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l ‐Methionine decarboxylase (MetDC) from Streptomyces sp. 590 is a vitamin B(6)‐dependent enzyme and catalyzes the non‐oxidative decarboxylation of l ‐methionine to produce 3‐methylthiopropylamine and carbon dioxide. We present here the crystal structures of the ligand‐free form of MetDC and of several enzymatic reaction intermediates. Group II amino acid decarboxylases have many residues in common around the active site but the residues surrounding the side chain of the substrate differ. Based on information obtained from the crystal structure, and mutational and biochemical experiments, we propose a key role for Gln64 in determining the substrate specificity of MetDC, and for Tyr421 as the acid catalyst that participates in protonation after the decarboxylation reaction.

Background: We previously reported the nutritional characteristics and effects of the DASH-JUMP diet, which is a WASHOKU-modified DASH diet, in Japanese participants with untreated high-normal blood pressure or stage 1 hypertension. The dietary adherence of the DASH diet in Japanese participants has never been evaluated before. Objective: We aimed to assess the relationships between dietary adherence, self-efficacy, and health behavior change among study participants who received the DASH-JUMP diet by home delivery. Methods: Participants were treated with the DASH-JUMP diet for 2 months and consumed their usual diets for the next 4 months. We conducted surveys using the stage of behavior change model questionnaire and the modified perceived health competence scale Japanese version questionnaire at baseline and 1, 2, 3, and 6 months to assess dietary adherence. Results: Forty-three participants (25 men, 18 women; mean age 53.6 ± 8.2 years) returned completed questionnaires, which we analyzed. Health behavior change was motivated by previous behavioral changes and improved biomarkers. The improvement and maintenance of self-efficacy were deeply related to health behavior change and previous self-efficacy. The experience of the DASH-JUMP study for participants included three processes to improve lifestyle habits: Phase 1, reflecting on previous lifestyle habits; Phase 2, learning through new experiences and the acquisition of knowledge; and Phase 3, desiring to maintain their own health. Conclusion: It indicated that the DASH-JUMP diet significantly increased self-efficacy and promoted health behavior change.

Oxidative stress, chronic inflammation, dyslipidemia, hyperglycemia, and shear stress (physical effect) are risk factors associated with the pathogenesis of atherosclerosis. Rice bran, a by-product of rice milling process, is known to house polyphenols and vitamins which exhibit potent antioxidant and anti-inflammatory properties. Through recent emerging knowledge of rice bran in health and wellness, the present study was aimed to assess the ameliorative effects of rice bran extracts (RBE) derived from Japanese colored rice varieties in modulating risk factors of atherosclerosis via in vitro and in vivo study models. Pre-treatment of lipopolysaccharide (LPS)-stimulated murine J774A.1 macrophage-like cells with RBE alleviated nitric oxide (NO) overproduction and downregulated gene expressions of pro-inflammatory modulators: tumor necrosis factor-α (TNF-α), interleukin (IL)-α (IL-1α), IL-1β, IL-6, and inducible nitric oxide synthase (iNOS). In addition, RBE also significantly attenuated LPS-stimulated protein expressions of iNOS, TNF-α, IL-1α, and IL-6 in J774A.1 macrophage-like cells as compared to non-treated LPS control group. In in vivo, 12 weeks of RBE dietary supplementations significantly reduced (p < 0.05) total cholesterol, triglycerides, and pro-atherogenic oxidized LDL/β2-glycoprotein I (oxLDL/β2GPI) complexes at plasma levels, in high fat diet (HFD) induced low density lipoprotein receptor knockout (Ldlr−/-) mice. En face pathological assessments of murine aortas also revealed significant reductions by 38% (p < 0.05) in plaque sizes of RBE-supplemented HFD mice groups as compared to non RBE-supplemented HFD control mice group. Moreover, gene expressions of aortic (iNOS, TNF-α, IL-1β) and hepatic (TNF-α, IL-1α, IL-1β) pro-inflammatory modulators were also downregulated in RBE-supplemented mice groups. Present study has revealed the potent health attributes and application of RBE as a dietary supplement to attenuate risks of inadvertent oxidative damage and chronic inflammation underlying the pathogenesis of atherosclerosis. Intrinsically, present preliminary findings may provide global health prospects for future dietary implementation of RBE in management of atherosclerosis., Food science; Food analysis; Rice bran extract (RBE); Functional food; Phytochemicals; Atherosclerosis; Oxidative stress; Inflammation; Antioxidant; Anti-inflammation; Oxidized lipoprotein (oxLDL).

For many years, clinical studies have suggested that blood levels of l-methionine and L-homocysteine correlate with health status or homocystinuria/hypermethioninemia. l-Methionine in a solution containing 0%, 10%, or 20% human serum was detected in 10–200 µM using l-methionine decarboxylase (MetDC). Spike and recovery tests showed that the enzymatic assay could accurately and reproducibly determine the increases in l-methionine in serum samples. These results suggest that our enzymatic method using MetDC is useful for primary screening of hypermethioninemia or homocystinuria based on serum l-methionine concentration. Additionally, we confirmed that l-methionine (100 nmol) in solution was degraded to less than the detection limit by incubation at 37ºC for 10 min using 2 U of MetDC. Therefore, l-homocysteine in serum samples can be detected with equivalent sensitivity using l-methionine γ-lyase (MGL), in solutions that either did not contain l-methionine or contained l-methionine preincubated with MetDC. Abbreviations DTT: dithiothreitol; IPTG: isopropyl-β-d-thiogalactopyranoside; KPB: potassium phosphate buffer; MBTH: 3-methyl-2-benzothiazolinonehydrazone; mdc: the gene coding l-methionine decarboxylase; MetDC: l-methionine decarboxylase; mgl: the gene coding l-methionine γ-lyase; MGL: l-methionine γ-lyase; PLP: pyridoxal 5ʹ-phosphate

