Aberrant activation of the intrarenal renin-angiotensin system in the developing kidneys of type 2 diabetic rats.

We have previously reported that intrarenal angiotensin II (Ang II) levels are increased long before diabetes becomes apparent in obese Otsuka-Long-Evans-Tokushima-Fatty (OLETF) rats, a model of type 2 diabetes. In this study, we examined the changes in intrarenal renin-angiotensin system (RAS) activity in the developing kidneys of OLETF rats. Ang II contents and mRNA levels of RAS components were measured in male OLETF and control Long-Evans Tokushima (LETO) rats at postnatal days (PND) 1, 5, and 15, and at 4-30 weeks of age. In both LETO and OLETF rats, kidney Ang II levels peaked at PND 1, then decreased during the pre- and post-weaning periods. However, Ang II levels and gene expression of RAS components, including angiotensinogen (AGT), renin, and angiotensin-converting enzyme (ACE), were not significantly different between LETO and OLETF rats. Intrarenal Ang IIcontents further decreased during puberty (from 7 to 11 weeks of age) in LETO rats, bur not in OLETF rats. At 11 weeks of age, kidney Ang II levels, urinary AGT excretion, and mRNA levels of AGT and renin were higher in OLETF rats than in LETO rats, while blood glucose levels were not significantly different between these groups of rats. These data indicate that continued intrarenal expression of Ang II during pubescence contributes to the increases in intrarenal Ang II levels in prediabetic OLETF rats, and is associated with increased intrarenal AGT and renin expression. Inappropriate activation of the intrarenal RAS in the prediabetic stage may facilitate the onset and development of diabetic nephropathy in later life.

Polymyxin-B hemoperfusion for acute exacerbation of idiopathic pulmonary fibrosis: serum IL-7 as a prognostic marker.

BACKGROUND: Acute exacerbation (AE) of idiopathic pulmonary fibrosis (IPF) has an extremely poor prognosis. Direct hemoperfusion with a polymyxin B-immobilized fiber column (PMX-DHP) has been used to improve oxygenation for acute respiratory distress syndrome. The study aim was to retrospectively determine the predictive factors affecting the prognosis of AE of IPF treated with PMX-DHP. METHODS: We studied patients suffering from AE of IPF, treated with PMX-DHP combined with high-dose corticosteroid therapy. Stored serum taken before and after PMX-DHP therapy was analyzed for 27 cytokines and chemokines. RESULTS: Nineteen patients with AE of IPF were studied. The median survival time after diagnosis of AE was 22 days. Survival rates after diagnosis of AE were 47.4% at 30 days, 31.6% at 60 days, and 26.3% at 90 days. Serum levels of Interleukin (IL)-7, an anti-fibrotic cytokine, in survivors at day 30 following PMX-DHP therapy ('Survivors') significantly increased after the treatment, compared to serum levels of non-survivors at day 30 after the therapy ('Nonsurvivors'), which did not demonstrate a significant change. Serum levels of IL-1beta, interferon-y and chemokine ligand (CCL) 2 levels were not significantly altered in 'Survivors', but were significantly changed in 'Nonsurvivors.' Multivariate Cox proportional-hazards analysis showed that an increase in IL-7 levels after PMX-DHP therapy and treatment without intubation (other than invasive positive-pressure ventilation) were significantly better prognostic factors. CONCLUSION: The results suggest that serum IL-7 may be a useful prognostic factor for patients with AE of IPF treated with PMX-DHP, possibly reflecting underlying anti-fibrotic mechanisms.

Subdose of fasudil suppresses myocardial fibrosis in aldosterone-salt-treated uninephrectomized rats.

Rho/Rho kinase (ROCK) pathway plays an important role in pathological cardiovascular conditions. In the present study, the effect of a subdose of fasudil, a selective ROCK inhibitor, on systemic hypertension and myocardium fibrosis induced by aldosterone was investigated in uninephrectomized Sprague-Dawley rats (SD). Treatment with a fasudil (10 mg/kg x day, s.c.) for 5 weeks decreased the activity of ROCK activity for more than 53% as determined by the expression of phosphorylated Myosin phosphatase target subunit 1 (MYPT1). Although this dose of fasudil did not signifantly prevent hypertension, it remarkably alleviated myocardium hypertrophy and fibrosis. The elevated transcriptional expression of transforming growth factors beta1 (TGF-beta1), atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and collagen I and III was also decreased. These results demonstrated that fasudil can protect the myocardium from injury by aldosterone at a subhypertensive dose.

