Role of spinal voltage-dependent calcium channel alpha 2 delta-1 subunit in the expression of a neuropathic pain-like state in mice

The present study was undertaken to investigate the role of spinal voltage-dependent calcium channel alpha(2)delta-1 subunit in the expression of a neuropathic pain-like state induced by partial sciatic nerve ligation in mice. In cultured spinal neurons, gabapentin (GBP), which displays the inhibitory effect of alpha(2)delta-1 subunit, suppressed the extracellular Ca(2+) influx induced by KCl, whereas it failed to inhibit the intracellular Ca(2+) release induced by inositol-1,4,5-triphosphate. Seven days after sciatic nerve ligation, the protein level of alpha(2)delta-1 subunit in the ipsilateral spinal cord was clearly increased compared to that observed in sham-operated mice. In addition, the mRNA level of alpha(2)delta-1 subunit was significantly increased in the dorsal root ganglion, but not in the spinal cord, of nerve-ligated mice. Under these conditions, a marked decrease in the latency of paw-withdrawal against a thermal stimulation and tactile stimulation, induced by sciatic nerve ligation was abolished by repeated intrathecal (i.t.) treatment with GBP. Additionally, the persistent reduction in the nociceptive threshold by i.t. treatment with GBP at the early stage of the neuropathic pain-like state was maintained for 7 days even after GBP withdrawal. It is of interest to note that a single i.t. post-injection of GBP showed a marked and transient inhibitory effect on the developed neuropathic pain-like state, whereas repeated i.t. post-treatment with GBP produced a persistent inhibitory effect during the treatment. In conclusion, we propose here that the neuropathic pain-like state with sciatic nerve ligation is associated with the increased level of the alpha(2)delta-1 subunit of Ca(2+) channels at the sensory nerve terminal in the spinal dorsal horn of mice. Furthermore, the present data provide evidence that the neuropathic pain may be effectively controlled by repeated treatment with GBP at the early stage.     

Tom20 recognizes mitochondrial presequences through dynamic equilibrium among multiple bound states

Most mitochondrial proteins are synthesized in the cytosol and imported into mitochondria. The N-terminal presequences of mitochondrial-precursor proteins contain a diverse consensus motif (phi chi chi phi phi, phi is hydrophobic and chi is any amino acid), which is recognized by the Tom20 protein on the mitochondrial surface. To reveal the structural basis of the broad selectivity of Tom20, the Tom20-presequence complex was crystallized. Tethering a presequence peptide to Tom20 through a disulfide bond was essential for crystallization. Unexpectedly, the two crystals with different linker designs provided unique relative orientations of the presequence with respect to Tom20, and neither configuration could fully account for the hydrophobic preference at the three hydrophobic positions of the consensus motif. We propose the existence of a dynamic equilibrium in solution among multiple states including the two bound states. In accordance, NMR 15N relaxation analyses suggested motion on a sub-millisecond timescale at the Tom20-presequence interface. We suggest that the dynamic, multiple-mode interaction is the molecular mechanism facilitating the broadly selective specificity of the Tom20 receptor toward diverse mitochondrial presequences.     

Cyclic peptide analysis of the biologically active loop region in the laminin alpha3 chain LG4 module demonstrates the importance of peptide conformation on biological activity

The laminin alpha3 chain LG4 module (alpha3LG4 module) has cell adhesion, heparin binding, migration, and neurite outgrowth activities. The LG4 module consists of a 14-stranded beta-sheet (A-N) sandwich structure. Previously, we identified the A3G756 sequence (KNSFMALYLSKGRLVFALG in the human laminin alpha3 chain 1411-1429) as a biologically active site in the alpha3LG4 module. The A3G756 sequence is located on the E and F strands based on a crystal structure-based sequence alignment. The Lys1421 and Arg1423 residues, critical amino acids for the biological activity of A3G756, are located on the E-F connecting loop region as a KGR sequence. In this study, we focused on the KGR sequence and investigated the structural requirements of the E-F connecting loop region in the alpha3LG4 module. We synthesized three linear peptides containing the KGR sequence at the middle and the N and C termini and also prepared three cyclic analogues corresponding to the linear peptides. cyclo-hEF3A (CLYLSKGRLVFAC), which is a cyclic peptide containing the KGR sequence at the middle, showed the strongest inhibitory effect on both the heparin binding and the cell attachment to the recombinant alpha3LG4 module protein. The cyclo-hEF3A peptide was more active for syndecan-4 binding and neurite outgrowth than the linear form. Furthermore, we found that the structure of cyclo-hEF3A is similar to that of the connecting E-F loop region in human laminin alpha3LG4 module by structural analysis using molecular dynamics simulations. These results suggest that the loop structure of the E-F connecting region of the alpha3LG4 module is important for its biological activities. The cyclo-hEF3A peptide may be useful for the development of therapeutic reagents especially for wound healing and nerve regeneration.     