Hyaluronan (HA) is an extracellular matrix (ECM) component of articular cartilage and has been used to treat patients with osteoarthritis (OA). A disintegrin and metalloproteinases with thrombospondin motifs (ADAMTSs) play an important role in cartilage degradation in OA. We have previously reported that ADAMTS4 and ADAMTS9 were induced by cytokine stimulation. However, the effect of HA on the cytokine-inducible ADAMTS9 has never been investigated. Moreover, it is unclear whether HA protects cartilage by suppressing aggrecan degradation. Here, we examined the effects of HA on ADAMTS expression in vitro and on cartilage degradation in vivo. ADAMTS9 expression was higher than that of the other aggrecanases (ADAMTS4 and 5) in human chondrocytes, chondrocytic cells, and rat cartilage. ADAMTS4 and 9 mRNA levels were upregulated in cytokine-stimulated chondrocytes and chondrocytic cells. Pre-incubation with HA significantly inhibited ADAMTS9 mRNA expression in cytokine-stimulated cells. In a rat OA model, Adamts5 and 9 mRNA levels were transiently increased after surgery; intra-articular HA injections attenuated the induction of Adamts5 and 9 mRNA. HA also blocked aggrecan cleavage by aggrecanase in OA rats in a molecular size-dependent manner. These results demonstrate that HA attenuates induced aggrecanases expression in OA and thereby protects articular cartilage degradation by this enzyme. Our findings provide insight into the molecular basis for the beneficial effects of HA in OA. © 2018 The Authors. Journal of Orthopaedic Research® Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 36:3247-3255, 2018.

Background We developed a WASHOKU-modified DASH diet named DASH-JUMP. We previously reported the hypotensive effect of the DASH-JUMP diet in Japanese participants with untreated high-normal Blood Pressure (BP) or stage 1 hypertension. Objective We aim to introduce the DASH-JUMP diet worldwide as a new lifestyle medicine. Accordingly, we prospectively assessed the nutritional characteristics of the DASH-JUMP diet. Methods Participants were treated with the DASH-JUMP diet for 2 months. Then, for 4 months after the intervention, they consumed their usual diets. We conducted a nutritional survey using the FFQg nutrient questionnaire at baseline and after 1, 2, 3, and 6 months. We received completed questionnaires from 55 participants (28 men and 27 women; mean age 54.2 ± 8.0 years) and analyzed them. Results The DASH-JUMP diet is rich in green-yellow vegetables, seaweed, milk, and mushrooms, while it has low contents of meat, eggs, confectionery, oils and fats, pickles, shellfish boiled in sweetened soy sauce, and fruits. Nutrients significantly associated with the observed change in systolic BP were niacin (P = 0.005) and carbohydrate (P = 0.033). The results of the FFQg questionnaire revealed that participants who had an increased BP at 1 month after ceasing the intervention had eating habits that broadly imitated the DASH-JUMP diet at 4 months after ceasing the intervention. Therefore, the systolic and diastolic BP values at 4 months after ceasing the intervention decreased significantly compared to those at baseline. Conclusion The DASH-JUMP diet may represent a new lifestyle medicine for reducing hypertension.

Idiopathic pulmonary fibrosis (IPF) is the most common and most deadly form of interstitial lung disease. Osteopontin (OPN), a matricellular protein with proinflammatory and profibrotic properties, plays a major role in several fibrotic diseases, including IPF; OPN is highly upregulated in patients' lung samples. In this study, we knocked down OPN in a bleomycin (BLM)-induced pulmonary fibrosis (PF) mouse model using small interfering RNA (siRNA) to determine whether the use of OPN siRNA is an effective therapeutic strategy for IPF. We found that fibrosing areas were significantly smaller in specimens from OPN siRNA-treated mice. The number of alveolar macrophages, neutrophils, and lymphocytes in bronchoalveolar lavage fluid was also reduced in OPN siRNA-treated mice. Regarding the expression of epithelial-mesenchymal transition (EMT)-related proteins, the administration of OPN-siRNA to BLM-treated mice upregulated E-cadherin expression and downregulated vimentin expression. Moreover, in vitro, we incubated the human alveolar adenocarcinoma cell line A549 with transforming growth factor (TGF)-beta 1 and subsequently transfected the cells with OPN siRNA. We found a significant upregulation of Col1A1, fibronectin, and vimentin after TGF-beta 1 stimulation in A549 cells. In contrast, a downregulation of Col1A1, fibronectin, and vimentin mRNA levels was observed in TGF-beta 1-stimulated OPN knockdown A549 cells. Therefore, the downregulation of OPN effectively reduced pulmonary fibrotic and EMT changes both in vitro and in vivo. Altogether, our results indicate that OPN siRNA exerts a protective effect on BLM-induced PF in mice. Our results provide a basis for the development of novel targeted therapeutic strategies for IPF.

A disintegrin and metalloproteinase with thrombospondin motif-1 (ADAMTS1) was initially cloned from a colon cachexia cell line. In the last 20 years, novel matrix metalloproteinase (MMP) genes were found, and in addition to their original members (MMPs and membrane-type MMPs), the current MMP family contains a disintegrin and metalloproteinases (ADAMs) and ADAMTS. ADAM and ADAMTS play essential roles in organogenesis as well as various diseases including osteoarthritis. ADAMTS has 19 members and can be divided into several groups according to their substrates. ADAMTS1, the first member of ADAMTS identified, is located on chromosome 21 very close to another ADAMTS member, ADAMTS5. Interestingly, ADAMTS1 is not highly expressed in normal tissues. One stimulation such as inflammation quickly induces ADAMTS1 expression. We found that hypoxia induced ADAMTS1 expression in endothelial cells, and serum ADAMTS1 levels were elevated in acute myocardial infarction patients. Once the artery was reperfused, the serum ADAMTS1 level quickly returned to the normal level. We also found that ADAMTS1 has specific roles in angiogenesis and lymphangiogenesis, and these functions were not related to its protease activity. It is also interesting that ADAMTS1 is likely to have a unique role in the tumor microenvironment. We also analyzed ADAMTS1-deficient mice and the results suggested that ADAMTS1 has diverse biological functions.