Leukotriene B4 limits the effectiveness of fish oil in an animal model of asthma.

This study aimed to evaluate the levels of eicosanoids derived from arachidonic acid (ARA) in the lungs of asthmatic rats supplemented with fish oil. The present data gives insight into the action of fish oil in asthma, related to its inability to modify the contractile capacity of tracheal smooth muscle reported previously in a model of asthma in rats. Male Wistar rats were supplemented daily with 1 g of fish oil/kg of body weight for 21 days. They were exposed to ovalbumin (OVA) after previous sensitization with OVA to induce asthma. Pulmonary levels of five eicosanoids were measured using immunoassay kits: PGE2, TXB2, LTB4, LXA4, and 8-iso PGF2α. In asthmatic rats, supplementation with fish oil resulted in lower concentrations of lung eicosanoids produced by cyclooxygenase-2 and 15-lipoxygenase: PGE2, TXB2, and LXA4, respectively. Fish oil supplementation also decreased the non-enzymatically produced eicosanoid 8-iso PGF2α. Fish oil supplementation did not affect LTB4, a metabolite of 5-lipoxygenase. The limited efficacy of fish oil supplementation in asthmatic rats is associated with a lack of action in reducing the levels of LTB4 in the lungs. Thus, fish oil differentially modulates the concentrations of eicosanoids derived from ARA via specific pathways in an animal model of asthma.

Platelet-activating factor and evidence of oxidative stress in the bronchoalveolar fluid of Thoroughbred colts during race training.

BACKGROUND: Inflammatory airway disease (IAD) is prevalent in young racehorses during training, being the 2nd most commonly diagnosed ailment interrupting training of 2-year-old Thoroughbred racehorses. HYPOTHESIS: That stabling and exercise cause oxidative stress, release of platelet-activating factor (PAF) and inflammation in airways of Thoroughbred colts. ANIMALS: Colts in breeding farms (NC, n = 45), stabled for 30 days (EC, n = 40), and race trained (EX, n = 34). METHODS: Cytological profile and parameters of bronchoalveolar lavage fluid (BALF) related to oxidative stress, bioactivity of the proinflammatory mediator PAF, catalase activity, and alveolar macrophage function. RESULTS: Percentages of neutrophils and eosinophils in the BALF of the EX group were higher (5.4 +/- 6.4% versus 0.9 +/- 1.2%) than the upper limits for normal horses (3-5%). BALF from the EX group (45.6 +/- 2.8 cells/microL of BALF) also displayed significantly (P = .017) higher total nucleated cell count. PAF bioactivity and the total protein concentration in the BALF were higher in the EX group (0.0683 +/- 0.076 versus 0.0056 +/- 0.007 340 : 380 nm ratio P = .0039, 0.36 +/- 0.30 versus 0.14 +/- 0.15 mg of proteins/mL of BALF P < .001). Concentration of BALF hydroperoxides was higher in the EC group (104.7 +/- 80.0 versus 35.2 +/- 28.0 nmol/mg of proteins, P = .013) and catalase activity was higher in the EX group (0.24 +/- 0.16 versus 0.06 +/- 0.02 micromol H2O2/min/mg of proteins, P = .0021). Alveolar macrophage phagocytosis (P = .048) as well as production of superoxide anion (P = .0014) and hydrogen peroxide (P = .0011) were significantly lower in EX group. CONCLUSIONS AND CLINICAL IMPORTANCE: Further studies should be performed to elucidate the role of PAF in the pathophysiology of IAD. Its presence in bronchoalveolar fluid of young athletic horses makes it a potential therapeutic target to be investigated.

Impact of Energy intake at One Week after Hospitalization on Prognosis for Older Adults with Pneumonia.

