Orally administered FPRL1 receptor agonist peptide MMK-1 inhibits etoposide-induced alopecia by a mechanism different from intraperitoneally administered MMK-1

Oral administration for 6 days of 100 mg/kg MMK-1, an agonist peptide selective for the FPRL1 receptor, suppressed alopecia induced by the anticancer drug etoposide in neonatal rats. The anti-alopecia effect of orally administered MMK-1 was not inhibited by pyrilamine or cimetidine, antagonists for histamine H1 and H2 receptors, respectively, which blocked the anti-alopecia effect of intraperitoneally administered MMK-1 at a dose of 10 mg/kg for 4 days. However, the anti-alopecia effect of orally administered MMK-1 was inhibited by indomethacin, an inhibitor of cyclooxygenase (COX), or AH-23848B, an antagonist of the EP4 receptor for prostaglandin (PG) E2, suggesting involvement of PGE2 release and the EP4 receptor in the oral MMK-1 anti-alopecia mechanism. The anti-alopecia effect of orally administered MMK-1 was also blocked by an inhibitor of nuclear factor-kappaB (NF-kappaB), pyrrolidine dithiocarbamate, suggesting that the oral anti-alopecia effect of MMK-1 may be mediated by activation of NF-kappaB. These results suggest that MMK-1 bound to FPRL1 receptor might suppress etoposide-induced apoptosis of hair follicle cells and alopecia by way of PGE2 release and NF-kappaB activation.     

Trp3, Arg5]-ghrelin(1-5) stimulates growth hormone secretion and food intake via growth hormone secretagogue (GHS) receptor

Ghrelin, a 28 amino acid peptide identified as an endogenous ligand for growth hormone secretagogue (GHS) receptor, stimulates food intake and growth hormone (GH) secretion. We designed low molecular weight peptides with affinity for the GHS receptor based on the primary structure of ghrelin. We found that [Trp3, Arg5]-ghrelin(1-5) (GSWFR), a novel pentapeptide composed of all L-amino acids, had affinity for the GHS receptor (IC50 = 10 microM). GSWFR stimulated GH secretion after intravenous or oral administration. Centrally administered GSWFR increased food intake in non-fasted mice. The orexigenic action of GSWFR was inhibited by a GHS receptor antagonist, [D-Lys3]-GH-releasing peptide-6, suggesting that GSWFR stimulated food intake through the GHS receptor. The orexigenic action of GSWFR was also inhibited by a neuropeptide Y (NPY) Y1 receptor antagonist, BIBO3304. These results suggest that the GSWFR-induced feeding is mediated by the NPY Y1 receptor.     

Structural dissection of the reaction mechanism of cellobiose phosphorylase

Cellobiose phosphorylase, a member of the glycoside hydrolase family 94, catalyses the reversible phosphorolysis of cellobiose into alpha-D-glucose 1-phosphate and D-glucose with inversion of the anomeric configuration. The substrate specificity and reaction mechanism of cellobiose phosphorylase from Cellvibrio gilvus have been investigated in detail. We have determined the crystal structure of the glucose-sulphate and glucose-phosphate complexes of this enzyme at a maximal resolution of 2.0 A (1 A=0.1 nm). The phosphate ion is strongly held through several hydrogen bonds, and the configuration appears to be suitable for direct nucleophilic attack to an anomeric centre. Structural features around the sugar-donor and sugar-acceptor sites were consistent with the results of extensive kinetic studies. When we compared this structure with that of homologous chitobiose phosphorylase, we identified key residues for substrate discrimination between glucose and N-acetylglucosamine in both the sugar-donor and sugar-acceptor sites. We found that the active site pocket of cellobiose phosphorylase was covered by an additional loop, indicating that some conformational change is required upon substrate binding. Information on the three-dimensional structure of cellobiose phosphorylase will facilitate engineering of this enzyme, the application of which to practical oligosaccharide synthesis has already been established.     

Association study on chromosome 20q11.21-13.13 locus and its contribution to type 2 diabetes susceptibility in Japanese

Several linkage studies have predicted that human chromosome 20q is closely related to type 2 diabetes, but there is no clear evidence that certain variant(s) or gene(s) have strong effects on the disease within this region. To examine disease susceptibility variant in Japanese, verified SNPs from the databases, with a minor allele frequency larger than 0.15, were selected at 10-kb intervals across a 19.31-Mb region (20q11.21-13.13), which contained 291 genes, including hepatocyte nuclear factor 4α (HNF4α). As a result, a total of 1,147 SNPs were genotyped with TaqMan assay using 1,818 Japanese samples. By searching for HNF4α as a representative disease-susceptible gene, no variants of HNF4α were strongly associated with disease. To identify other genetic variant related with disease, we designed an extensive two-stage association study (725 first and 1,093 second test samples). Although SNP1146 (rs220076) was selected as a landmark within the 19.31 Mb region, the magnitude of the nominal P value (P = 0.0023) was rather weak. Subsequently, a haplotype-based association study showed that two common haplotypes were weakly associated with disease. All of these tests resulted in non-significance after adjusting for Bonferroni’s correction and the false discovery rate to control for the impact of multiple testing. Contrary to the initial expectations, we could not conclude that certain SNPs had a major effect on this promising locus within the framework presented here. As a way to extend our observations, we emphasize the importance of a subsequent association study including replication and/or meta-analysis in multiple populations.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Carnitine/xenobiotics transporters in the human mammary gland epithelia, MCF12A