β2-Glycoprotein I (β2GPI) is a highly-glycosylated plasma protein composed of five homologous domains which regulates coagulation, fibrinolysis, and/or angiogenesis by interacting to negatively charged hydrophobic molecules and/or with plasminogen and its metabolites. The present study focused on structural and functional characterization of β2GPI's domain I (DI) and V (DV). Through N-terminal amino acid sequencing, a novel plasmin-cleaved site at K287 C288 was identified in DV. We further modified the intact DV by altering two amino acids at specific proteolytic cleavage sites to generate three stable DV mutants: DV(PP), (PE), and (AA). Results of both SDS-PAGE and MALDI-TOF-MS showed that all three DV mutants were more stable than the intact DV, and DV(PE) was predominantly resistant to proteolysis. Competitive ELISA assessed affinities of intact β2GPI and those mutants to cardiolipin. In culture system, all DV and DI mutants potently inhibited HUVEC's proliferation by 18–30% as compared to control. Only DI and nicked β2GPI showed significant inhibition in HUVEC's tube formation. Moreover, DV(PE)-coated affinity columns demonstrated its binding property towards anionic lipids and could substantially isolate anionic DOPS from zwitterionic DOPC as a purification model. In summary, the proteolytic resistant and unhindered phospholipid (PL) binding properties of DV(PE) have made it an appealing element for subsequent prospective studies. Future in-depth characterization and optimized applications of cleavage-resistant DV(PE) would complement its full capacity as a novel clinical modality in the field of vascular imaging and/or lipidomics studies.

The Dietary Approaches to Stop Hypertension (DASH) diet is recommended by the American Heart Association to lower blood pressure (BP); however, its effects in Japanese participants have not been rigorously studied. We assessed the effects of the DASH-Japan Ube Modified diet Program (DASH-JUMP), a modified DASH diet, on cardiometabolic and inflammatory biomarkers in Japanese participants with untreated high-normal BP or stage 1 hypertension. Fifty-eight participants (30 men and 28 women; mean age 54.1±8.1 years) with untreated high-normal BP or stage 1 hypertension followed the DASH-JUMP (salt 8.0 g per day) for 2 months. After the intervention period, they resumed their usual diets for 4 months. The DASH-JUMP significantly decreased the participants' body mass index values (24.6±3.5 kg m−2 at baseline→23.2±3.3 kg m−2 at 2 months, P=0.000), BP (153±14/91±11 mm Hg at baseline→130±16/80±9 mm Hg at 2 months, P=0.000 and 139±16/85±10 mm Hg at 6 months, P=0.000), fasting serum glucose level (100±26 mg dl−1→94±15 mg dl−1 at 2 months, P=0.003) and fasting insulin level (6.9±5.9 μIU ml−1→4.4±2.7 μIU ml−1 at 2 months, P=0.000). The mean compliance of the participants for the DASH-JUMP diet was 88.5%. The DASH-JUMP diet reduced cardiovascular risk factors and may be an effective nutritional strategy for preventing cardiovascular events.

In patients with osteoarthritis (OA), there is a decrease in both the concentration and molecular size of hyaluronan (HA) in the synovial fluid and cartilage. Cell migration-inducing hyaluronidase 1 (CEMIP), also known as hyaluronan (HA)-binding protein involved in HA depolymerization (HYBID), was recently reported as an HA depolymerization-related molecule expressed in the cartilage of patients with OA. However, the underlying mechanism of CEMIP regulation is not well understood. We found that CEMIP expression was transiently increased by interleukine-1&beta, (IL-1&beta, ) stimulation in chondrocytic cells. We also observed that ERK activation and NF-&kappa, B nuclear translocation were involved in the induction of CEMIP by IL-1&beta, In addition, both administration of HA and mechanical strain attenuated the CEMIP induction in IL-1&beta, stimulated chondrocytes. In conclusion, we clarified the regulatory mechanism of CEMIP in chondrocytes by inflammatory cytokines and suggested the potential involvement in osteoarthritis development.

In Japan, the number of elderly people who require long-term care is increasing as a result of the country’s aging population. Consequently, the burden experienced by caregivers who provide end-of-life care at home has become a social problem. This study aimed to confirm the factor structure of such caregiver burden by analyzing the Japanese version of the Zarit Caregiver Burden Interview (J-ZBI). The J-ZBI was administered to 389 caregivers providing end-of-life care, and 247 answers were analyzed, with exploratory factor analysis performed on the results. Consequently, a four-factor structure emerged (sacrificing life, personal strain, severe anxiety, and captivity); these four factors, constituting 15 items, were cumulatively named “J-ZBI_15.” In regard to reliability, Cronbach’sαcoefficient for each factor was high; in terms of validity, a confirmatory factor analysis was conducted to examine the four-factor structure, and the goodness of model fit was determined to be satisfactory. Further, the convergent validity was also high. The care burden experienced by those providing end-of-life care at home differs from the burden of caregivers of individuals with other diseases, such as Alzheimer’s. For assessing the burden felt by this population, the 15-item four-factor ZBI model is more appropriate than the single-factor 22-item ZBI, and we also determined that J-ZBI_8 is unsuitable for this task. Thus, measurement of family caregivers’ burden in regard to providing end-of-life care at home should be performed using the 15-item four-factor J-ZBI model.

The proteoglycan versican is implicated in growth and metastases of several cancers. Here we investigated a potential contribution of stromal versican to tumor growth and angiogenesis. We initially determined versican expression by several cancer cell lines. Among these, MDA-MB231 and B16F10 had none to minimal expression in contrast to Lewis lung carcinoma (LLC). Notably, tumors arising from these cell lines had higher versican levels than the cell lines themselves suggesting a contribution from the host-derived tumor stroma. In LLC-derived tumors, both the tumor and stroma expressed versican at high levels. Thus, tumor stroma can make a significant contribution to tumor versican content. Versican localized preferentially to the vicinity of tumor vasculature and macrophages in the tumor. However, an ADAMTS protease-generated versican fragment uniquely localized to vascular endothelium. To specifically determine the impact of host/stroma-derived versican we therefore compared growth of tumors from B16F10 cells, which produced littleversican, in Vcan hdf/+ mice and wild-type littermates. Tumors in Vcan hdf/+ mice had reduced growth with a lower capillary density and accumulation of capillaries at the tumor periphery. These findings illustrate the variability of tumor cell line expression of versican, and demonstrate that versican is consistently contributed by the stromal tissue, where it contributes to tumor angiogenesis.

