Complex formation by the mrpABCDEFG gene products, which constitute a principal Na+/H+ antiporter in Bacillus subtilis

The Bacillus subtilis Mrp (also referred to as Sha) is a particularly unusual Na(+)/H(+) antiporter encoded by mrpABCDEFG. Using His tagging of Mrp proteins, we showed complex formation by the mrpABCDEFG gene products by pull-down and blue native polyacrylamide gel electrophoresis analyses. This is the first molecular evidence that the Mrp is a multicomponent antiporter in the cation-proton antiporter 3 family.     

Both mammalian PIG-M and PIG-X are required for growth of GPI14-disrupted yeast

GPI mannosyltransferase I (GPI-MT-I) transfers the first mannose to a GPI-anchor precursor, glucosamine-(acyl)phosphatidylinositol [GlcN-(acyl)PI]. Mammalian GPI-MT-I consists of two components, PIG-M and PIG-X, which are homologous to Gpi14p and Pbn1p in Saccharomyces cerevisiae, respectively. In the present study, we disrupted yeast GPI14 and analysed the phenotype of gpi14 yeast. The gpi14 haploid cells were inviable and accumulated GlcN-(acyl)PI. We cloned PIG-M homologues from human, Plasmodium falciparum (PfPIG-M) and Trypanosoma brucei (TbGPI14), and tested whether they could complement gpi14-disrupted yeast. None of them restored GPI-MT-I activity and cell growth in gpi14-disrupted yeast. However, gpi14-disrupted yeast cells with human PIG-M, but not with PfPIG-M or TbGPI14, grew slowly but significantly when they were supplemented with rat PIG-X. This suggests that the association of PIG-X and PIG-M for GPI-MT-I activity is not interchangeable between mammals and the other lower eukaryotes.     

Production of a recombinant chitin oligosaccharide deacetylase from Vibrio parahaemolyticus in the culture medium of Escherichia coli cells

An open reading frame (ORF) encoding chitin oligosaccharide deacetylase (Pa-COD) gene and its signal sequence was cloned from the Vibrio parahaemolyticus KN1699 genome and its sequence was analyzed. The ORF encoded a 427 amino acid protein, including the 22 amino acid signal sequence. The deduced amino acid sequence was highly similar to several bacterial chitin oligosaccharide deacetylases in carbohydrate esterase family 4. An expression plasmid containing the gene was constructed and inserted into Escherichia coli cells and the recombinant enzyme was secreted into the culture medium with the aid of the signal peptide. The concentration of the recombinant enzyme in the E. coli culture medium was 150 times larger than that of wild-type enzyme produced in the culture medium by V. parahaemolyticus KN1699. The recombinant enzyme was purified to homogeneity from culture supernatant in an overall yield of 16%. Substrate specificities of the wild-type and the recombinant enzymes were comparable.     

Putative mechanism of the itch-scratch circle: repeated scratching decreases the cutaneous level of prostaglandin D2, a mediator that inhibits itching

In atopic dermatitis, scratching of the skin as a reaction to itching causes injury to the skin, which, in turn, further increases the itching resulting in the establishment of the so-called itch-scratch circle. We have shown that prostaglandin (PG) D2 plays an inhibitory role against pruritus in mice with atopic-like dermatitis; therefore, we examined the relationship between scratching and the cutaneous PGD2 level using an artificial scratching model with a wire brush. Mechanical scratching induced a temporary increase of the skin PGs levels (PGE2, PGD2, 6-ketoPGF1alpha, PGF2alpha). The skin PGD2 level and the ability of PGD2 production decreased at 48 h after repeated scratch, compared to that of normal skin, not so after single scratch. Immunohistochemical analysis and Western blotting revealed a decrease in the levels of cyclooxygenase-1 (COX-1) and hematopoietic PGD synthase in mechanically scratched skin. The reduced ability of the skin for PGD2 production following mechanical scratching could be caused by this decrease in the expression levels of COX-1 and PGD2 synthase. The results suggest that repeated scratching in mice decreases the ability of the skin to produce PGD2, which is an endogenous mediator that inhibits pruritus, resulting in the establishment of the itch-scratch circle.     

Design and synthesis of opioidmimetics containing 2',6'-dimethyl-L-tyrosine and a pyrazinone-ring platform

Twelve 2',6'-dimethyl-L-tyrosine (Dmt) analogues linked to a pyrazinone platform were synthesized as 3- or 6-[H-Dmt-NH(CH(2))(n)],3- or 6-R-2(1H)-pyrazinone (n=1-4). 3-[H-Dmt-NH-(CH(2))(4)]-6-beta-phenethyl-5-methyl-2(1H)-pyrazinone 11 bound to mu-opioid receptors with high affinity (K(i)mu=0.13 nM; K(i)delta/K(i)mu=447) with mu-agonism (GPI IC(50)=15.9 nM) and weak delta-antagonism (MVD pA(2)=6.35). Key factors affecting opioid affinity and functional bioactivity are the length of the aminoalkyl chain linked to Dmt and the nature of the R residue. These data present a simplified method for the formation of pyrazinone opioidmimetics and new lead compounds.     

Taxodistines A and B, abietane-type diterpenes from Taxodium distichum

Two new abietane-type diterpenes, taxodistines A (1) and B (2), have been isolated by the guidance of inhibitory effect of tubulin polymerization from the fruits of Taxodium distichum and the structures were elucidated by using 2D NMR data. Taxodistine B (2) showed inhibition of tubulin polymerization.