The barrier function of the human mammary gland collapses if challenged with cationic drugs, causing their accumulation in milk. However, underlying molecular mechanisms are not well understood. To gain insight into the mechanism, we characterized transport of organic cations in the MCF12A human mammary gland epithelial cells, using carnitine and tetraethylammonium (TEA) as representative nutrient and xenobiotics probes, respectively. Our results show that the mammary gland cells express mRNA and proteins of human (h) novel organic cation transporters (OCTN) 1 and hOCTN2 (a Na+-dependent carnitine carrier with Na+-independent xenobiotics transport function), which belong to the solute carrier superfamily (SLC) of transporters. Other SLC OCTs such as hOCT1 and extraneuronal monoamine transporter (EMT)/hOCT3 are also expressed at mRNA levels, but hOCT2 was undetectable. We further showed mRNA expression of ATB0+ (an amino acid transporter with a Na+/Cl(-)-dependent carnitine transport activity), and Fly-like putative transporter 2/OCT6 (a splice variant of carnitine transporter 2: a testis-specific Na+-dependent carnitine transporter). TEA uptake was pH dependent. Carnitine uptake was dependent on Na+, and partly on Cl-, compatible with hOCTN2 and ATB0+ function. Modeling analyses predicted multiplicity of the uptake mechanisms with the high-affinity systems characterized by K(m) of 5.1 microM for carnitine and 1.6 mM for TEA, apparently similar to the reported hOCTN2 parameter for carnitine, and that of EMT/hOCT3 for TEA. Verapamil, cimetidine, carbamazepine, quinidine, and desipramine inhibited the carnitine uptake but required supratherapeutic concentrations, suggesting robustness of the carnitine uptake systems against xenobiotic challenge. Our findings suggest functional roles of a network of multiple SLC organic cation/nutrient transporters in human mammary gland drug transfer.     

Ghrelin concentrations and cardiac vagal tone are decreased after pharmacologic and cognitive-behavioral treatment in patients with bulimia nervosa

Patients with bulimia nervosa (BN) have bulimic and depressive symptoms, which have been associated with abnormalities in the neuroendocrine and vagal systems. Subjects included twenty-four female drug-free outpatients with BN that were selected from patients seeking treatment for eating behavior in our hospital along with twenty-five age-matched healthy females who served as controls. We investigated ghrelin and leptin levels, cardiac vagal tone and sympathovagal balance, frequency of sets of binge-eating and vomiting episodes per week and the Profile of Mood States (POMS) depression scale in BN before and after a 16-week administration of the serotonin selective reuptake inhibitor (SSRI) paroxetine combined with cognitive-behavioral therapy. Compared to controls, the BN group had higher ghrelin levels and resting cardiac vagal tone, and lower leptin levels and resting cardiac sympathovagal balance before treatment, although there was a significant difference between the two groups for the body mass index (BMI). The elevated ghrelin levels (301.7 +/- 18.9 pmol/l, mean +/- SEM vs. 202.8 +/- 15.6 pmol/l, P < 0.01), cardiac vagal tone (2246.4 +/- 335.5 ms(2) vs. 1128.5 +/- 193.3 ms(2), P < 0.01), frequency of sets of binge-eating and purging episodes and T scores for the POMS depression scale were all significantly decreased after treatment despite similar BMI, percent body fat and leptin levels. In close association with cardiac vagal function and ghrelin recoveries, abnormal eating behavior and depressive symptoms improved, indicating the usefulness of these indexes in the assessment of clinical condition and therapeutic efficacy in BN.     

Syntheses, characterization, and DFT investigation of new mononuclear acetonitrile- and chloro-ruthenium(II) terpyridine complexes

A series of mononuclear acetonitrile complexes of the type [Ru(CH₃CN)(L)(terpy)]²⁺ {L = phen (1), dpbpy (3), and bpm (5)}, and their reference complexes [RuCl(L)(terpy)]⁺ {L = phen (2), dpbpy (4), and dpphen (6)} were prepared and characterized by electrospray ionization mass spectrometry, UV-vis spectroscopy, and cyclic voltammograms (CV). Abbreviations of the ligands (Ls) are phen = 1,10-phenanthroline, dpbpy = 4,4′-diphenyl-2,2′-bipyridine, bpm = 2,2′-bipyrimidine, dpphen = 4,7-diphenyl-1,10-phenanthroline, bpy = 2,2′-bipyridine, and terpy = 2,2′:6′,2″-terpyridine. The X-ray structures of the two complexes 2 and 3 were newly obtained. The metal-to-ligand charge transfer (MLCT) bands in the visible region for 1, 3, and 5 in acetonitrile were blue shifted relative to those of the reference complexes [RuCl(L)(terpy)]⁺. CV for all the [Ru(CH₃CN)(L)(terpy)]²⁺ complexes showed the first oxidation wave at around 0.95 V, being more positive than those of [RuCl(L)(terpy)]⁺. The time-dependent-density-functional-theory approach (TDDFT) was used to interpret the absorption spectra of 1 and 2. Good agreement between computed and experimental absorption spectra was obtained. The DFT approach also revealed the orbital interactions between Ru(phen)(terpy) and CH₃CN or Cl⁻. It is demonstrated that the HOMO-LUMO energy gap of the acetonitrile ligand is larger than that of the Cl⁻ one.     

