Paxillin is the target of c-Jun N-terminal kinase in Schwann cells and regulates migration

During development of the peripheral nervous system (PNS), Schwann cells migrate along axons, wrapping individual axons to form a myelin sheath. This process is all mediated by the intercellular signaling between neurons and Schwann cells. As yet, little is known about the intracellular signaling mechanisms controlling these morphological changes including Schwann cell migration. We previously showed that c-Jun N-terminal kinase (JNK) plays a key role in Schwann cell migration before the initiation of myelination. Here we show that JNK, acting through phosphorylation of the cytoskeletal protein paxillin, regulates Schwann cell migration and that it mediates dorsal root ganglion (DRG) neuronal conditioned medium (CM). Phosphorylation of paxillin at the Ser-178 position, the JNK phosphorylation site, is observed following stimulation with neuronal CM. Phosphorylation is also detected as a result of stimulation with each of growth factors contained in neuronal CM. Knockdown of paxillin with the specific small interfering RNA (siRNA) inhibits migration. The reintroduction of paxillin reverses siRNA-mediated inhibition of migration, whereas paxillin harboring the Ser-178-to-Ala mutation fails to reverse it. In addition, while JNK binds to the N-terminal region (called LD1), the deletion of LD1 blocks migration. Together, JNK binds and phosphorylates paxillin to regulate Schwann cell migration, illustrating that paxillin provides one of the convergent points of intracellular signaling pathways controlling Schwann cell migration.     

Molecular cloning and characterization of plasma membrane- and vacuolar-type Na+/H+ antiporters of an alkaline-salt-tolerant monocot, Puccinellia tenuiflora

A better understanding of salt tolerance in plants might lead to the genetic engineering of crops that can grow in saline soils. Here we cloned and characterized plasma membrane and vacuolar Na?/H? antiporters of a monocotyledonous alkaline-tolerant halophyte, Puccinellia tenuiflora. The predicted amino acid sequence of the transporters were very similar to those of orthologs in monocotyledonous crops. Expression analysis showed that (1) NHA was more strongly induced by NaCl in the roots of P. tenuiflora while in rice it was rather induced in the shoots, suggesting that the role of NHA in salt excretion from the roots partly accounts for the difference in the tolerance of the two species, and that (2) NHXs were specifically induced by NaHCO? but not by NaCl in the roots of both species, suggesting that vacuolar-type Na?/H? antiporters play roles in pH regulation under alkaline salt conditions. Overexpression of the antiporters resulted in increased tolerance of shoots to NaCl and roots to NaHCO?. Overexpression lines exhibited a lower Na? content and a higher K? content in shoots under NaCl treatments, leading to a higher Na?/H? ratio.     

Rapid evolution of major histocompatibility complex class I genes in primates generates new disease alleles in humans via hitchhiking diversity

A plausible explanation for many MHC-linked diseases is lacking. Sequencing of the MHC class I region (coding units or full contigs) in several human and nonhuman primate haplotypes allowed an analysis of single nucleotide variations (SNV) across this entire segment. This diversity was not evenly distributed. It was rather concentrated within two gene-rich clusters. These were each centered, but importantly not limited to, the antigen-presenting HLA-A and HLA-B/-C loci. Rapid evolution of MHC-I alleles, as evidenced by an unusually high number of haplotype-specific (hs) and hypervariable (hv) (which could not be traced to a single species or haplotype) SNVs within the classical MHC-I, seems to have not only hitchhiked alleles within nearby genes, but also hitchhiked deleterious mutations in these same unrelated loci. The overrepresentation of a fraction of these hvSNV (hv1SNV) along with hsSNV, as compared to those that appear to have been maintained throughout primate evolution (trans-species diversity; tsSNV; included within hv2SNV) tends to establish that the majority of the MHC polymorphism is de novo (species specific). This is most likely reminiscent of the fact that these hsSNV and hv1SNV have been selected in adaptation to the constantly evolving microbial antigenic repertoire.     

