance of Rho-kinase-mediated control and endothelial control of pulmonary vascular tone in PAH. Indeed, an effective therapeutic strategy for treating PAH should target both the smooth muscle Rho-kinase and endothelial pathways.     

Characterization of the locus of genes encoding enzymes producing heptadepsipeptide micropeptin in the unicellular cyanobacterium Microcystis

The gene cluster involved in producing the cyclic heptadepsipeptide micropeptin was cloned from the genome of the unicellular cyanobacterium Microcystis aeruginosa K-139. Sequencing revealed four genes encoding non-ribosomal peptide synthetases (NRPSs) that are highly similar to the gene cluster involved in cyanopeptolins biosynthesis. According to predictions based on the non-ribosomal consensus code, the order of the mcnABCE NPRS modules was well consistent with that of the biosynthetic assembly of cyclic peptides. The biochemical analysis of a McnB(K-139) adenylation domain and the knock-out of mcnC in a micropeptin-producing strain, M. viridis S-70, revealed that the mcn gene clusters were responsible for the production of heptadepsipeptide micropeptins. A detailed comparison of nucleotide sequences also showed that the regions between the mcnC and mcnE genes of M. aeruginosa K-139 retained short stretches of DNA homologous to halogenase genes involved in the synthesis of halogenated cyclic peptides of the cyanopeptolin class including anabaenopeptilides. This suggests that the mcn clusters of M. aeruginosa K-139 have lost the halogenase genes during evolution. Finally, a comparative bioinformatics analysis of the congenial gene cluster for depsipetide biosynthesis suggested the diversification and propagation of the NRPS genes in cyanobacteria.     

Cloning and characterization of a new hetero-gene cluster of nonribosomal peptide synthetase and polyketide synthase from the cyanobacterium Microcystis aeruginosa K-139

Two nonribosomal peptide synthetase genes responsible for the biosynthesis of microcystin and micropeptin in Microcystis aeruginosa K-139 have been identified. A new nonribosomal peptide synthetase gene, psm3, was identified in M. aeruginosa K-139. The gene is a cluster extending 30 kb and comprising 13 bidirectionally transcribed open reading frames arranged in two putative operons. psm3 encodes four adenylation proteins, one polyketide synthase, and several unique proteins, especially Psm3L consisting of halogenase, acyl-CoA binding protein-like protein, and acyl carrier protein. Alignment of the binding pocket of the adenylation domain and an ATP-PPi exchange analysis using a recombinant protein with the adenylation domain of Psm3B showed that Psm3G and Psm3B activate aspartic acid and tyrosine, respectively. Although disruption of psm3 did not reveal the product produced by Psm3, we identified microviridin B and aeruginosin K139 in the cells of M. aeruginosa K-139. The above-mentioned results indicated that M. aeruginosa possesses at least five nonribosomal peptide synthetase gene clusters.     

N-glycolylneuraminic acid deficiency in mice: implications for human biology and evolution

Humans and chimpanzees share >99% identity in most proteins. One rare difference is a human-specific inactivating deletion in the CMAH gene, which determines biosynthesis of the sialic acid N-glycolylneuraminic acid (Neu5Gc). Since Neu5Gc is prominent on most chimpanzee cell surfaces, this mutation could have affected multiple systems. However, Neu5Gc is found in human cancers and fetuses and in trace amounts in normal human tissues, suggesting an alternate biosynthetic pathway. We inactivated the mouse Cmah gene and studied the in vivo consequences. There was no evidence for an alternate pathway in normal, fetal, or malignant tissue. Rather, null fetuses accumulated Neu5Gc from heterozygous mothers and dietary Neu5Gc was incorporated into oncogene-induced tumors. As with humans, there were accumulation of the precursor N-acetylneuraminic acid and increases in sialic acid O acetylation. Null mice showed other abnormalities reminiscent of the human condition. Adult mice showed a diminished acoustic startle response and required higher acoustic stimuli to increase responses above the baseline level. In this regard, histological abnormalities of the inner ear occurred in older mice, which had impaired hearing. Adult animals also showed delayed skin wound healing. Loss of Neu5Gc in hominid ancestors approximately 2 to 3 million years ago likely had immediate and long-term consequences for human biology.     