L-Lysine α-oxidase (LysOX) from Trichoderma viride is a homodimeric 112 kDa flavoenzyme that catalyzes the oxidative deamination of L-lysine to form α-keto-ε-aminocaproate. LysOX severely inhibited growth of cancer cells but showed relatively low cytotoxicity for normal cells. We have determined the cDNA nucleotide sequence encoding LysOX from T. viride. The full-length cDNA consists of 2,119 bp and encodes a possible signal peptide (Met1-Arg77) and the mature protein (Ala78-Ile617). The LysOX gene have been cloned and heterologously expressed in Streptomyces lividans TK24 with the enzyme activity up to 9.8 U/ml. The enzymatic properties of the purified recombinant LysOX, such as substrate specificity and thermal stability, are same as those of native LysOX. The crystal structure of LysOX at 1.9 Å resolution revealed that the overall structure is similar to that of snake venom L-amino acid oxidase (LAAO), and the residues involved in the interaction with the amino or carboxy group of the substrate are structurally conserved. However, the entrance and the inner surface structures of the funnel to the active site, as well as the residues involved in the substrate side-chain recognition, are distinct from LAAOs. These structural differences well explain the unique substrate specificity of LysOX.

Hyaluronan (HA) is an extracellular matrix (ECM) component of articular cartilage and has been used to treat patients with osteoarthritis (OA). A disintegrin and metalloproteinases with thrombospondin motifs (ADAMTSs) play an important role in cartilage degradation in OA. We have previously reported that ADAMTS4 and ADAMTS9 were induced by cytokine stimulation. However, the effect of HA on the cytokine‐inducible ADAMTS9 has never been investigated. Moreover, it is unclear whether HA protects cartilage by suppressing aggrecan degradation. Here, we examined the effects of HA on ADAMTS expression in vitro and on cartilage degradation in vivo. ADAMTS9 expression was higher than that of the other aggrecanases (ADAMTS4 and 5) in human chondrocytes, chondrocytic cells, and rat cartilage. ADAMTS4 and 9 mRNA levels were upregulated in cytokine‐stimulated chondrocytes and chondrocytic cells. Pre‐incubation with HA significantly inhibited ADAMTS9 mRNA expression in cytokine‐stimulated cells. In a rat OA model, Adamts5 and 9 mRNA levels were transiently increased after surgery; intra‐articular HA injections attenuated the induction of Adamts5 and 9 mRNA. HA also blocked aggrecan cleavage by aggrecanase in OA rats in a molecular size‐dependent manner. These results demonstrate that HA attenuates induced aggrecanases expression in OA and thereby protects articular cartilage degradation by this enzyme. Our findings provide insight into the molecular basis for the beneficial effects of HA in OA. © 2018 The Authors. Journal of Orthopaedic Research® Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 36:3247–3255, 2018.

The synovial fluids of patients with osteoarthritis (OA) contain elevated levels of inflammatory cytokines, which induce the expression of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) and of the matrix metalloproteinase (MMP) in chondrocytes. Mechanical strain has varying effects on organisms depending on the strength, cycle, and duration of the stressor; however, it is unclear under inflammatory stimulation how mechanical strain act on. Here, we show that mechanical strain attenuates inflammatory cytokine-induced expression of matrix-degrading enzymes. Cyclic tensile strain (CTS), as a mechanical stressor, attenuated interleukin (IL)-1β and tumor necrosis factor (TNF)-α-induced mRNA expression of ADAMTS4, ADAMTS9, and MMP-13 in normal chondrocytes (NHAC-kn) and in a chondrocytic cell line (OUMS-27). This effect was abolished by treating cells with mechano-gated channel inhibitors, such as gadolinium, transient receptor potential (TRP) family inhibitor, ruthenium red, and with pharmacological and small interfering RNA-mediated TRPV1 inhibition. Furthermore, nuclear factor κB (NF-κB) translocation from the cytoplasm to the nucleus resulting from cytokine stimulation was also abolished by CTS. These findings suggest that mechanosensors such as the TRPV protein are potential therapeutic targets in treating OA.

l-Methionine decarboxylase (MetDC) from Streptomyces sp. 590 depends on pyridoxal 5'-phosphate and catalyzes the non-oxidative decarboxylation of l-methionine to produce 3-methylthiopropylamine and carbon dioxide. MetDC gene (mdc) was determined to consist of 1,674 bp encoding 557 amino acids, and the amino acid sequence is similar to that of l-histidine decarboxylases and l-valine decarboxylases from Streptomyces sp. strains. The mdc gene was cloned and recombinant MetDC was heterologously expressed by Escherichia coli. The purification of recombinant MetDC was carried out by DEAE-Toyopearl and Ni-NTA agarose column chromatography. The recombinant enzyme was homodimeric with a molecular mass of 61,000 Da and showed optimal activity between 45 to 55 °C and at pH 6.6, and the stability below 30 °C and between pH 4.6 to 7.0. l-Methionine and l-norleucine were good substrates for MetDC. The Michaelis constants for l-methionine and l-norleucine were 30 and 73 mM, respectively. The recombinant MetDC (0.50 U/ml) severely inhibited growth of human tumour cells A431 (epidermoid ovarian carcinoma cell line) and MDA-MB-231 (breast cancer cell line), however showed relatively low cytotoxicity for human normal cell NHDF-Neo (dermal fibroblast cell line from neonatal foreskin). This study revealed the properties of the gene and the protein sequence of MetDC for the first time.

Citation Inagaki J, Hao L, Nakatsuka M, Yasuda T, Hiramatsu Y, Shoenfeld Y, Matsuura E. A possible mechanism of autoimmune-mediated infertility in women with endometriosis. Am J Reprod Immunol 2011; 66: 90–99 Problem Endometriosis has been proposed to be an autoimmune disease because of the presence of a variety of autoantibodies specific for endometrial or ovarian antigens. The object of the present study is to characterize binding specificity of anti-laminin-111 autoantibodies in infertile patients with endometriosis and to investigate whether these autoantibodies affect the in vitro embryo development. Method of study An ELISA analysis using overlapping synthesized peptides that covered the entire G domain of laminin-α1 chain was performed in infertile patients with endometriosis (n = 45). Mouse blastocysts were cultured in media containing the purified IgG from one antibody-positive serum on laminin-111-coated dishes. Results Anti-laminin-111 autoantibodies were directed to several particular biologically functional peptide sequences in laminin-α 1 chain G domain. The tested IgG significantly inhibited the extent of in vitro trophoblast outgrowth. Conclusion Anti-laminin-111 autoantibodies may have major pathogenic roles on early reproductive failure including endometriosis-associated infertility.

