Nitric oxide-induced increase of excitatory amino acid levels in the trigeminal nucleus caudalis of the rat with tactile hypersensitivity evoked by the loose-ligation of the inferior alveolar nerves

To investigate whether or not N-methyl-D-aspartate (NMDA)/nitric oxide (NO) pathway in the trigeminal system is involved in the development and/or maintenance of such pathological pain states as the hyperalgesia and allodynia observed after dental surgery, we examined the alteration patterns of excitatory amino acid (EAA) level in the superficial layer of subnucleus caudalis of the brain-stem trigeminal sensory nuclear complex (SpVc-I,II) by in vivo microdialysis. A very high EAA release response was observed immediately after the start of the perfusion in ligated animals compared with sham-operated rats. The EAA level evoked by application of the 40-V tooth pulp-stimulation or 1% capsaicin cream was significantly higher in the ligated animals than those in the sham-operated animals. This increase of EAA level induced by capsaicin cream was inhibited by adding carboxy-PTIO (100 microM) to the perfusate. The applications of SNAP (2 mM) into the perfusate enhanced the level of EAAs in ligated animals and sham-operated animals. However, SNAP-evoked EAA levels in ligated animals were not significantly different compared with those of sham-operated animals. These results suggest that alterations in the stimulus-evoked raised EAA levels that occur in the site of the first synaptic relay of the dental pain pathway and which are expressed via endogenous NO, and which play an important role in development and/or maintenance of pathological pain states following dental peripheral nerve injury.     

Lhx2 mediates the activity of Six3 in zebrafish forebrain growth

The telencephalon shows the greatest degree of size variation in the vertebrate brain. Understanding the genetic cascade that regulates telencephalon growth is crucial to our understanding of how evolution of the normal human brain has supported such a variation in size. Here, we present a simple and quick approach to analyze this cascade that combines caged-mRNA technology and the use of antisense morpholino oligonucleotides in zebrafish embryos. Lhx2, a LIM-homeodomain protein, and Six3s (Six3b and Six3a), another homeodomain proteins, show very similar expression patterns early in forebrain development, and these are known to be involved in the growth of this part of the brain. The telencephalon of six3b and six3a double morphant (six3 morphant) embryos is markedly reduced in size due to impaired cellular proliferation. Head-specific overexpression of Lhx2 by photoactivation of a caged-lhx2 mRNA completely rescued this size reduction, whereas similar head-specific activation of Six3b could not rescue the knockdown effect of lhx2. In the forebrain of medaka embryos, Six3 facilitates cellular proliferation by sequestration of Geminin from Cdt1, a key component in the assembly of the prereplication complex. Our results suggest that Lhx2 may mediate an alternative or parallel pathway for control of cellular proliferation in the developing forebrain via Six3.     

Cooperative effect of hydrophobic and electrostatic forces on alcohol-induced alpha-helix formation of alpha1-acid glycoprotein

Alpha1-acid glycoprotein (AGP) is a serum glycoprotein that mainly binds basic drugs. Previous reports have shown that AGP converts from a beta-sheet to an alpha-helix upon interaction with biomembranes. In the current studies, we found that alkanols, diols, and halogenols all induce this conformational change. Increased length and bulkiness of the hydrocarbon group and the presence of a halogen atom promoted this conversion, whereas the presence of a hydroxyl group inhibited it. Moreover, the effect was dependent on the hydrophobic and electrostatic properties of the alcohols. These results indicate that, in a membrane environment, hydrophobic and electrostatic factors cooperatively induce the transition of AGP from a beta-sheet to an alpha-helix.     

Characterization of human stellate cell activation-associated protein and its expression in human liver

This study cloned cDNA of human homologue (hSTAP) of rat stellate cell activation-associated protein (rSTAP). hSTAP gene is on chromosome 17q and is composed of four exons. Various types of cells including hepatic stellate cells expressed hSTAP mRNA. Recombinant hSTAP was a heme protein with the activity of peroxidase. hSTAP can be used as a marker of quiescent stellate cells in human liver.     

Urinary Stone Analysis in a Patient with Hyperuricemia to Determine the Mechanism of Stone Formation

In order to determine the mechanism of urinary stone formation in patients with hyperuricemia, we analyzed the crystal components and matrix proteins in a urinary stone from such a patient. Micro-area X-ray spectrometry and infrared (IR) spectroscopy suggested that the outside of the stone was composed of calcium oxalate monohydrate (COM) and the inside of uric acid (UA). Proteomic analysis identified 37 and 14 proteins from the inside and outside of the stone, respectively, as matrix proteins. The proteins that were identified in an ethylenediaminetetraacetic acid (EDTA) fraction were able to bind calcium ions. Thus, calcium-binding proteins may play a significant role in the formation of urinary stones in patients with hyperuricemia.     

