核磁共振成像（NMRI）在化学领域中的应用

本文对核磁共振成像（ＮＭＲＩ）与核磁共振波谱（ＮＭＲ）进行了概括性的比较，重点介绍了当前ＮＭＲＩ在化学领域的最新应用的情况。同时对ＮＭＲＩ的发展也做了展望。

核磁共振成像（NMRI）在非医学领域中的应用

对核磁共振成像（ＮＭＲＩ）与核磁共振（ＮＭＲ）波谱进行了概括性的比较。重点介绍了当前ＮＭＲＩ在非医学领域最新应用的进展情况，同时对ＮＭＲＩ的发展也做了回顾与展望。

The effects of organic environmental toxicants on hard tissue formation in developing tooth : An in vitro study in mice

Dioxins are organic toxicants that are known to impair tooth development, especially dental hard tissue formation. The most toxic dioxin congener is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Further, clinical studies suggest that maternal smoking during pregnancy can affect child s tooth development. One of the main components of tobacco smoke is the group of non-halogenated polycyclic aromatic hydrocarbons (PAHs), a representative of which is 7,12-dimethylbenz[a]anthracene (DMBA). Tributyltin (TBT), an organic tin compound, has been shown to impair bone mineralization in experimental animals. In addition to exposure to organic toxicants, a well-established cause for enamel hypomineralization is excess fluoride intake. The principal aim of this thesis project was to examine in vitro if, in addition to dioxins, other organic environmental toxicants, like PAHs and organic tin compounds, have adverse effects on tooth development, specifically on formation and mineralization of the major dental hard tissues, the dentin and the enamel. The second aim was to investigate in vitro if fluoride could intensify the manifestation of the detrimental developmental dental effects elicited by TCDD. The study was conducted by culturing mandibular first and second molar tooth germs of E18 NMRI mouse embryos in a Trowell-type organ culture and exposing them to DMBA, TBT, and sodium fluoride (NaF) and/or TCDD at various concentrations during the secretory and mineralization stages of development. Specific methods used were HE-staining for studying cell and tissue morphology, BrdU-staining for cell proliferation, TUNEL-staining for apoptosis, and QPCR, in situ hybridization and immunohistochemistry for the expressions of selected genes associated with mineralization. This thesis work showed that DMBA, TBT, TCDD and NaF interfere with dentin and enamel formation of embryonic mouse tooth in vitro, and that fluoride can potentiate the harmful effect of TCDD. The results suggested that adverse effects of TBT involve altered expression of genes associated with mineralization, and that DMBA and TBT as well as NaF and TCDD together primarily affect dentin mineralization. Since amelogenesis does not start until mineralization of dentin begins, impaired enamel matrix secretion could be a secondary effect. Dioxins, PAHs and organotins are all liposoluble and can be transferred to the infant by breast-feeding. Since doses are usually very low, developmental toxicity on most of the organs is difficult to indentify clinically. However, tooth may act as an indicator of exposure, since the major dental hard tissues, the dentin and the enamel, are not replaced once they have been formed. Thus, disturbed dental hard tissue formation raises the question of more extensive developmental toxicity.

Antitumor activity of Titanocene Y in xenografted PC3 tumors in mice

Chemotherapeutic options for androgen-independent prostate cancer are extremely limited with minimum survival advantage. The benzyl-substituted unbridged titanocene bis-[(p-methoxybenzyl) cyclopentadienyl] titanium(IV) dichloride (Titanocene Y) was tested in vitro against the human prostate cancer androgen-independent cell, PC-3, which demonstrated an IC50 value of 56 x 10(-6) mol/L compared to 5.6 x 10(-6) mol/L for cisplatin. Then Titanocene Y was given at the maximum tolerable dose of 40 mg/kg/d on five consecutive days to one cohort of eight PC3 tumor-bearing male NMRI: nu/nu mice, while a second cohort was treated similarly with 3 mg/kg/d of cisplatin. Both of these mouse cohorts showed a statistically significant tumor growth reduction with respect to the third solvent-treated control group, which led to T/C values of 42% for cisplatin and 52% for Titanocene Y at the end of the experiment. This encouraging activity of Titanocene Y against prostate tumors in vivo, which is almost comparable with respect to cisplatin hopefully leads to further development of Titanocene Y in the future.