Purification and biochemical characterization of a membrane-bound [NiFe]-hydrogenase from a hydrogen-oxidizing, lithotrophic bacterium, Hydrogenophaga sp. AH-24

Membrane-bound [NiFe]-hydrogenase from Hydrogenophaga sp. AH-24 was purified to homogeneity. The molecular weight was estimated as 100±10 kDa, consisting of two different subunits (62 and 37 kDa). The optimal pH values for H₂ oxidation and evolution were 8.0 and 4.0, respectively, and the activity ratio (H₂ oxidation/H₂ evolution) was 1.61 × 10² at pH 7.0. The optimal temperature was 75 °C. The enzyme was quite stable under air atmosphere (the half-life of activity was c . 48 h at 4 °C), which should be important to function in the aerobic habitat of the strain. The enzyme showed high thermal stability under anaerobic conditions, which retained full activity for over 5 h at 50 °C. The activity increased up to 2.5-fold during incubation at 50 °C under H₂. Using methylene blue as an electron acceptor, the kinetic constants of the purified membrane-bound homogenase (MBH) were V _max=336 U mg⁻¹, k _cat=560 s⁻¹, and k _cat/ K _m=2.24 × 10⁷ M⁻¹ s⁻¹. The MBH exhibited prominent electron paramagnetic resonance signals originating from [3Fe–4S]⁺ and [4Fe–4S]⁺ clusters. On the other hand, signals originating from Ni of the active center were very weak, as observed in other oxygen-stable hydrogenases from aerobic H₂-oxidizing bacteria. This is the first report of catalytic and biochemical characterization of the respiratory MBH from Hydrogenophaga .     

Alkaloids inhibiting l-histidine decarboxylase from Sinomenium acutum

Chromatographic separation of an extract of the stems of Sinomenium acutum resulted in the isolation of two new alkaloids, 2-O-demethyl-acutumine (4), and 6-O-methyl-laudanosoline-1-O-glucoside (5), together with three known alkaloids, sinomenine (1), sinomenine N-oxide (2), and magnoflorine (3). Sinomenine was found to show good inhibitory activity toward l-histidine decarboxylase from Lactobacillus 30a with an IC₅₀ value of 969 μM but its N-oxide showed no inhibition of this enzyme. Sinomenine inhibited this enzyme in a noncompetitive manner with a K_i of 762 μM.     

Expression in Escherichia coli, refolding, and purification of the recombinant mature form of human matrix metalloproteinase 7 (MMP-7)

In the latent pro-form of matrix metalloproteinase 7 (MMP-7), the cysteine residue in the pro-peptide binds the active-site zinc ion. Hence, recombinant active MMP-7 was prepared from pro-MMP-7 by modification of this cysteine residue with a mercuric reagent. In this study, mature MMP-7 was expressed in Escherichia coli as inclusion bodies, solubilized, and refolded with 1 M L-arginine. The purified product was indistinguishable from the one prepared from pro-MMP-7 as assessed by hydrolysis of (7-methoxycoumarin-4-yl)acetyl-L-Pro-L-Leu-Gly-L-Leu-[N(3)-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl]-L-Ala-L-Arg-NH(2).     

Topographic pattern of the rearrangement of the dystrophin gene in Japanese Duchenne muscular dystrophy

To compare the frequency and distribution of rearrangements in the dystrophin gene in Duchenne muscular dystrophy (DMD) between Japanese DMD patients and those in North America and Europe, Southern blot analyses of the dystrophin gene were carried out in 88 probands classified as DMD. Gene rearrangements were found in 61 (69%) subjects, and they were composed of partial gene deletions in 53 (60%) probands and partial duplications in 7 (8%) probands. A total deletion of the gene was found in 1 (1%) patient. Among 53 patients with deletions, 34 (64%) had breakpoints between introns 44 and 52 and 7 (13%) had breakpoints between introns 2 and 11. Both the frequency and the distribution of gene rearrangements found in this study were similar to those reported in North America and Europe. These data suggest that there are no ethnic or racial differences in the frequency and distribution of rearrangements thought to be caused by similar mechanisms in the dystrophin gene in all human racial groupings.     

Biocatalyst collection and heterologous expression of sesquiterpene synthases from basidiomycetous fungi: Discovery of a novel sesquiterpene hydrocarbon

Basidiomycetes produce a wide variety of sesquiterpenoids, which attract significant interest in pharmaceutical and industrial applications. Structural diversification of sesquiterpenoids is performed by sesquiterpene synthases (STSs), which produce a wide array of backbone structures; therefore, functional characterization and increased biocatalyst collection of STSs are important for expanding scientific knowledge and meeting the needs of advanced biotechnology. Gene identification and functional annotation of STSs from the basidiomycetous fungi Agaricus bisporus, Auriscalpium vulgare, Lepista nuda, Pleurotus ostreatus and Trametes versicolor were conducted. Through these investigations, the catalytic functions of 30 STSs were revealed using recombinant enzymes heterologously expressed in Saccharomyces cerevisiae. Furthermore, the unique function of an STS from P. ostreatus, PoSTS-06, was revealed to be the production of a novel sesquiterpene hydrocarbon that we named pleostene. The absolute structure of pleostene was determined by NMR spectroscopy and X-ray crystallography using the crystalline sponge method.