ADAMTS1 (a disintegrin and metalloproteinase with thrombospondin motifs 1) is an early immediate gene. We have previously reported that ADAMTS1 was strongly induced by hypoxia. In this study, we investigated whether ADAMTS1 promoter-driven reporter signal is detectable by acute hypoxia. We constructed the GFP (green fluorescent protein) expression vector [AHR (acute hypoxia-response sequence)-GFP] under the control of ADAMTS1 promoter and compared it with the constitutive GFP-expressing vector under the control of CMV (cytomegalovirus promoter-GFP). We transduced AHR-GFP and examined whether GFP signals can be detected under the acute hypoxia. When the human umbilical vein [HUVEC (human umbilical vein endothelial cells)] was transduced under normoxia, there were few GFP signals, while CMV-GFP showed considerable GFP signals. When HUVEC was stimulated with hypoxia, GFP signals from AHR-GFP gene were induced under hypoxic conditions. Notably, the GFP signals peaked at 3 h under hypoxia. In ischaemic hind limb model, transduced AHR-GFP showed hypoxic induction of GFP signals. In summary, we have demonstrated that the AHR system induced the reporter gene expression by acute hypoxia, and its induction is transient. This is the first report showing the unique acute hypoxia-activated gene expression system.

Beta2-glycoprotein I (beta2-GPI) is a major antigen for anti-cardiolipin antibodies and their epitopes are cryptic. Conformation of each domain of beta2-GPI was optimized from its crystal structure by energy minimization and by molecular dynamics simulation. Three electrostatic interactions, i.e. D193-K246, D222-K317 and E228-K308, were observed between domains IV and V in the optimized structure that was constructed based on the consensus sequences obtained by the phage-displayed random peptide library. Antigenic structures determined by the epitope mapping mainly consisted of hydrophobic amino acids located on two discontinuous sequences in domain IV. These amino acid clusters, as an epitope, were covered by domain V and were of a hidden nature. A similar but incomplete counterpart to the epitopic clusters was found in domain I but was not in domains II or III. Binding of anti-beta2-GPI auto-antibodies to solid-phase beta2-GPI was significantly reduced either by L replacement for W235, a common amino acid component for the epitopes, or by V replacement for all of D193, D222 and E228. Structural analysis indicated a hypothesis that these electrostatic interactions between domains IV and V retained exposure to W235 and that epitope spreading occurred in the region surrounding W235. Thus, epitopic structures recognized by anti-beta2-GPI auto-antibodies are cryptic and inter-domain electrostatic interactions are involved in their in exposure.

Percentage finger systolic blood pressure (%FSBP) in response to finger cooling is used to assess vascular components of the hand-arm vibration syndrome and the measurement method is under discussion for standardization. It has been suggested that measurement circumstances including room temperature may affect %FSBP. We investigated the effect of room temperature on %FSBP response to finger cooling in healthy subjects. Six healthy male subjects who were medical students volunteered for the study. Multi-channel plethysmograph was used for simultaneous multi-finger FSBP measurements. The examination room was kept at 21 +/- 1 degrees C and 25 +/- 1 degrees C, and the subjects were randomly assigned. Percentage finger systolic blood pressures for the index, middle, ring and little fingers at 15 degrees C and 10 degrees C cuff-water temperatures were calculated. Four-way analysis of variance was performed to determine the independent effect of subject, room temperature, finger and cuff-water temperature factors on %FSBP. The room temperature as an independent factor affecting %FSBP was statistically significant (P0.01). From the results, it can be concluded that %FSBP response to finger cooling in healthy subjects may be affected by room temperature. Therefore, room temperature is expected to be controlled when assessing peripheral vascular components of the upper extremities using %FSBP response to finger cooling.

Laminin-1 is a major multifunctional glycoprotein that forms an integral part of the scaffolding network of basement membranes, and is the earliest synthesized component during embryogenesis. This protein (alpha1beta1gamma1) plays an important role in basement membrane assembly and epiblast differentiation during embryonic development. Anti-laminin-1 autoantibodies are known to cause infertility and recurrent spontaneous abortion in animals. Recently, we reported that the presence of IgG anti-laminin-1 antibodies (Abs) in the blood is significantly associated with recurrent first-trimester miscarriages and subsequent negative pregnancy outcomes. Interestingly, these antibodies are also strongly associated with infertility, especially infertility caused by endometriosis. Laminin-alpha1, laminin-beta1, and laminin-gamma1 mRNAs were also detected in 90% of endometriotic lesions, and all laminin-alpha1, laminin-beta1, and laminin-gamma1 chains were localized to the basement membranes of glandular epithelium in endometriotic peritoneal lesions. ELISA showed specific reactivity of the autoantibodies to a particular region of the laminin-1 molecule, that is, the alpha1 chain G domain. IgM monoclonal anti-laminin-1 Abs, which we recently established, also recognized the G domain and cross-reacted with human alpha1 chain located in the basement membrane of the glandular epithelium of human endometrium. We also established an animal model that produced high titers of anti-laminin-1 Abs after immunization with mouse laminin-1. Anti-laminin-1 Abs from the immunized mice caused a higher fetal resorption rate with lower embryonic and placental weights. Thus, anti-laminin-1 Abs may be important in the development of autoimmune-mediated reproductive failures, and the assessment of the such antibodies may provide a novel means for noninvasive diagnosis of endometriosis.

Laminin-1 is a structural glycoprotein that forms an integral part of the scaffolding of basement membranes, and plays an important role during embryonic development. We have recently demonstrated a significant association between anti-laminin-1 antibodies (Abs) and reproductive failure, such as recurrent spontaneous abortions and infertility-associated endometriosis in both human and mouse studies. In the present study, we established an IgM (μ,κ) monoclonal anti-laminin-1 Ab (AK8) by immunizing mice with mouse Engelbreth-Holm-Swarm sarcoma (EHS)-derived laminin-α1. The AK8 monoclonal antibody (mAb) reacted with particular peptide sequences from the globular G domain of mouse laminin-α1 chain of using ELISA and Western blot techniques. The peptide tertiary structure of the epitope recognized by AK8 mAb was predicted using eight synthesized domain peptide sequences and three consensus sequences obtained by phage displayed random peptide library. Basement membranes of endometrium of pregnant mice and humans were immunostained with AK8 mAb. Thus, AK8 mAb recognized a common structure present in the G domain of the laminin-1 chain in both mice and humans. The passive immunization of mice with AK8 mAb may represent a suitable animal model for anti-laminin-1 Ab-mediated reproductive failure.

