Characterization of a homologue of mammalian serine racemase from Caenorhabditis elegans: the enzyme is not critical for the metabolism of serine in vivo.

Free d-serine (d-Ser) plays a crucial role in regulating brain function in mammals. In various organisms, including mammals, d-Ser is biosynthesized by Ser racemase, a synthetic enzyme that produces d-Ser from l-Ser. Ser racemase also exhibits dehydratase activity toward several hydroxyamino acids. Thus, this enzyme is unique in that it possesses the capability to both synthesize and degrade d-Ser; however, the physiological significance of its degradative activity remains unclear. In contrast to the physiological roles of d-Ser in mammals, little is known about the role of this amino acid in lower organisms, including the nematode Caenorhabditis elegans. It is known that a mammalian Ser racemase homologue (T01H8.2) from C. elegans exhibits racemase activity. Here, the enzymatic properties of recombinant T01H8.2 were characterized and compared with those of recombinant human Ser racemase. Furthermore, the levels of several d- and l-amino acids were measured in wild-type C. elegans and in a mutant in which the T01H8.2 gene is partially deleted and thereby inactivated. The results indicate that T01H8.2 also shows dehydratase activity toward several hydroxyamino acids, although the enzyme is not critical for Ser metabolism in vivo. The possible physiological roles of T01H8.2 are discussed.

Development of nitrilase promoter-derived inducible vectors for Streptomyces.

An inducible expression vector, pSH19, which harbors regulatory expression system PnitA-NitR, for streptomycetes was constructed previously. Here, we have modified pSH19 to obtain shuttle vectors for Streptomyces-E. coli by introducing the replication origin of a plasmid for E. coli (ColE1) and an antibiotic-resistant gene. Six inducible shuttle vectors, pESH19cF, pESH19cR, pESH19kF, pESH19kR, pESH19aF, and pESH19aR, for Streptomyces-E. coli, were successfully developed. The stability of these vectors was examined in five different E. coli strains and Streptomyces lividans TK24. The stability test showed that the pSH19-derived shuttle vectors were stable in E. coli Stbl2 and S. lividans TK24. Heterologous expression experiments involving each of the catechol 2,3-dioxygenase, nitrilase, and N-substituted formamide deformylase genes as a reporter gene showed that pESH19cF, pESH19kF, and pESH19aF possess inducible expression ability in S. lividans TK24. Thus, these vectors were found to be useful expression tools for experiments on both Gram-negative and Gram-positive bacterial genes.

Characterization of the enzymatic and structural properties of human D-aspartate oxidase and comparison with those of the rat and mouse enzymes.

D-Aspartate (D-Asp), a free D-amino acid found in mammals, plays crucial roles in the neuroendocrine, endocrine, and central nervous systems. Recent studies have implicated D-Asp in the pathophysiology of infertility and N-methyl-D-Asp receptor-related diseases. D-Asp oxidase (DDO), a degradative enzyme that is stereospecific for acidic D-amino acids, is the sole catabolic enzyme acting on D-Asp in mammals. Human DDO is considered an attractive therapeutic target, and DDO inhibitors may be potential lead compounds for the development of new drugs against the aforementioned diseases. However, human DDO has not been characterized in detail and, although preclinical studies using experimental rodents are prerequisites for evaluating the in vivo effects of potential inhibitors, the existence of species-specific differences in the properties of human and rodent DDOs is still unclear. Here, the enzymatic activity and characteristics of purified recombinant human DDO were analyzed in detail. The kinetic and inhibitor-binding properties of this enzyme were also compared with those of purified recombinant rat and mouse DDOs. In addition, structural models of human, rat, and mouse DDOs were generated and compared. It was found that the differences among these DDO proteins occur in regions that appear involved in migration of the substrate/product in and out of the active site. In summary, detailed characterization of human DDO was performed and provides useful insights into the use of rats and mice as experimental models for evaluating the in vivo effects of DDO inhibitors.

Estimation of 1999-2004 dietary daily intake of PCDDs, PCDFs and dioxin-like PCBs by a total diet study in metropolitan Tokyo, Japan.

A study of the dietary intake of dioxins, consisting of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and dioxin-like polychlorinated biphenyls (dioxin-like PCBs) through foods retailed in the metropolitan Tokyo area from 1999 to 2004 was carried out by the total diet-market basket method on the basis of food classification (14 groups) and the data on food consumption in the Tokyo region obtained from the Japan Nutrition Survey. The daily intake of dioxins per kg of body weight for a 50 kg average adult body was 2.18 pg TEQ/kg/day in 1999, 1.87 pg TEQ/kg/day in 2000, 1.25 pg TEQ/kg/day in 2001, 1.60 pg TEQ/kg/day in 2002 and 2003 and 1.55 pg TEQ/kg/day in 2004, respectively. These amounts were less than the tolerable daily intake (TDI) of 4 pg TEQ/kg/day for dioxins established in Japan. The dioxins taken daily through fish and shellfish (group 10) accounted for more than 50% of sum WHO-TEQs. In addition, more than 90% of the daily intake of dioxins was taken through fish and shellfish (group 10), meat and eggs (group 11), milk and dairy products (group 12). Also, this study clearly showed that the ratio of dioxin-like PCBs in the daily intake of dioxins was increasing yearly because the reduction rate of dioxin-like PCBs was lower than that of PCDDs and PCDFs in foods.