Parasporin-2Ab,a Newly Isolated Cytotoxic Crystal Protein from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

A novel crystal protein that exhibited potent cytotoxicity against human leukemic T-cells was cloned from the Bacillus thuringiensis TK-E6 strain. The protein, designated as parasporin-2Ab (PS2Ab), was a polypeptide of 304 amino acid residues with a predicted molecular weight of 33,017. The deduced amino acid sequence of PS2Ab showed significant homology (84% identitiy) to parasporin-2Aa (PS2Aa) from the B. thuringiensis A1547 strain. Upon processing of PS2Ab with proteinase K, the active form of 29 kDa was produced. The activated PS2Ab showed potent cytotoxicity against MOLT-4 and Jurkat cells and the EC₅₀ values were estimated as 0.545 and 0.745 ng/mL, respectively. The cytotoxicity of PS2Ab was significantly higher than that of PS2Aa reported elsewhere. Although both cytotoxins were structurally related, it was thought that the minor differences found were responsible for the different cytotoxicities of PS2Ab and PS2Aa.     

Design, synthesis, and evaluation of isoindolinone-hydroxamic acid derivatives as histone deacetylase (HDAC) inhibitors

We designed and synthesized hydroxamic acid derivatives bearing a 4-(3-pyridyl)phenyl group as a cap structure, and found that they exhibit potent histone deacetylase (HDAC) inhibitory activity. A representative compound, 17a, showed more potent growth-inhibitory activity against pancreatic cancer cells and greater upregulation of p21(WAF1/CIP1) expression than the clinically used HDAC inhibitor suberoylanilide hydroxamic acid (Zolinza).     

Discovery of (-)-6-[2-[4-(3-fluorophenyl)-4-hydroxy-1-piperidinyl]-1-hydroxyethyl]-3,4-dihydro-2(1H)-quinolinone--a potent NR2B-selective N-methyl D-aspartate (NMDA) antagonist for the treatment of pain

(-)-6-[2-[4-(3-Fluorophenyl)-4-hydroxy-1-piperidinyl]-1-hydroxyethyl]-3,4-dihydro-2(1H)-quinolinone was identified as an orally active NR2B-subunit selective N-methyl-d-aspartate (NMDA) receptor antagonist. It has very high selectivity for NR2B subunits containing NMDA receptors versus the HERG-channel inhibition (therapeutic index=4200 vs NR2B binding IC(50)). This compound has improved pharmacokinetic properties compared to the prototype CP-101,606.     

New oligosaccharyltransferase assay method

We developed a new in vitro assay for oligosaccharyltransferase (OST), which catalyzes the transfer of preassembled oligosaccharides on lipid carriers onto asparagine residues in polypeptide chains. The asparagine residues reside in the sequon, Asn-X-Thr/Ser, where X can be any amino acid residue except Pro. We demonstrate the potency of our assay using the OST from yeast. In our method, polyacrylamide gel electrophoresis is used to separate the glycopeptide products from the peptide substrates. The substrate peptide is fluorescently labeled and the formation of glycopeptides is analyzed by fluorescence gel imaging. Two in vitro OST assay methods are now widely used, but both the methods depend on previous knowledge of the oligosaccharide moiety: One method uses lectin binding as the separation mechanism and the other method uses biosynthetically or chemoenzymatically synthesized lipid-linked oligosaccharides as donors. N-linked protein glycosylation is found in all three domains of life, but little is known about the N-glycosylation in Archaea. Thus, our new assay, which does not require a priori knowledge of the oligosaccharides, will be useful in such cases. Indeed, we have detected the OST activity in the membrane fraction from a hyperthermophilic archaeon, Pyrococcus furiosus.     

Comparison of stream benthic invertebrate assemblages among forest types in the temperate region of Japan

We compared benthic invertebrate assemblages among headwater streams in several forest types in Japan. Forests were divided into three clusters based on vegetation composition: old-growth broad-leaved forest, planted coniferous forest, and mixed forest. The numbers of individuals and families and the diversity (Shannon-Wiener) of benthic invertebrate assemblages did not differ significantly among the three forest clusters. However, principal components analysis of family abundance showed differences in the benthic invertebrate assemblages among the three forest clusters. No environmental factors were correlated with these differences. Benthic invertebrate assemblages differed depending on forest composition. The abundances of Taeniopterygidae and Athericidae in old-growth broad-leaved forest were significantly greater than in planted coniferous forest. The abundances of Heptageniidae, Baetiidae, Stenopsychidae, Uenoidae, Chironomidae, and Potamidae in planted coniferous forest were significantly greater than in old-growth broad-leaved forest. If the remaining old-growth broad-leaved forest were to be converted to coniferous plantation, species that inhabit old-growth forest may become extinct.