Laminin-1 is a major component and multifunctional glycoprotein of basement membranes that consists of three different subunits, α1, β1 and γ1 chains. It is the earliest synthesized network-forming protein during embryogenesis and plays an important role in embryonic development, embryonic implantation and placentation. We have recently shown that IgG anti-laminin-1 antibodies were significantly associated with recurrent first-trimester miscarriages and with subsequent pregnancy outcome. Interestingly, these antibodies were also observed in patients with endometriosis-associated infertility but not in patients with other causes of infertility, including tubal factors, hormonal and uterine abnormalities. Laminin-α1, -β1 and -γ1 mRNAs have been detected in 90% of endometriotic lesions and all laminin-α1, -β1 and -γ1 chains were localized in the basement membranes of glandular epithelium in endometriotic peritoneal lesions. Western blot analysis showed that anti-laminin-1 antibodies from those patients reacted with all laminin-1's chains. ELISA also confirmed that one of the target epitopes for these antibodies was located in a particular region of the laminin-1 molecule, i.e. the carboxyl-terminal globular G domain of α1 chain. IgM monoclonal anti-laminin-1 autoantibody, that we recently established, also recognized the G domain. Anti-laminin-1 antibodies from mice immunized with –mouse— laminin-1, caused a higher fetal resorption rate with lower embryonic and placental weights. Thus, anti-laminin-1 antibodies may be important in development of autoimmune-mediated reproductive failures and the assessment of the antibodies may provide a novel non-invasive diagnosis of endometriosis.

The purpose of this study was to examine the influence of waterproof covering on finger skin temperature (FST) and hand pain during immersion test for diagnosing hand-arm vibration syndrome complying with the proposal of the International Organization for Standardization (ISO/ CD14835-1, 2001) for measurement procedure. Six healthy male subjects took part in the immersion tests and immersed their both hands into water at 12 degrees C for 5 min, repeatedly with two types of waterproof covering (polyethylene and natural rubber gloves) or without hand covering (bare hands) during immersion. The FST data from middle fingers and subjective pain scores for hand pain were analyzed. Statistically significant differences in FST among three conditions were observed showing the highest FST with natural rubber gloves, followed by the FST with polyethylene gloves and the lowest with bare hands. Significant differences in pain score among three conditions were observed during immersion showing the lowest pain score with natural rubber gloves, followed by the pain score with polyethylene gloves and the highest with bare hands. Immersion test with polyethylene gloves instead of bare hands during immersion seems to be suitable for reducing subject suffering.

PROBLEM: Laminins have important roles during placental and embryonic development. The aim of our study was to determine if active immunization of mice with laminin-1 could elicit an autoimmune response, and induce features of reproductive failure. METHOD OF STUDY: BALB/c mice were immunized with mouse laminin-1. Autoantibodies to laminin-1 were measured by enzyme-linked immunosorbent assay. Pregnant mice were killed on day 14 of pregnancy and examined for pregnancy outcome. RESULTS: Mice immunized with laminin-1 developed elevated levels of anti-laminin-1 auto-antibodies contrary to the control group. A higher fetal resorption rate was found in the laminin-1 immunized group (23.8%) compared with that of the control group (12.2%), and was even higher in the subgroup of those animals with very high levels of anti-laminin-1 (P

beta(2)-Glycoprotein I (beta(2)-GPI) is a major antigen for anticardiolipin antibodies (aCL, Abs) present in patients with antiphospholipid syndrome. We recently reported that beta(2)-GPI specifically binds to oxidized LDL (oxLDL) and that the beta(2)-GPI's major ligand, oxLig-1 is 7-ketocholesteryl-9-carboxynonanoate (Kobayashi, K., E. Matsuura, Q. P. Liu, J. Furukawa, K. Kaihara, J. Inagaki, T. Atsumi, N. Sakairi, T. Yasuda, D. R. Voelker, and T. Koike. 2001. A specific ligand for beta(2)-glycoprotein I mediates autoantibody-dependent uptake of oxidized low density lipoprotein by macrophages. J. Lipid Res. 42: 697-709). In the present study, we demonstrate that omega-carboxylated 7-ketocholesteryl esters are critical for beta(2)-GPI binding. A positive ion mass spectrum of a novel ligand, designated oxLig-2, showed fragmented ions at m/z 383 and 441 in the presence of acetone, which share features of oxLig-1 and 7-ketocholesterol. In the negative ion mode, ions at m/z 627, 625, and 243 were observed. oxLig-2 was most likely 7-ketocholesteryl-12-carboxy (keto) dodecanoate. These ligands were recognized by beta(2)-GPI. Liposome binding to macrophages was significantly increased depending on the ligand's concentration, in the presence of beta(2)-GPI and an anti-beta(2)-GPI Ab. Synthesized variant, 7-ketocholesteryl-13-carboxytridecanoate (13-COOH-7KC), also showed a significant interaction with beta(2)-GPI and a similar binding profile with macrophages. Methylation of the carboxyl function diminished all of the specific ligand interactions with beta(2)-GPI. Thus, omega-carboxyl variants of 7-ketocholesteryl esters can mediate anti-beta(2)-GPI Ab-dependent uptake of oxLDL by macrophages, and autoimmune atherogenesis linked to beta(2)-GPI interaction with oxLDL.

beta(2)-Glycoprotein I (beta(2)-GPI) is a major antigen for antiphospholipid antibodies (Abs) present in patients with the antiphospholipid syndrome (APS). We previously reported that beta(2)-GPI specifically binds to oxidized low density lipoprotein (oxLDL), but not to native low density lipoprotein (LDL). In the present study, a ligand specific for beta(2)-GPI, oxLig-1, was purified from the extracted lipids of oxLDL. The structure of oxLig-1 was shown to be identical to that of synthesized 7-ketocholesteryl-9-carboxynonanoate by mass spectroscopy and nuclear magnetic resonance analyses. Both purified and synthesized oxLig-1 were recognized by beta(2)-GPI and subsequently by anti-beta(2)-GPI auto-Abs, either in enzyme-linked immunosorbent assay (ELISA) or in ligand blot analysis. Binding of liposomes containing oxLig-1 (oxLig-1-liposomes) to mouse macrophages, J774A.1 cells, was relatively low, as compared with that of phosphatidylserine (PS)-liposomes. In contrast, binding of oxLig-1-liposomes was enhanced more than 10-fold in the presence of both beta(2)-GPI and an anti-beta(2)-GPI auto-Ab (WB-CAL-1), derived from (NZW x BXSB) F1 mouse, an animal APS model. Anti-beta(2)-GPI auto-Abs derived from APS patients with episodes of arterial thrombosis were detected in ELISA, using a solid phase oxLig-1 complexed with beta(2)-GPI. We suggest that autoimmune atherogenesis linked to beta(2)-GPI interaction with oxLDL and Abs may be present in APS.