Estrogen receptor-negative and human epidermal growth factor receptor 2-negative breast cancer tissue have the highest Ki-67 labeling index and EGFR expression: gene amplification does not contribute to EGFR expression.

Estrogen receptor (ER) and human epidermal growth factor 2 (HER2) are well-investigated molecules and the focus of many breast cancer therapies. There is a group of breast cancers lacking ER and HER2, but it is not fully understood. Treatment for these patients is limited to cytotoxic chemotherapy. The purpose of present study is to examine ER(-)/HER2(-) breast cancers, with a particular focus on epidermal growth factor receptor (EGFR). EGFR is a target molecule for which novel medicines have been recently developed for other organ cancers, however biological significance in breast cancer is not yet well demonstrated. Breast cancer specimens (n=58) were categorized into four groups: i) ER(+)/HER2(-) (51.7%); ii) ER(+)/HER2(+) (8.6%); iii) ER(-)/HER2(+) (20.7%); and iv) ER(-)/HER2(-) (19.0%). They were immunohistochemically (IHC) examined using antibodies for EGFR, platelet derived growth factor receptor (PDGFR)alpha, PDGFRbeta, parathyroid hormone (PTH) receptor, Ki-67, cyclinD1, p53, and vimentin. The Ki-67 labeling index (LI) was highest in ER(-)/HER2(-) (36.5%), and decreased in order from ER(-)/HER2(+) (31.4%), ER(+)/HER2(+) (17.7%), to (ER(+)/HER2(-) (15.9%) (p=0.001). EGFR, p53 and vimentin were highly expressed in ER(-)/HER2(-) breast cancer cells (p<0.01). CyclinD1 was inversely expressed to Ki-67 LI (p<0.001). Gene amplification of EGFR was examined by two in situ hybridization techniques, fluorescence in situ hybridization (FISH) and chromogenic in situ hybridization (CISH) in serial sections to IHC. Only 1 of 14 EGFR-positive breast cancers showed gene amplification at low levels by CISH. Overall, the ER(-)/HER2(-) breast cancer showed the highest Ki-67 LI, the most frequent expression of EGFR, p53 and vimentin, as well as the lowest expression of cyclinD1. It is unlikely that gene amplification contributes to EGFR expression. ER(-)/HER2(-) breast cancers have potential in the development of novel therapeutics, including targeted medicines.

Increased expression of matrix metalloproteinase-7 in invasive early gastric cancer.

BACKGROUND: Matrix metalloproteinase-7 (MMP-7), a proteolytic enzyme, is suspected to play an important role in the progression of various cancers. To clarify the clinical importance of MMP-7, we retrospectively analyzed MMP-7 expression in gastric epithelial tumors. METHODS: We tested 201 lesions (from 172 patients) of surgically or endoscopically resected gastric epithelial tumors (gastric cancer, 158 lesions; gastric adenoma, 32 lesions; hyperplastic polyp, 11 lesions). MMP-7 expression was immunohistochemically examined. Sections with immunostaining signals in more than 30% of tumor cells were judged to show positive expression. RESULTS: MMP-7 was expressed in 33.3% (67/201) of all lesions. MMP-7-positive tumors were significantly more frequent in diffuse-type adenocarcinomas (62.2%; 28/45) compared with intestinal-type lesions (31.9%; 36/113; P < 0.001). Cancers invading the submucosa or deeper (60.5%; 46/76) were showed positivity significantly more frequently than mucosal cancers (22.0%; 18/82; P < 0.001). MMP-7-positive lesions increased with the progression of gastric epithelial tumors, including adenomas, mucosal cancers, and cancers invading the submucosal layer or deeper (P < 0.001). MMP-7 expression occurred significantly more often in lymphatic invasion-positive cancers (65.1%; 41/63) than in lymphatic invasion-negative cancers (24.2%; 23/95; P < 0.001). CONCLUSIONS: The MMP-7-positive rate increased with the progression of gastric epithelial tumors, such as adenoma, mucosal cancer, and cancer invading the submucosal layer or deeper. MMP-7 was significantly associated with aggressive pathological phenotypes of cancer. The detection of the MMP-7 protein may be useful in pretherapeutic diagnosis.

Relationship between matrix metalloproteinase-7 and pit pattern in early stage colorectal cancer.

BACKGROUND: The surface pit pattern of early stage colorectal cancer changes with tumor growth and invasion. It was postulated by us that the expression of matrix metalloproteinase-7 is related to tumor invasiveness and disturbance of the pit pattern. METHODS: Sixty-eight colorectal epithelial tumors were examined, and the pit pattern was classified by stereoscopic microscopy. Immunostaining for matrix metalloproteinase-7 and its substrate laminin were performed. RESULTS: The rate of matrix metalloproteinase-7 positive staining was significantly higher for mucosal (70.6%) and submucosal cancer (80.0%) than for adenoma (18.6%) (p<0.0083). The rate of matrix metalloproteinase-7 positive specimens was significantly higher for type IV and type V compared with type III pit patterns. Where the tumor surface was positive for matrix metalloproteinase-7, expression of laminin was negative in 40% of specimens with a type IV pit pattern and 100% of those with the type V pit pattern. CONCLUSIONS: The results of this study indicate that expression of matrix metalloproteinase-7 is related to both the invasiveness of colorectal epithelial tumors and the disturbance of the pit pattern on the tumor surface.