OBJECTIVES: This study objectives to investigate the influence of average energy intake at 1 week of hospitalization on prognosis for older adults with pneumonia. DESIGN: Retrospective observational cohort study. SETTING: The Japan Rehabilitation Nutrition Database comprise those with pneumonia in acute care hospitals. PARTICIPANTS: The study included 329 pneumonia patients (aged over 65 years) who entered into the Japan Rehabilitation Nutrition Database (JRND) from November 2015 to March 2018. MEASUREMENTS: Logistic regression analysis was performed to confirm the relationship of energy intake with the rate of mortality, discharge home, and pneumonia recurrence during hospitalization. Variables included in the multiple regression analysis model were age, sex, Mini Nutritional Assessment-Short Form score (MNA-SF) at hospitalization, A-DROP, Charlson comorbidity index (CCI), and presence or absence of rehabilitation. RESULTS: Of 315 patients with pneumonia (median age 85 years), 63.8% were men. 57.7% were assigned to the lack of energy intake (LEI) at 1 week after admission. Patients in the LEI group were older (p = 0.033), had higher A-DROP score (p < 0.001), and showed higher malnutrition rate in MNA-SF at hospitalization (p < 0.001) than those in the control group. Mortality, pneumonia recurrence (p = 0.001), median body mass index (p = 0.012), and low malnutrition in MNA-SF (p < 0.001) at discharge were significantly higher in the LEI group than in the control group. Logistic regression analysis showed that LEI was an independent risk factor for mortality (Odds ratio: 5.07, p = 0.002), discharge home (Odds ratio: 0.33, p = 0.007), and pneumonia recurrence (Odds ratio: 3.26, p = 0.007). CONCLUSIONS: LEI at 1 week after hospitalization in older adults with pneumonia was an independent risk factor for mortality, difficult at-home recovery, and pneumonia recurrence. These findings suggest the importance of adequate energy intake from the early days of hospitalization.

Differentiation and death of premyelinating oligodendrocytes in developing rodent brain.

Previous studies have indicated that newly formed oligodendrocytes are dynamic cells whose production, survival, and differentiation depend upon axonal influences. This study has characterized the appearance and fate of newly formed oligodendrocytes in developing rat brain. Oligodendrocytes appear in predictable locations and radially extend DM-20-positive processes that cover 80-microm domains in the cortex and 40-microm domains in the corpus callosum. These premyelinating oligodendrocytes have one of two fates: they myelinate axons or degenerate. Between 7 and 21 d after birth, approximately 20% of premyelinating oligodendrocytes identified in the cerebral cortex were degenerating. Oligodendrocytes that ensheathed axons expressed and selectively targeted proteolipid protein to compact myelin and did not degenerate. These observations support the hypothesis that axonal influences affect oligodendrocyte survival, differentiation, and expression of proteolipid protein gene products.

The primary structure of NG2, a novel membrane-spanning proteoglycan.

The complete primary structure of the core protein of rat NG2, a large, chondroitin sulfate proteoglycan expressed on O2A progenitor cells, has been determined from cDNA clones. These cDNAs hybridize to an mRNA species of 8.9 kbp from rat neural cell lines. The total contiguous cDNA spans 8,071 nucleotides and contains an open reading frame for 2,325 amino acids. The predicted protein is an integral membrane protein with a large extracellular domain (2,224 amino acids), a single transmembrane domain (25 amino acids), and a short cytoplasmic tail (76 amino acids). Based on the deduced amino acid sequence and immunochemical analysis of proteolytic fragments of NG2, the extracellular region can be divided into three domains: an amino terminal cysteine-containing domain which is stabilized by intrachain disulfide bonds, a serine-glycine-containing domain to which chondroitin sulfate chains are attached, and another cysteine-containing domain. Four internal repeats, each consisting of 200 amino acids, are found in the extracellular domain of NG2. These repeats contain a short sequence that resembles the putative Ca(++)-binding region of the cadherins. The sequence of NG2 does not show significant homology with any other known proteins, suggesting that NG2 is a novel species of integral membrane proteoglycan.

Dystrophin nonsense mutations can generate alternative rescue transcripts in lymphocytes.

Secondary alterations in splicing have been reported to produce semi-functional mRNA from several nonsense mutations in the dystrophin gene. Disruptions of exonic splicing enhancers by single nucleotide changes are thought to underlie such alterations. The precise frequencies of such nonsense mutation-dependent splicing alterations, however, remain unknown. Here we analyzed the splicing patterns of dystrophin mRNA in lymphocytes from 38 patients with dystrophinopathies due to nonsense mutations in the dystrophin gene. In seven of the cases (18%), we observed partial skipping of the nonsense-encoding exon. Two of the seven cases, however, exhibited complex activation of a nonsense mutation-created splice site, which resulted in the generation of novel transcripts. Examination of cis-regulatory splicing elements through calculation of splicing probability scores and identification of potential splicing enhancer or silencer sequences failed to disclose a single cause for exon skipping. Remarkably, individual differences in splicing patterns were observed for cells from patients with identical nonsense mutations (C.5899C>T). Although five cases produced semi-functional dystrophin mRNAs, only one of these exhibited a mild clinical course. These results provide important insights about targets for exon skipping induced by candidate antisense oligonucleotides and for ribosomal read-through of nonsense mutations.