Experimental comparative study on vortex-induced motion (VIM) of a monocolumn platform

An analysis methodology is presented as well as a comparison of results obtained from vortex-induced motion (VIM) model tests of the MonoGoM platform, a monocolumn floating unit designed for the Gulf of Mexico. The choice of scale between the model and the platform in which the tests took place was a very important issue that took into account the basin dimensions and mooring design. The tests were performed in three different basins: the IPT Towing Tank in Brazil (Sept. 2005), the NMRI Model Ship Experimental Towing Tank in Japan (Mar. 2007), and the NMRI Experimental Tank in Japan (Jun. 2008). The purpose is to discuss the most relevant issues regarding the concept, execution, and procedures to comparatively analyze the results obtained from VIM model tests, such as characteristic motion amplitudes, motion periods, and forces. The results pointed out the importance of considering the 2DOF in the model tests, i.e., the coexistence of the motions in both in-line and transverse directions. The approach employed in the tests was designed to build a reliable data set for comparison with theoretical and numerical models for VIM prediction, especially that of monocolumn platforms. [DOI: 10.1115/1.4003494]

Correlation of individual papilloma latency time with DNA adducts, 8-hydroxy-2'-deoxyguanosine, and the rate of DNA synthesis in the epidermis of mice treated with 7,12-dimethylbenz[alpha]anthracene.

The question was addressed whether the risk of cancer of an individual in a heterogeneous population can be predicted on the basis of measurable biochemical and biological variables postulated to be associated with the process of chemical carcinogenesis. Using the skin tumor model with outbred male NMRI mice, the latency time for the appearance of a papilloma was used as an indicator of the individual cancer risk. Starting at 8 weeks of age, a group of 29 mice was treated twice weekly with 20 nmol of 7,12-dimethylbenz[alpha]anthracene (DMBA) applied to back skin. The individual papilloma latency time ranged from 13.5 to 25 weeks of treatment. Two weeks after the appearance of the first papilloma in each mouse, an osmotic minipump delivering 5-bromo-2'-deoxyuridine was s.c. implanted and the mouse was killed 24 hr later. Levels of DMBA-DNA adducts, of 8-hydroxy-2'-deoxyguanosine, and various measures of the kinetics of cell division were determined in the epidermis of the treated skin area. The levels of 8-hydroxy-2'-deoxyguanosine and the fraction of cells in DNA replication (labeling index for the incorporation of 5-bromo-2'-deoxyuridine) were significantly higher in those mice that showed short latency times. On the other hand, the levels of DMBA-DNA adducts were lowest in animals with short latency times. The latter finding was rather unexpected but can be explained as a consequence of the inverse correlation seen for the labeling index: with each round of cell division, the adduct concentration is reduced to 50% because the new DNA strand is free of DMBA adducts until the next treatment. Under the conditions of this bioassay, therefore, oxygen radical-related genotoxicity and the rate of cell division, rather than levels of carcinogen-DNA adducts, were found to be of predictive value as indicators of an individual cancer risk.

