B. Yukihiro Hiraoka

Multidrug resistance gene 1 expression in salivary gland adenocarcinomas and oral squamous-cell carcinomas

In combined chemotherapy for head‐and‐neck cancer (HNC), salivary gland‐cell adenocarcinoma (SGA) shows insufficient clinical outcome, and it has been suggested that the sensitivity and/or the mechanism of resistance to anti‐cancer drugs are different between SGA and oral squamous‐cell carcinoma (SCC). The aim of our study was to clarify whether P‐glycoprotein (P‐gp) expression is associated with multidrug resistance (MDR) in HNC and the difference in the process of its development between SGA and SCC. In immunohistochemical analysis, P‐gp expression was found in the ductal cells of salivary glands but not in oral mucosal epithelium. In cancer tissues, a few SCC cells in 12 of 37 and most cells in all SGAs expressed P‐gp. The intensive P‐gp expression was significantly found in SGA compared with SCC. In an in vivo chemotherapeutic model using tumor‐bearing nude mice, P‐gp expression in counterparts was observed in only a few cells of the HSY line, while no P‐gp expression was observed in Hepd cells. However, P‐gp expression was developed in both HSY and Hepd cell lines after vincristine (VCR) treatment. RT‐PCR showed that the mean ratios of mdr1 mRNA expression levels in HSY clones were 3.7‐fold higher than those in Hepd clones after VCR treatment, while each cell line exhibited both induction and activated production of P‐gp. These results suggest that P‐gp–related MDR in SGA is an inherent phenotype caused by both high levels of P‐gp induction and activated P‐gp production during VCR treatment, while that in SCC is an acquired phenotype chiefly caused by induction of P‐gp.

Depth of side-chain pocket in the S2 subsite of dipeptidyl peptidase IV.

Kinetic studies of pig kidney dipeptidyl peptidase IV (dipeptidyl-peptide hydrolase, EC 3.4.14.5) were carried out using substrates possessing a side-chain of different length at the P2 position (or amino-terminal position in this case) such as Lys-, Arg-, Phe-, Met-, Ser-, His-, Glu- and Gly-Pro-pNA. The hydrolytic coefficient (Kcat/Km) has determined in the order Met- greater than Glu- greater than Ser- greater than His- greater than Phe- greater than Lys- greater than Gly- greater than Arg-, indicating a gradual increase with elongation of the side-chain from 0.03 to 0.60 nm followed by a decline when side-chain length approached 0.70 nm. Thus, the most probable depth of the side-chain pocket at the S2 subsite of the enzyme is proposed to be 0.50-0.60nm.

Bone Formation Is Coupled to Resorption Via Suppression of Sclerostin Expression by Osteoclasts

Bone formation is coupled to bone resorption throughout life. However, the coupling mechanisms are not fully elucidated. Using Tnfrsf11b‐deficient (OPG–/–) mice, in which bone formation is clearly coupled to bone resorption, we found here that osteoclasts suppress the expression of sclerostin, a Wnt antagonist, thereby promoting bone formation. Wnt/β‐catenin signals were higher in OPG–/– and RANKL‐transgenic mice with a low level of sclerostin. Conditioned medium from osteoclast cultures (Ocl‐CM) suppressed sclerostin expression in UMR106 cells and osteocyte cultures. In vitro experiments revealed that osteoclasts secreted leukemia inhibitory factor (LIF) and inhibited sclerostin expression. Anti‐RANKL antibodies, antiresorptive agents, suppressed LIF expression and increased sclerostin expression, thereby reducing bone formation in OPG–/– mice. Taken together, osteoclast‐derived LIF regulates bone turnover through sclerostin expression. Thus, LIF represents a target for improving the prolonged suppression of bone turnover by antiresorptive agents.

High-performance liquid chromatographic determination of prolylcarboxypeptidase activity in monkey kidney

A simple HPLC procedure for the determination of prolylcarboxypeptidase activity in monkey kidney was established with Cbz-Pro-Ala used as substrate. Decrease of the substrate and increase of the product were stoichiometrically related to each other. Heat treatment at 60 degrees C freed the enzyme preparation of contaminating activities. Data on substrate specificity and influence of inhibitors suggested this method was sensitive for the determination of prolylcarboxypeptidase without the use of a radioactive substrate.