Identification of oxidized methionine sites in erythrocyte membrane protein by liquid chromatography/electrospray ionization mass spectrometry peptide mapping

In this study, we used peptide mapping combined with liquid chromatography/electrospray ionization mass spectrometry (LC/ESI MS) to examine the methionine oxidation of band 3 of erythrocyte membrane protein. Initially, we identified the methionine sites oxidized by chloramine T (N-chloro-p-toluenesulfoamide), a hydrophilic reagent. There were three oxidized methionines (Met 559, Met 741, and Met 909) in band 3, and these methionines were located in a hydrophilic region determined by previous topological studies of band 3. In addition, we found that C12E8, a polyoxyethylene detergent, leads to the oxidation of methionines in a transmembrane segment in band 3, and this oxidation occurs in a C12E8 preincubation time-dependent manner. In a previous study, it was found that peroxides accumulate in a polyoxyethylene detergent. Thus, our method enabled the direct and quantitative detection of protein damage due to detergent peroxides. Furthermore, we examined methionine oxidation in the presence of 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS) or diethyl pyrocarbonate (DEPC), which induced either an outward or an inward conformation in band 3, respectively. Our results indicated that the location of Met 741 was associated with the band 3 conformation induced by band 3-mediated anion transport. In conclusion, we found that methionine oxidation can be applied to examine membrane protein structures as follows: (1) for topological studies of membrane proteins, (2) for assessing the quality of proteins in detergent solubilization studies, and (3) for the detection of conformational changes in membrane proteins.     

Proton pumping mechanism of bovine heart cytochrome c oxidase

X-ray structures of bovine heart cytochrome c oxidase at 1.8/1.9 A resolution in the oxidized/reduced states exhibit a redox coupled conformational change of an aspartate located near the intermembrane surface of the enzyme. The alteration of the microenvironment of the carboxyl group of this aspartate residue indicates the occurrence of deprotonation upon reduction of the enzyme. The residue is connected with the matrix surface of the enzyme by a hydrogen-bond network that includes heme a via its propionate and formyl groups. These X-ray structures provide evidence that proton pumping occurs through the hydrogen bond network and is driven by the low spin heme. The function of the aspartate is confirmed by mutation of the aspartate to asparagine. Although the amino acid residues of the hydrogen bond network and the structures of the low spin heme peripheral groups are not completely conserved amongst members of the heme-copper terminal oxidase superfamily, the existence of low spin heme and the hydrogen bond network suggests that the low spin heme provides the driving element of the proton-pumping process.     

Translesion synthesis past tamoxifen-derived DNA adducts by human DNA polymerases eta and kappa

The long-term treatment of tamoxifen (TAM), widely used for adjuvant chemotherapy and chemoprevention for breast cancer, increases a risk of developing endometrial cancer. A high frequency of K-ras mutations has been observed in the endometrium of women treated with TAM. Human DNA polymerase (pol) eta and pol kappa are highly expressed in the reproductive organs and are associated with translesion synthesis past bulky DNA adducts. To explore the miscoding properties of alpha-(N2-deoxyguanosinyl)tamoxifen (dG-N2-TAM), a major TAM-DNA adduct, site-specifically modified oligodeoxynucleotides containing a single diastereoisomer of trans or cis forms of dG-N2-TAM were prepared by phosphoramidite chemical procedure and used as templates. The primer extension reaction catalyzed by pol kappa deltaC, a truncated form of pol kappa, extended more efficiently past the adduct than that of pol eta by incorporating dCMP, a correct base, opposite the adduct. With pol eta, all diastereoisomers of dG-N2-TAM promoted small amounts of direct incorporation of dAMP and deletions. With pol kappa deltaC, dG-N2-TAM promoted small amounts of dTMP and/or dAMP incorporations and deletions. The miscoding properties varied depending on the diastereoisomer of dG-N2-TAM adducts and the DNA pol used. Steady-state kinetic studies were also performed using either the nonspecific sequence or the K-ras gene sequence containing a single dG-N2-TAM at the second base of codon 12. With pol eta, the bypass frequency past the dA x dG-N2-TAM pair positioned in the K-ras sequence was only 2.3 times lower than that for the dC x dG-N2-TAM pair, indicating that dG-N2-TAM in the K-ras sequence has higher miscoding potential than that in the nonspecific sequence. However, with pol kappa deltaC, the bypass frequency past the dC x dG-N2-TAM pair was higher than that of the dT x dG-N2-TAM pair in both sequences. The properties of pol eta and pol kappa are consistent with the mutagenic events attributed to TAM-DNA adducts.