Dock6, a Dock-C subfamily guanine nucleotide exchanger, has the dual specificity for Rac1 and Cdc42 and regulates neurite outgrowth

Small GTPases of the Rho family, Rho, Rac, and Cdc42, are critical regulators of the changes in the actin cytoskeleton. Rho GTPases are typically activated by Dbl-homology (DH)-domain-containing guanine nucleotide exchange factors (GEFs). Recent genetic and biochemical studies revealed a new type of GEF for the Rho GTPases. This family is composed of 11 genes, designated as Dock1 to Dock11, and is structurally divided into four classes Dock-A, -B, -C, and -D. Dock-A and -B subfamilies are typically GEFs specific for Rac1, while the Dock-D subfamily is specific for Cdc42. Here we show that Dock6, a member of the Dock-C subfamily, exchanges GDP for GTP for Rac1 and Cdc42 in vitro and in vivo. Furthermore, we find that, in mouse N1E-115 neuroblastoma cells, expression of Dock6 is increased following differentiation. Transfection of the catalytic Dock Homology Region-2 (DHR-2) domain of Dock6 promotes neurite outgrowth mediated by Rac1 and Cdc42. Conversely, knockdown of endogenous Dock6 by small interference RNA reduces activation of Rac1 and Cdc42 and neurite outgrowth. Taken together, these results suggest that Dock6 differs from all of the identified Dock180-related proteins, in that it is the GEF specific for both Rac1 and Cdc42 and may be one of physiological regulators of neurite outgrowth.     

Divergence times in the termite genus Macrotermes (Isoptera: Termitidae)

The evolution of fungus-growing termites is supposed to have started in the African rain forests with multiple invasions of semi-arid habitats as well as multiple invasions of the Oriental region. We used sequences of the mitochondrial COII gene and Bayesian dating to investigate the time frame of the evolution of Macrotermes, an important genus of fungus-growing termites. We found that the genus Macrotermes consists of at least 6 distantly related clades. Furthermore, the COII sequences suggested some cryptic diversity within the analysed African Macrotermes species. The dates calculated with the COII data using a fossilized termite mound to calibrate the clock were in good agreement with dates calculated with COI sequences using the split between Locusta and Chortippus as calibration point which supports the consistency of the calibration points. The clades from the Oriental region dated back to the early Tertiary. These estimates of divergence times suggested that Macrotermes invaded Asia during periods with humid climates. For Africa, many speciation events predated the Pleistocene and fall in range of 6-23 million years ago. These estimates suggest that savannah-adapted African clades radiated with the spread of the semi-arid ecosystems during the Miocene. Apparently, events during the Pleistocene were of little importance for speciation within the genus Macrotermes. However, further investigations are necessary to increase the number of taxa for phylogenetic analysis.     

In vivo knockdown of ErbB3 in mice inhibits Schwann cell precursor migration

The myelin sheath insulates neuronal axons and markedly increases the nerve conduction velocity. In the peripheral nervous system (PNS), Schwann cell precursors migrate along embryonic neuronal axons to their final destinations, where they eventually wrap around individual axons to form the myelin sheath after birth. ErbB2 and ErbB3 tyrosine kinase receptors form a heterodimer and are extensively expressed in Schwann lineage cells. ErbB2/3 is thought to be one of the primary regulators controlling the entire Schwann cell development. ErbB3 is the bona fide Schwann cell receptor for the neuronal ligand neuregulin-1. Although ErbB2/3 is well known to regulate both Schwann cell precursor migration and myelination by Schwann cells in fishes, it still remains unclear whether in mammals, ErbB2/3 actually regulates Schwann cell precursor migration. Here, we show that knockdown of ErbB3 using a Schwann cell-specific promoter in mice causes delayed migration of Schwann cell precursors. In contrast, littermate control mice display normal migration. Similar results are seen in an in vitro migration assay using reaggregated Schwann cell precursors. Also, ErbB3 knockdown in mice reduces myelin thickness in sciatic nerves, consistent with the established role of ErbB3 in myelination. Thus, ErbB3 plays a key role in migration, as well as in myelination, in mouse Schwann lineage cells, presenting a genetically conservative role of ErbB3 in Schwann cell precursor migration.