Muscle catabolism in cancer and its attenuation by eicosapentaenic acid

This work examines skeletal muscle catabolism in cancer and its attenuation by Eicosapentaenoic Acid (EPA). In vivo studies in mice bearing a cachexia inducing murine colon adenocarcinoma - MAC16, demonstrated an elevation in the gastrocnemius muscle in the activity and expression of regulatory components of the ubiquitin-proteasome proteolytic pathway. This was accompanied by an accelerated loss of muscle tissue correlating with an increase in overall weight loss, all of which were attenuated by prior daily dosing with EPA. Recently a proteolysis inducing factor (PIF) has been isolated from the MAC16 tumour, and from the serum and urine of cachectic cancer patients. Previous studies have shown that PIF induces protein degradation in vitro, and that this is possibly mediated through 15-hydroxyeicosatetraenoic acid (15-HETE), a metabolite of the n-6 polyunsaturated fatty acid- arachidonate. Employing the murine myoblast cell line C2C12, it was shown that both PIF and 15-HETE increased protein degradation and expression of proteasome subunits, processes which were again attenuated by prior incubation in EPA. Similarly, in NMRI mice which had been fasted for 24hours, EPA and the lipoxygenase inhibitor CV-6504 (but not structurally related fatty acids) inhibited skeletal muscle proteolysis and expression of various proteasome subunits, showing that firstly, EPA may be anti-cachexic partly through its ability to influence 15-HETE production; and secondly that the effect is specific for EPA as other fatty acids had no effect. Previous studies have suggested the involvement of the signal transduction family NFKB in response to PIF in the liver. It has been demonstrated here that both PIF and 15-HETE increased nuclear translocation of NFKB in the skeletal muscle of tumour bearing mice and that EPA inhibited this process by its ability to prevent the degradation of the NFKB inhibitor protein IKB. When an NFKB inhibitor was added to C2C12 myotubes, prior to the addition of PIF, proteasome activity and protein degradation was inhibited, showing that NFKB is responsible for the increased proteasome activity and muscle catabolism induced by PIF. Taken together this work suggests that 15-hydroxyeicosatetraenoic acid is the intracellular mediator for PIF induced protein degradation in skeletal muscle and that elevated muscle catabolism is accomplished through an increased functioning of the ubiquitin-proteasome pathway, a process possibly mediated through an NFKB dependent mechanism. The anticachectic (and possibly the anti-tumourigenic) effects of EPA appear to be achieved in part by its ability to inhibit the degradation of IKB and possibly by its ability to interfere with 15-HETE production.

Mechanism of action of a tumour derived lipid mobilising factor

Cancer cachexia comprises unintentional and debilitating weight loss associated with certain tumour types. Fat loss in cachexia is mediated by a 43kDa Lipid Mobilising Factor (LMF) sharing homology with endogenous Zinc-α2-Glycoprotein (ZAG). LMF and ZAG induced significant lipolysis in isolated epidydimal adipose tissue. This is attenuated by co-incubation with 10μM of antagonist SR59230A and partially attenuated by 25μM PD098059 (indicating β3-AR and MAPK involvement respectively). LMF/ZAG induced in vitro lipid depletion in differentiated 3T3-L1 adipocytes that seen to comprise a significant increase in lipolysis (p<0.01), with only a modest decrease in lipid synthesis (p=0.09). ZAG significantly increased in vitro protein synthesis (p<0.01) in C2C12 myotubes (without an effect on protein degradation). This increase was activated at transcription and attenuated by co-incubation with 10μM SR59230A. Proteolytic digestion of ZAG and LMF followed by sephadex G50 chromatography yielded active fragments of 6-15kDa, indication the entire molecule was not required for bioactivity. Cachexigenic MAC16 cells demonstrated significant in vitro ZAG expression over non-cachexigenic MAC13 cells (p<0.001). WAT and BAT excised from MAC16 mice of varying weight loss demonstrated increased ZAG expression compared to controls. Dosing of NMRI mice with s/c ZAG failed to reproduce this up-regulation, thus another cachectic factor is responsible. 0.58nM LMF conferred significant protection against hydrogen peroxide, paraquat and bleomycin-induced oxidative stress in the non-cachexigenic MAC13 cell line. This protection was attenuated by 10μM SR59230A indicating a β3-AR mediated effect. In addition, 0.58nM LMF significantly up regulated UCP2 expression (p<0.001), (a mitochondrial protein implicated in the detoxification of ROS) implying this to be the mechanism by which survival was achieved. In vitro, LMF caused significant up-regulation of UCP1 in BAT and UCP2 and 3 in C2C12 myotubes. This increase in uncoupling protein expression further potentiates the negative energy balance and wasting observed in cachexia.