Conversion of the metal-specific activity of Escherichia coli Mn-SOD by site-directed mutagenesis of Gly165Thr

Glycine 165, which is located near the active site metal, is mostly conserved in aligned amino acid sequences of manganese-containing superoxide dismutase (Mn-SOD) proteins, but is substituted to threonine in most iron-containing SODs (Fe-SODs). Because threonine 165 is located between Trp128 and Trp130, and Trp128 is one of the metal-surrounding aromatic amino acids, the conversion of this amino acid may affect the metal-specific activity of Escherichia coli Mn-SOD. In order to clarify this possibility, we prepared a mutant of E. coli Mn-SOD with the replacement of Gly165 by Thr. The ratio of the specific activities of Mn- to Fe-reconstituted enzyme increased from 0.006 in the wild-type to 0.044 in the mutant SOD; therefore, the metal-specific SOD was converted to a metal-tolerant SOD. The visible absorption spectra of the Fe- and Mn-reconstituted mutant SODs indicated the loss of Mn-SOD character. It was concluded that Gly at position 165 plays a catalytic role in maintaining the integrity of the metal specificity of Mn-SOD.

Purification and characterization of tripeptide aminopeptidase from bovine dental follicles

Tripeptide aminopeptidase (EC 3.4.11.4) was purified from bovine dental follicles by a series of chromatographies. Purified enzyme had a specific activity of 59.5 units per mg protein with L-prolyl-glycylglycine as substrate. The pH optimum was 8.0. The purified native enzyme had a Mr of 230,000 and was shown to be a tetramer of subunit Mr of 58,000. The isoelectric point was pH 7.0. The enzyme was inhibited 5-5′-dithio-bis (2-nitrobenzoic acid),o-phenanthroline, and bestatin. Substrate specificity studies indicated that the enzyme specifically hydrolyzes the N-terminal amino acid residue from tripeptides only.

Porphyromonas gingivalis hydrogen sulfide enhances methyl mercaptan-induced pathogenicity in mouse abscess formation

Porphyromonas gingivalis produces hydrogen sulfide (H2S) from l-cysteine. However, the role of H2S produced by P. gingivalis in periodontal inflammation is unclear. In this study, we identified the enzyme that catalyses H2S production from l-cysteine and analysed the role of H2S using a mouse abscess model. The enzyme identified was identical to methionine γ-lyase (PG0343), which produces methyl mercaptan (CH3SH) from l-methionine. Therefore, we analysed H2S and CH3SH production by P. gingivalis W83 and a PG0343-deletion mutant (ΔPG0343) with/without l-cysteine and/or l-methionine. The results indicated that CH3SH is produced constitutively irrespective of the presence of l-methionine, while H2S was greatly increased by both P. gingivalis W83 and ΔPG0343 in the presence of l-cysteine. In contrast, CH3SH production by ΔPG0343 was absent irrespective of the presence of l-methionine, and H2S production was eliminated in the absence of l-cysteine. Thus, CH3SH and H2S production involves different substrates, l-methionine or l-cysteine, respectively. Based on these characteristics, we analysed the roles of CH3SH and H2S in abscess formation in mice by P. gingivalis W83 and ΔPG0343. Abscess formation by P. gingivalis W83, but not ΔPG0343, differed significantly in the presence and absence of l-cysteine. In addition, the presence of l-methionine did not affect the size of abscesses generated by P. gingivalis W83 and ΔPG0343. Therefore, we conclude that H2S produced by P. gingivalis does not induce inflammation; however, H2S enhances inflammation caused by CH3SH. Thus, these results suggest the H2S produced by P. gingivalis plays a supportive role in inflammation caused by methionine γ-lyase.

Membrane Pro-X carboxypeptidase

Publisher Summary This chapter focuses on the structural chemistry and the biological aspects of membrane Pro-X carboxypeptidase. The enzyme catalyzes the hydrolysis of C-terminal amino acid residues from peptides bearing a penultimate (P1) praline residue. The purified kidney enzyme can hydrolyze peptide bonds, such as Pro–Phe, Pro–Met, Pro–Thr, Pro–Ala, Pro–Ile and Pro–Gly; whereas Pro–Pro, Pro–Hyp, Hyp–Gly and Pro–Leu bonds are not recognized as substrates. Membrane Pro-X carboxypeptidase has an M r of 240,000 and is a dimer of subunits of M r 135,000. The chemical analysis data indicates the enzyme to be a glycoprotein containing 33.2% (w/w) carbohydrate, with each subunit having one zinc atom. Membrane Pro-X carboxypeptidase cleaves the C-terminal Pro–Phe bond in angiotensins II (Asp-Arg-Val-Tyr-Ile-His-Pro–Phe) and III (des-Asp-angiotensin II). It has also been suggested to hydrolyze enterostatin, a powerfully anorectic pentapeptide that is produced by the tryptic cleavage of procolipase in the small intestine. Membrane Pro-X carboxypeptidase is distinguishable from serine-type carboxypeptidases and lysosomal Pro-X carboxypeptidase by its stability to heating and by its pH optimum. Unlike the serine enzymes, it has not been shown to be inhibited by DFP.