Problem The present study assesses the clinical significance of anti-laminin-1 auto-antibodies (auto-Abs) in recurrent miscarriages. Method of study A total of 207 recurrent aborters with a history of two or more consecutive first-trimester miscarriages were tested for the presence of anti-laminin-1 Abs, beta2-glycoprotein I-dependent anticardiolipin Abs, lupus anticoagulants, anti-DNA Abs, and anti-nuclear Abs, before they had conceived again. Recurrent aborters then were followed up during subsequent pregnancies and their outcomes were evaluated relative to their blood test results prior to pregnancy. Results Fifty-five (31.1%) women out of 177 recurrent aborters were positive for IgG anti-laminin-1 auto-Abs. The levels of IgG anti-laminin-1 auto-Abs in recurrent aborters were significantly higher than those in healthy pregnant women and in healthy non-pregnant women (P = 0.0043 and 0.0073, respectively). The live birth rate of subsequent pregnancies in IgG anti-laminin-1 auto-Abs-positive recurrent aborters was significantly lower than the IgG anti-laminin-1 auto-Abs-negative recurrent aborters (P = 0.0320). There were no specifically significant relationships observed between IgG anti-laminin-1 auto-Abs and other tested auto-Abs. Conclusion IgG anti-laminin-1 auto-Abs are associated with recurrent miscarriages and the subsequent pregnancy outcome of recurrent aborters.

Hemoglobin A2 (alpha2delta2), which is present at low concentration (1-2%) in the circulating red cells of normal individuals, has two important features that merit its study, i.e., it inhibits polymerization of sickle HbS and its elevated concentration in some thalassemias is a useful clinical diagnostic. However, reports on its functional properties regarding O2 binding are conflicting. We have attempted to resolve these discrepancies by expressing, for the first time, recombinant hemoglobin A2 and systematically studying its functional properties. The construct expressing HbA2 contains only alpha and delta genes so that the extensive purification required to isolate natural HbA2 is circumvented. Although natural hemoglobin A2 is expressed at low levels in vivo, the amount of recombinant alpha2delta2 expressed in yeast is similar to that found for adult hemoglobin A and for fetal hemoglobin F when the alpha + beta or the alpha + gamma genes, respectively, are present on the construct. Recombinant HbA2 is stable, i.e., not easily oxidized, and it is a cooperative functional hemoglobin with tetramer-dimer dissociation properties like those of adult HbA. However, its intrinsic oxygen affinity and response to the allosteric regulators chloride and 2,3-diphosphoglycerate are lower than the corresponding properties for adult hemoglobin. Molecular modeling studies which attempt to understand these properties of HbA2 are described.

Angiogenesis and lymphangiogenesis play roles in malignant tumor progression, dissemination, and metastasis. ADAMTS1, a member of the matrix metalloproteinase family, is known to inhibit angiogenesis. Recombinant ADAMTS1 was shown to strongly inhibit angiogenesis. We investigated whether ADAMTS1 inhibited lymphangiogenesis in the present study. We examined cell proliferation and cell migration in normal human dermal lymphatic microvascular endothelial cells (HMVEC-dLy) transduced with or without adenoviral human ADAMTS1 gene therapy. We then examined the VEGFC/VEGFR3 signal transduction pathway in ADAMTS1-transduced HMVEC-dLy. Cell proliferation and tube formation in Matrigel were significantly lower with transduced ADAMTS1 than with control (non-transduced HMVEC-dLy). The phosphorylation of VEGFR3 was also attenuated by ADAMTS1 gene therapy in HMVEC-dLy. Immunoprecipitation assays revealed that ADAMTS1 formed a complex with VEGFC. Our results demonstrated that ADAMTS1 inhibited lymphangiogenesis in vitro. The data highlight the new function of ADAMTS1 in the regulation of lymphangiogenesis and the therapeutic potential of ADAMTS1 in cancer therapy.

In Euglena gracilis Z, a considerably high activity of mono-ADP-ribosyltransferase occurred and change of it was accompanied by a cell cycle induced by a light-dark cycle. The enzyme activity was strongly inhibited by L-arginine and supported in the presence of poly-L-arginine as a substrate, indicating that ADP-ribosylated amino acid is an arginine residue. Arginine: mono-ADP-ribosyltransferase activity was found in the chloroplasts, mitochondria, microsomes and cytosol as judged from marker enzyme activities and the activity in each organelle fluctuated with the cell cycle.

Angiogenesis plays an important role in tumor progression. Several reports have demonstrated that a disintegrin and metalloproteinase with thrombospondin motifs1 (ADAMTS1) inhibited angiogenesis via multiple mechanisms. The aim of this study was to investigate the effect of ADAMTS1 on endothelial cells in vitro and on tumor growth with regard to angiogenesis in vivo. We examined the effects of the transfection of ADAMTS1 using two constructs, full-length ADAMTS1 (full ADAMTS1) and catalytic domain-deleted ADAMTS1 (delta ADAMTS1). Transfection of both the full ADAMTS1 and delta ADAMTS1 gene constructs demonstrated the secretion of tagged-ADAMTS1 protein into the conditioned medium, so we examined the effects of ADAMTS1-containing conditioned medium on endothelial cells. Both types of conditioned media inhibited endothelial tube formation, and this effect was completely abolished after immunoprecipitation of the secreted protein from the medium. Both types of conditioned media also inhibited endothelial cell migration and proliferation. We then examined the impact of ADAMTS1 on endothelial cell apoptosis. Both conditioned media increased the number of Annexin V-positive endothelial cells and caspase-3 activity and this effect was attenuated when z-vad was added. These results indicated that ADAMTS1 induced endothelial cell apoptosis. We next examined the effects of ADAMTS1 gene transfer into tumor-bearing mice. Both full ADAMTS1 and delta ADAMTS1 significantly inhibited the subcutaneous tumor growth. Collectively, our results demonstrated that ADAMTS1 gene transfer inhibited angiogenesis in vitro and in vivo, likely as a result of the induction of endothelial cell apoptosis by ADAMTS1 that occurs independent of the protease activity.