Free radicals as potential antitumour agents

The aim of this work was to use extremely low concentrations of free radical generating compounds as a 'catalyst' to trigger endogenous free radical chain reactions in the host and to selectively eliminate neoplastic cells in the host. To test the hypothesis, a number of free radical generating compounds were screened on several malignant cell lines in vitro to select model compounds that were used against tumour models in vivo. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and its derivatives were selected at the model compounds for in vivo experiments in view of their high cytotoxic potency against several malignant cell lines in vitro. The water soluble derivative, 2,2-diphenyl-1-(2', 4'-dinitro-6'-sulphophenyl) hydrazyl (DDSH) given by subcutaneous injections demonstrated significant antitumour activities against the MAC 16 murine colon adenocarcinoma implanted subcutaneously in male NMRI mice at nanomolar concentration range. 40-60% of long term survival of over 60 days was achieved (compared with control survival of 20 days) with total tumour elimination. This compound was also active against both P388 leukaemia in male BDF1 mice and TLX5 lymphoid tumour in male CBA/CA mice at a similar concentration range. However, some of these animals died suddenly after treatment with no evidence of disease present at post mortem. The cause of death was unknown but thought to be related to the treatment. There was significant increase in serum level of malondialdehyde (MDA) following treatment, but did not correlate to the antitumour activities of these compounds. Induction of supcroxide dismutase (SOD), and glutathione peroxidase (GPx) occurred around day 8 after the administration of DDSH. Histological sections of MAC16 tumours showed areas of extensive massive haemorrhagic necrosis and vascular collapse associated with perivascular cell death following the administration of nanomolar concentration of DDSH which was probably compatible with the effects of free radicals. It was concluded that the antitumour activities of these compounds may be related to free radical and cytokine production.

Microencapsulation strategies for islet transplantation

A variety of islet microencapsulation techniques have been investigated to establish which method provides the least occlusive barrier to net insulin release in vitro, and optimum biocompatibility for islet implantation in vivo. NMRI mouse islets have been microencapsulated with Na+ -alginate-poly-L-lysine (PLL)/poly-L-ornithine (PLO)-alginate, Ba2+ -alginate and agarose gels. Both free and microencapsulated islets responded to glucose challenge in static incubation and perifusion by significantly increasing their rate of insulin release and theophylline significantly potentiated the insulin response to glucose. While little insulin was released from microencapsulated islets after short term (2 hours) static incubation, significantly greater amounts were released in response to glucose challenge after extended (8-24 hours) incubation. However, insulin release from all types of microencapsulated islets was significantly reduced compared with free islets. Na+ -alginate-PLO-alginate microencapsulated islets were significantly more responsive to elevated glucose than Na+ -alginate-PLL-alginate microencapsulated islets, due to the enhanced porosity of PLO membranes. The outer alginate layer created a significant barrier to glucose/insulin exchange and reduced the insulin responsiveness of microencapsulated islets to glucose. Ba2+ -alginate membrane coated islets, generated by the density gradient method, were the most responsive to glucose challenge. Low concentrations of NG-monomethyl L-arginine (L-NMMA) had no significant effect on glucose stimulated insulin release from either free or microencapsulated islets. However, 1.0 mmol/1 L-NMMA significantly inhibited the insulin response of both free and microencapsulated islets to glucose challenge. In vivo work designed to evaluate the extent of pericapsular fibrosis after 28 days ip. and sc. implantation of microencapsulated islets into STZ-diabetic recipients, revealed that the inclusion of islets within microcapsules increased their immunogenicity and markedly increased the extent of pericapsular fibrosis. When the outer alginate layer was omitted from microcapsules, little or no pericapsular mononuclear cell deposition was observed. The subcutaneous site was not suitable for microencapsulated islet transplantation in NMRI recipient mice. Systemic immunosuppression using cyclosporin A was effective in preventing pericapsular mononuclear cell deposition, while L-NMMA loading into microcapsules had no significant effect on pericapsular fibrosis, although it did maintain the integrity of microencapsulated islets.