Patterns of missing occlusal units and oral health-related quality of life in SDA patients.

The aim of this study was to explore the relationship between patterns of missing occlusal units (OUs) and oral health-related quality of life (OHRQoL) in subjects with the shortened dental arches (SDAs). Subjects with SDAs were recruited consecutively for 1 month from six university-based prosthodontic clinics. In total, 115 SDA subjects participated (mean age, 58.5 +/- 10.0 years; 71% female). The location and number of missing teeth were examined and the number of missing OUs was calculated. To evaluate OHRQoL, the Japanese version of the Oral Health Impact Profile (OHIP-J) was administered and the summary score of OHIP-J was calculated. The SDA subjects were categorized depending upon the anterior-posterior lengths of the missing or remaining OUs. Regression analyses were performed to investigate the OHIP-J differences between groups of subjects with various anterior-posterior SDA lengths. The analyses revealed that subjects who only lost the second molar contact exhibited significantly better OHRQoL than those who lost more teeth [coefficient: 11.1, 95% confidence interval (CI): 2.8-19.2, P = 0.02]. Furthermore a statistically significant group difference was observed between the groups with and without the first molar occlusal contact (coefficient: 12.8, 95% CI: 1.4 to 24.1, P = 0.03). In conclusion, although our results are of exploratory nature and need validation, patterns of missing OUs are likely to be related to the OHRQoL impairment in SDA subjects with the presence of first molar contact having a particularly important role.

Effect of direct retainer and major connector designs on RPD and abutment tooth movement dynamics.

Designs of removable partial dentures are suggested to affect the mobility of abutment teeth and removable partial denture (RPD) during oral functions. This study aimed to examine the effect of direct retainer and major connector designs on RPD dynamics under simulated loading. Six different Kennedy class II maxillary RPDs were fabricated on a maxillary model. These dentures involved 3 different direct retainers (wrought-wire clasp, RPA clasp, and conical crown telescopic retainer) and 2 different major connectors (Co-Cr major connector and heat-cured acrylic resin with a metal strengthener). Using an experimental model with simulated periodontal ligaments and mucosa that were fabricated using silicone impression material, three-dimensional displacements of the RPDs were measured under a simulated 30-N loading with a displacement transducer type M-3. Significant effects of "direct retainer design" on bucco-palatal displacements and "major connector" on mesio-distal displacements were revealed by 2 x 3 two-way analysis of variance of abutment teeth movements (P < 0.001 and P = 0.002, respectively). Additionally, analysis of variance of RPD displacements revealed significant effects of "direct retainer design" on corono-apical displacements and "major connector" on mesio-distal displacements (P = 0.001 and P = 0.028, respectively). Rigid direct retainers and rigid major connectors decrease the movements of both abutment tooth and RPD.

Effect of angiotensin-converting enzyme blockade, alone or combined with blockade of soluble epoxide hydrolase, on the course of congestive heart failure and occurrence of renal dysfunction in Ren-2 transgenic hypertensive rats with aorto-caval fistula.

We showed recently that increasing kidney epoxyeicosatrienoic acids (EETs) by blocking soluble epoxide hydrolase (sEH), an enzyme responsible for EETs degradation, retarded the development of renal dysfunction and progression of aorto-caval fistula(ACF)-induced congestive heart failure (CHF) in Ren-2 transgenic hypertensive rats (TGR). In that study the final survival rate of untreated ACF TGR was only 14 % but increased to 41 % after sEH blockade. Here we examined if sEH inhibition added to renin-angiotensin system (RAS) blockade would further enhance protection against ACF-induced CHF in TGR. The treatment regimens were started one week after ACF creation and the follow-up period was 50 weeks. RAS was blocked using angiotensin-converting enzyme inhibitor (ACEi, trandolapril, 6 mg/l) and sEH with an sEH inhibitor (sEHi, c-AUCB, 3 mg/l). Renal hemodynamics and excretory function were determined two weeks post-ACF, just before the onset of decompensated phase of CHF. 29 weeks post-ACF no untreated animal survived. ACEi treatment greatly improved the survival rate, to 84 % at the end of study. Surprisingly, combined treatment with ACEi and sEHi worsened the rate (53 %). Untreated ACF TGR exhibited marked impairment of renal function and the treatment with ACEi alone or combined with sEH inhibition did not prevent it. In conclusion, addition of sEHi to ACEi treatment does not provide better protection against CHF progression and does not increase the survival rate in ACF TGR: indeed, the rate decreases significantly. Thus, combined treatment with sEHi and ACEi is not a promising approach to further attenuate renal dysfunction and retard progression of CHF.