A flow-injection system with a co-immobilized enzyme reactor is described for the determination of 3-hydroxybutyrate. 3-Hydroxybutyrate dehydrogenase and NADH oxidase were immobilized on aminated poly(vinyl alcohol) beads and packed into a stainless-steel column (5 cm × 4 mm i.d.). Serum was diluted 10-fold with borate buffer (pH 9.4) and ultrafiltered. Filtrate (20 μl) was injected into the carrier stream. The hydrogen peroxide produced was detected chemiluminometrically via a luminol-hexacyanoferrate(III) reaction. The calibration graph was linear for 2 × 10−7 M-5 × 10−4 M; the detection limit was 8 × 10−8 M and the sample throughput was 30 h−1 without carryover.

To investigate the influence of room temperature on finger blood flow (FBF) change in healthy subjects exposed to short-term grasping of a vibrating handle under different room temperatures.FBF was measured using a blood flowmeter in six male subjects on the dorsum of the middle phalanx of third finger in both hands once at the end of every minute for an equal duration of 5 min at pre-exposure, during exposure to grasping of vibrating handle with sinusoidal vibration and after exposure. Vibration was generated with a frequency of 125 Hz and an rms acceleration of 40 m/s(2). Measurements were conducted in four room temperatures of 15+/-1, 20+/-1, 25+/-1 and 30+/-1 degrees C.Compared with the baseline measurements in the exposed hand during grasping of vibrating handle most significant increase in FBF was observed at 15+/-1 degrees C (P0.001) and least at 30+/-1 degrees C (P0.05), and after vibration least significant FBF was found at 25+/-1 degrees C (P0.05). In case of the unexposed hand significant increase in FBF was exhibited at 20+/-1 degrees C (P0.01) and 30+/-1 degrees C (P0.01) during vibration, and only at 15+/-1 degrees C (P0.05) after vibration.Response in FBF due to grasping of vibrating handle was of different patterns from the baseline measurement under different room temperature conditions in both exposed and unexposed hands and it was influenced by room temperature. Overall, the influence was greater at lower test room temperature, inducing more significant increase in FBF.

This study examined relationships between different job types of shift work and hypertension, obesity, and dyslipidemia. Male blue-collar workers 50-59 years of age (n = 210) on the same three-shift schedule in a pulp and paper mill were divided into two groups; 118 in paper manufacturing (group 1) and 92 in the chemical products section (group 2). Only the frequency of hypertension differed significantly (p = 0.012) between the groups, 52.2% (n = 48) in group 2 vs 33.9% (n = 40) in group 1. The odds ratio for group 2 in relation to hypertension was 2.3 (95% CI 1.2-4.2). These results indicate a positive association between job type of shift work and hypertension and suggest that different job types of shift workers should not be combined when the effects of shift work on blood pressure are being examined.

BEta(2)-glycoprotein I (beta(2)-GPI) is proteolytically cleaved by plasmin in domain V (nicked beta(2)-GPI), being unable to bind to phospholipids. This cleavage may occur in vivo and elevated plasma levels of nicked beta(2)-GPI were detected in patients with massive plasmin generation and fibrinolysis turnover. In this study, we report higher prevalence of elevated ratio of nicked beta(2)-GPI against total beta(2)-GPI in patients with ischemic stroke (63%) and healthy subjects with lacunar infarct (27%) when compared to healthy subjects with normal findings on magnetic resonance imaging (8%), suggesting that nicked beta(2)-GPI might have a physiologic role beyond that of its parent molecule in patients with thrombosis. Several inhibitors of extrinsic fibrinolysis are known, but a negative feedback regulator has not been yet documented. We demonstrate that nicked beta(2)-GPI binds to Glu-plasminogen with K(D) of 0.37 x 10(-6) M, presumably mediated by the interaction between the fifth domain of nicked beta(2)-GPI and the fifth kringle domain of Glu-plasminogen. Nicked beta(2)-GPI also suppressed plasmin generation up to 70% in the presence of tissue plasminogen activator, plasminogen, and fibrin. Intact beta(2)-GPI lacks these properties. These data suggest that beta(2)-GPI/plasmin-nicked beta(2)-GPI controls extrinsic fibrinolysis via a negative feedback pathway loop.

To determine if the type of work performed should be considered in research on shift work and cardiovascular disease, we compared the heart rates, total number of steps walked, and blood pressures of 12 shift workers on the same rotating 3-shift schedule in a pulp and paper mill. Six workers were selected from the paper manufacturing section (group 1) and six workers from the chemical products section (group 2). Average heart rate (in beats per min) monitored during duty time was 84.3 in group 1 and 87.4 in group 2. Average heart rate during work was not significantly higher than that during rest in both groups 1 (work 85.8, rest 75.3) and 2 (work 87.9, rest 83.1). There was no significant difference in the total number of steps walked. A non-significant decrease in systolic blood pressure value was found in group 1 compared with that in group 2. Although future studies will be needed to explain the relation between different work styles and their effects on the health of shift workers, our results suggest no significant difference in heart rates among workers engaged in different kinds of work on the same shift work schedule.

It is currently thought that chloroplasts of higher plants were derived from endosymbiont oxygenic photosynthetic bacteria (primary endosymbiosis), while Euglena, a photosynthetic protista, gained chloroplasts by secondary endosymbiosis (i.e., incorporation of a photosynthetic eukaryote into heterotrophic eukaryotic host). To examine if the protein transport inside chloroplasts is similar between these organisms, we carried out heterologous protein import experiments with Euglena precursor proteins and spinach chloroplasts. The precursor of a 30-kDa subunit of the oxygen-evolving complex (OEC30) from the thylakoid lumen of Euglena chloroplasts contained the N-terminal signal, stroma targeting, and thylakoid transfer domains. Truncated preOEC30s lacking the N-terminal domain were post-translationally imported into spinach chloroplasts, transported into the thylakoid lumen, and processed to a mature protein. These results showed that protein translocations within chloroplasts in Euglena and higher plants are similar and supported the hypothesis that Euglena chloroplasts are derived from the ancestral Chlorophyta.