Inhibitors of lipolysis in tumor-induced cachexia

The MAC16 tumour produces a factor which exhibits lipid-mobilizing activity in vitro in addition to causing extensive depletion of host lipid stores. The mechanism of the anti-lipolytic effect of two anti-cachectic agents, eicosapentaenoic acid, an ω-3 polyunsaturated fatty acid (PUFA), and N-(3-phenoxycinnamyl)acetohydroxamic acid (BW A4C), a 5-lipoxygenase inhibitor, has been investigated. These two agents reduce tumour growth and reverse the weight loss which accompanies transplantation of the MAC16 murine colon adenocarcinoma into NMRI mice. Mice transplanted with the MAC16 tumour exhibited weight loss which was directly proportional to the serum lipolytic activity measured in vitro up to a weight loss corresponding to 16% of the original body weight. After this time, an inverse relationship between weight loss and lipolytic activity was observed. Body composition analysis revealed a large decrease in body fat relative to other body compartments. The anti-tumour/anti-cachectic effect of EPA did not appear to be due to its ability to inhibit the production of prostaglandin E2. The MAC16 lipolytic factor increased adenylate cyclase activity in adipocyte plasma membranes in a concentration-dependent manner. EPA inhibited the production of cAMP attributed to this lipid-mobilizing factor. EPA produced alterations in Gi , the guanine nucleotide binding protein which mediates hormonal inhibition of adenylate cyclase, in addition to altering cAMP production in adipocyte plasma membranes in response to hormonal stimulation. The alterations in adenylate cyclase activity were complex and not specific to EPA. EPA stimulated adenylate cyclase activity when in a relatively high fatty acid : membrane ratio and inhibited activity when this ratio was lowered. The inhibitory effect of EPA on adenylate cyclase activity may be the underlying mechanism which explains its anti-lipolytic and anti-cachectic effect. The inability of the related ω-3 PUFA, docosahexaenoic acid (DHA), to inhibit cachexia may be due to a difference in the metabolic fates of these two fatty acids. BW A4C inhibited lipolysis in isolated adipocytes which suggests that this compound may possess the potential for an anti-cachectic effect which is independent of its inhibitory effect on tumour growth.

Mechanism of protein catobolism in skeletal muscle during cancer cachexia

Cancer cachexia is characterised by selective depletion of skeletal muscle protein reserves. The ubiquitin-proteasome proteolytic pathway has been shown to be responsible for muscle wasting in a range of cachectic conditions including cancer cachexia. To establish the importance of this pathway in muscle wasting during cancer (and sepsis), a quantitative competitive RT-PCR (QcRT-PCR) method was developed to measure the mRNA levels of the proteasome sub units C2a and C5ß and the ubiquitin-conjugating enzyme E214k. Western blotting was also used to measure the 20S proteasome and E214k protein expression. In vivo studies in mice bearing a cachexia inducing murine colon adenocarcinoma (MAC16) demonstrated the effect of progressive weight loss on the mRNA and protein expression for 20S proteasome subunits, as well as the ubiquitin-conjugating enzyme, E214k, in gastrocnemius and pectoral muscles. QcRT-PCR measurements showed a good correlation between expression of the proteasome subunits (C2 and CS) and the E214k enzyme mRNA and weight loss in gastrocnemius muscle, where expression increased with increasing weight loss followed by a decrease in expression at higher weight losses (25-27%). Similar results were obtained in pectoral muscles, but with the expression being several fold lower in comparison to that in gastrocnemius muscle, reflecting the different degrees of protein degradation in the two muscles during the process of cancer cachexia. Western blot analysis of 20S and E214k protein expression followed a similar pattern with respect to weight loss as that found with mRNA. In addition, mRNA and protein expression of the 20S proteasome subunits and E214k enzyme was measured in biopsies from cachectic cancer patients, which also showed a good correlation between weight loss and proteasome expression, demonstrating a progressive increase in expression of the proteasome subunits and E214k mRNA and protein in cachectic patients with progressively increasing weight loss.The effect of the cachexia-inducing tumour product PIF (proteolysis inducing factor) and 15-hydroxyeicosatetraenoic acid (15-HETE), the arachidoinic acid metabolite (thought to be the intracellular transducer of PIF action) has also been determined. Using a surrogate model system for skeletal muscle, C2C12 myotubes in vitro, it was shown that both PIF and 15-HETE increased proteasome subunit expression (C2a and C5ß) as well as the E214k enzyme. This increase gene expression was attenuated by preincubation with EPA or the 15-lipoxygenase inhibitor CV-6504; immunoblotting also confirmed these findings. Similarly, in sepsis-induced cachexia in NMRI mice there was increased mRNA and protein expression of the 20S proteasome subunits and the E214k enzyme, which was inhibited by EPA treatment. These results suggest that 15-HETE is the intracellular mediator for PIF induced protein degradation in skeletal muscle, and that elevated muscle catabolism is accomplished through upregulation of the ubiquitin-proteasome-proteolytic pathway. Furthermore, both EPA and CV -6504 have shown anti-cachectic properties, which could be used in the future for the treatment of cancer cachexia and other similar catabolic conditions.