Crystal Structures of Copper-depleted and Copper-bound Fungal Pro-tyrosinase: INSIGHTS INTO ENDOGENOUS CYSTEINE-DEPENDENT COPPER INCORPORATION*

Tyrosinase, a dinuclear copper monooxygenase/oxidase, plays a crucial role in the melanin pigment biosynthesis. The structure and functions of tyrosinase have so far been studied extensively, but the post-translational maturation process from the pro-form to the active form has been less explored. In this study, we provide the crystal structures of Aspergillus oryzae full-length pro-tyrosinase in the holo- and the apo-forms at 1.39 and 2.05 Å resolution, respectively, revealing that Phe⁵¹³ on the C-terminal domain is accommodated in the substrate-binding site as a substrate analog to protect the dicopper active site from substrate access (proteolytic cleavage of the C-terminal domain or deformation of the C-terminal domain by acid treatment transforms the pro-tyrosinase to the active enzyme (Fujieda, N., Murata, M., Yabuta, S., Ikeda, T., Shimokawa, C., Nakamura, Y., Hata, Y., and Itoh, S. (2012) ChemBioChem. 13, 193–201 and Fujieda, N., Murata, M., Yabuta, S., Ikeda, T., Shimokawa, C., Nakamura, Y., Hata, Yl, and Itoh, S. (2013) J. Biol. Inorg. Chem. 18, 19–26). Detailed crystallographic analysis and structure-based mutational studies have shown that the copper incorporation into the active site is governed by three cysteines as follows: Cys⁹², which is covalently bound to His⁹⁴ via an unusual thioether linkage in the holo-form, and Cys⁵²² and Cys⁵²⁵ of the CXXC motif located on the C-terminal domain. Molecular mechanisms of the maturation processes of fungal tyrosinase involving the accommodation of the dinuclear copper unit, the post-translational His-Cys thioether cross-linkage formation, and the proteolytic C-terminal cleavage to produce the active tyrosinase have been discussed on the basis of the detailed structural information.     

Enzymatic characterization of an extracellular glucoamylase from Tricholoma matsutake and its cloning and secretory expression in Pichia pastoris

ABSTRACT

A glucoamylase from the ectomycorrhizal fungus Tricholoma matsutake (TmGLA) was purified 33.2-fold to homogeneity as a single monomeric glycoprotein with a molecular mass of 63.9 kDa. Maximum activity was observed at 60°C and pH 5.0. The enzyme is active down to 50°C and in the pH range of 4.0–6.0, and its activity is strongly inhibited by Ag⁺. It degrades α-1,4- and α-1,6-glycosidic linkages in various polysaccharides. Its gene (TmGlu1) was cloned using information from the enzyme’s internal amino acid sequences and the whole genome sequence of T. matsutake NBRC 30605. The deduced amino acid sequence showed clear homology with those of GH family 15 proteins. Pichia pastoris transformed with TmGlu1 secreted the active enzyme in a glycosylated form, and its characteristics were the same as the native enzyme.     

Properties of Two Inhibitors of Plant Virus Infection from Fruiting Bodies of Lentinus edodes and from Leaves of Yucca recurvifolia Salisb.

One of the inhibitors, named “fruiting body protein (FBP),” was purified from fruiting bodies of Lentinus edodes, and the other, named “yucca leaf protein (YLP),” from leaves of Yucca recurvifolia Salisb. The properties of these inhibitors were investigated, and the concentration of substances for a 50% inhibition ratio of TMV infection were measured. The inhibition ratios of YLP, FBP, Poly-Lys, Poly-Orn, Poly-Arg and cytochrome c were 0.6, 6.3, 14.1, 31.6, 44.7 and 100 ppm, respectively. Two inhibitors had no RNA hydrolyzing activity and no activity to TMV aggregation. FBP and YLP prevented infection of the plant by TMV when treated within 3 days before TMV inoculation, but not when treated within 1 hr for FBP or 3 hr for YLP after TMV inoculation. It seems that these two inhibitors had a preventive effect on plant virus infection, but no curative effect.