Redox control of resistance to cis-diamminedichloroplatinum (II) (CDDP): protective effect of human thioredoxin against CDDP-induced cytotoxicity.

Thioredoxin is a small ubiquitous protein with multiple biological functions, including cellular defense mechanisms against oxidative stress. In the present study, we investigated the role of human thioredoxin (hTRX) in the acquisition of cellular resistance to cis-diamminedichloroplatinum (II) (CDDP). The expression and activity of hTRX in Jurkat T cells was dose-dependently enhanced by exposure to CDDP, as determined by immunoblot analysis and insulin reducing assay. Furthermore, chloramphenicol acetyltransferase analysis using the hTRX promoter-reporter gene construct revealed that treatment of Jurkat cells with CDDP caused transcriptional activation of the hTRX gene, which might be mediated through increased generation of intracellular reactive oxygen intermediates. To examine the biological significance of hTRX induction, we established hTRX-overexpressing derivatives of L929 fibrosarcoma cells by stable transfection with the hTRX cDNA. The clones, which constitutively expressed the exogenous hTRX, displayed increased resistance to CDDP-induced cytotoxicity, compared with the control clones. After exposure to CDDP, the control cells showed a significant increase in the intracellular accumulation of peroxides, whereas the hTRX-transfected cells did not. Taken together, these results suggest that overexpressed hTRX is responsible for the development of cellular resistance to CDDP, possibly by scavenging intracellular toxic oxidants generated by this anticancer agent.

Expression of NG2 proteoglycan causes retention of type VI collagen on the cell surface.

NG2 is a membrane-associated chondroitin sulfate proteoglycan with a core protein of 300 kD. Previously it was shown immunochemically that the core protein of NG2 can bind type VI collagen (Stallcup, W., Dahlin, K., and P. Healy. 1990. J. Cell Biol. 111:3177-3188). We have extended our studies on the interaction of NG2 and type VI collagen by transfecting cells with the full-length rat NG2 cDNA. B28 rat neural cells and U251MG human glioma cells used for transfection do not synthesize NG2. Both cell lines secrete type VI collagen into tissue culture medium but do not anchor it at the cell surface. Upon transfection of these cells with the NG2 cDNA, NG2 was correctly localized to the cell surface. Furthermore, type VI collagen could now be detected on the surface of NG2-positive cells in a pattern that coincided with that of NG2. This ability of NG2 to anchor type VI collagen to the cell surface could be abolished by incubating the cells in the presence of anti-NG2 monoclonal antibodies. These findings indicate that NG2 functions as a cell surface receptor for type VI collagen and may play a role in modulating the assembly of pericellular matrix.

Generation of truncated forms of the NG2 proteoglycan by cell surface proteolysis.

NG2 is a chondroitin sulfate proteoglycan that is expressed on dividing progenitor cells of several lineages including glia, muscle, and cartilage. It is an integral membrane proteoglycan with a core glycoprotein of 300 kDa. In the present study we have characterized three molecular forms of the NG2 core protein expressed by different cell lines. Many cell lines that express the full length 300-kDa NG2 core protein also release a 290-kDa form into the medium. This species lacks the cytoplasmic domain but contains almost the entire ectodomain. Two core protein species, the intact 300-kDa form and a truncated 275-kDa form, are expressed at the surface of an NG2-transfected cell line U251NG52. The 275-kDa species lacks the cytoplasmic domain and at least 64 amino acids of the ectodomain. Mild trypsinization of B49 cells also generates the 275-kDa species, suggesting that this component is produced by proteolysis of the 300-kDa form. Conversion of the 300-kDa species to the 275-kDa form in U251NG52 cells is stimulated by reagents such as phorbol esters, which activate protein kinase C. Phorbol esters are also known to induce expression of metalloproteinases such as collagenase and stromelysin, which could be responsible for cleavage of the 300-kDa core protein. Although B49 cells do not spontaneously produce the truncated 275-kDa species, use of monoclonal antibodies against NG2 to block the interaction between NG2 and type VI collagen results in the appearance of the 275-kDa component in these cells. Thus the interaction between NG2 and type VI collagen, which contains a Kunitz-type proteinase inhibitor sequence in the alpha 3 chain, may protect the proteoglycan against proteolysis. This is consistent with the observed deficiency of U251NG52 cells in anchoring type VI collagen at the surface.

Cytoskeletal reorganization induced by engagement of the NG2 proteoglycan leads to cell spreading and migration.