Effect of a tumour-derived lipid-mobilising factor on glucose and lipid metabolism in vivo

Treatment of ex-breeder male NMRI mice with lipid mobilising factor isolated from the urine of cachectic cancer patients, caused a significant increase in glucose oxidation to CO2, compared with control mice receiving phosphate buffered saline. Glucose utilisation by various tissues was determined by the 2-deoxyglucose tracer technique and shown to be elevated in brain, heart, brown adipose tissue and gastrocnemius muscle. The tissue glucose metabolic rate was increased almost three-fold in brain, accounting for the ability of lipid mobilising factor to decrease blood glucose levels. Lipid mobilising factor also increased overall lipid oxidation, as determined by the production of 14CO2 from [14C carboxy] triolein, being 67% greater than phosphate buffered saline controls over a 24 h period. There was a significant increase in [14C] lipid accumulation in plasma, liver and white and brown adipose tissue after administration of lipid mobilising factor. These results suggest that changes in carbohydrate metabolism and loss of adipose tissue, together with an increased whole body fatty acid oxidation in cachectic cancer patients, may arise from tumour production of lipid mobilising factor. © 2002 Cancer Research UK.

Effect of inulin supplementation in male mice fed with high fat diet on biochemical profile and α-amylase gene expression

Purpose: To evaluate the preventive and therapeutic effects of inulin supplementation in Naval Medical Research Institute (NMRI) male mice fed with high fat diet. Methods: NMRI male mice (n = 36) were divided into three groups. Control (C1), obese (O1) and experimental mice (E1) were fed during 8 weeks as follows: C1 with normal rodent pellet, O1 with high fat diet, and E1 with high fat diet plus 20 % inulin. C2, O2, and E2 were fed as follows: C2 with normal rodent pellets for 12 weeks; O2 with high fat diet during 8 weeks and switched to normal rodent pellet during next 4 weeks; and E2 with high fat diet over a period of 8 weeks and switched to normal rodent pellet plus 20 % inulin for 4 weeks. Body weight, serum glucose, triglycerides, total cholesterol, high density lipoprotein (HDL), low density lipoprotein (LDL), and hepatic α-amylase gene expression were measured. Results: Groups receiving high fat diet showed higher weight (30.71 ± 0.66 g in O2, p < 0.001), nonfasting blood glucose levels (257.69 ± 5.10 mg/dl in O2, p < 0.001), TG (282.15 ± 1.83 mg/dl in O2, (p < 0.001)), and cholesterol levels (335.72 ± 2.23 mg/dl in O2, (p < 0.001)), compared with control. In C2 group, mean body weight was 25.71 ± 0.54 g, non-fasting blood level 161.54 ± 4.48 mg/dl, TG level 214.29 ± 5.54 mg/dl, and cholesterol level 164.29 ±4.57 mg/dl. Compared to obese group, mice receiving inulin showed lower blood glucose levels (223.10 ± 8.7 mg/dl in E2, p < 0.001), body weight (27.86 ± 0.57 g in E2, p < 0.001), TG (232.14 ± 4.02 mg/dl in E2, p < 0.001) and cholesterol (249.97 ± 2.28 in E2, p < 0.001). A slight decrease in hepatic α-amylase gene expression was observed only in E1. Conclusion: Besides its sweetening properties, inulin may also find use as a potential anti-obesity compound.