Cells expressing the NG2 proteoglycan can attach, spread, and migrate on surfaces coated with NG2 mAbs, demonstrating that engagement of NG2 can trigger the cytoskeletal rearrangements necessary for changes in cell morphology and motility. Engagement of different epitopes of the proteoglycan results in distinct forms of actin reorganization. On mAb D120, the cells contain radial actin spikes characteristic of filopodial extension, whereas on mAb N143, the cells contain cortical actin bundles characteristic of lamellipodia. Cells that express NG2 variants lacking the transmembrane and cytoplasmic domains are unable to spread or migrate on NG2 mAb-coated surfaces, indicating that these portions of the molecule are essential for NG2-mediated signal transduction. Cells expressing an NG2 variant lacking the C-terminal half of the cytoplasmic domain can still spread normally on mAbs D120 and N143, suggesting that the membrane-proximal cytoplasmic segment is responsible for this process. In contrast, this variant migrates poorly on mAb D120 and exhibits abnormal arrays of radial actin filaments decorated with fascin during spreading on this mAb. The C-terminal portion of the NG2 cytoplasmic domain, therefore, may be involved in regulating molecular events that are crucial for cell motility.

Cloning and characterization of six highly similar endo-1,3-beta-glucanase genes in hexaploid wheat.

Pathogenesis-related (PR) proteins are often used as a marker of plant defense reactions. Some endo-1,3-beta-glucanase (Gns) genes encode proteins that belong to the PR protein family 2 (PR-2). Although the number of homologous family member genes is significantly greater in hexaploid wheat (Triticum aestivum L.) compared to other model plants, earlier studies did not evaluate the possible contribution of their homologs to hybridization signals in Northern blot analysis. In this study, we have examined whether routine transcriptional analyses of a PR gene is of high reliability or not by isolating six highly similar Gns genes (TaGlb2a, TaGlb2b, TaGlb2c, TaGlb2d, TaGlb2e, and TaGlb2f) and characterizing their expression patterns in detail. While TaGlb2b was shown to be a PR-2 gene, transcription of TaGlb2c and TaGlb2d was not induced upon infection with either powdery mildew (Erysiphe graminis) or head blight (Fusarium graminearum) pathogens; their transcripts were most abundant in healthy spikes (lemmas and in particular paleae). Therefore, in some cases, the conventional analyses do not necessarily provide accurate information on expression pattern of a PR gene in hexaploid wheat. This is also the first report of wheat genes that are specifically expressed in lemma/palea tissues of flowering spikelets.

Angiogenesis inhibitor TNP-470 inhibits human breast cancer osteolytic bone metastasis in nude mice through the reduction of bone resorption.

Angiogenesis inhibitor TNP-470, 6-O-(N-chloroacetyl-carbamoyl)-fumagillol, semisynthetic analogue of fumagillin, has strong inhibitory activities against in vivo tumor growth and metastasis in a wide variety of tumors. However, it is still unknown whether this agent inhibits bone metastasis. We examined the effects of TNP-470 in a bone metastasis model in nude mice in which intracardiac injection of the human breast cancer cell line MDA-MB-231 (MDA-231) produced osteolytic bone metastasis. After inoculation of MDA-231 cells into the left heart ventricle, TNP-470 (30 mg/kg, three times a week) or PBS was s.c. administrated for 4 weeks. After this period, the TNP-470 had reduced not only the number and area of osteolytic bone metastases (approximately 60 and 70%, respectively) but also their radiolucency. Histological examination of the femurs of the untreated group revealed that most of the cancellous bone had been replaced by the metastatic cancer. Numerous active osteoclasts were present along the trabecular bone surface surrounded by the metastatic MDA-231 cancer cells aggressively invading the bone marrow. In contrast, in the bone from TNP-470-treated mice, bone destruction was markedly inhibited, and there were much fewer osteoclasts. In a murine bone marrow culture under 1,25-dihydroxyvitamin D3 in which mature functional osteoclasts formed in vitro, TNP-470 significantly inhibited the formation of tartrate-resistant acid phosphatase-positive multinucleated osteoclast-like cells. And also, TNP-470 suppressed the in vivo bone resorption in calvaria treated with interleukin-1beta, an osteoclast stimulator. These data suggested that TNP-470 inhibited bone metastasis through not only antitumor action by its angiogenesis inhibition but also by the inhibition of osteoclastic bone resorption. Our results indicate that TNP-470 should be a potentially beneficial drug to be used in the treatment of osteolytic metastasis.