Successful prolonged treatment with sofosbuvir/velpatasvir for a hepatitis C patient with decompensated cirrhosis and treatment failure after 12-week therapy.

There is no established rescue therapy for hepatitis C patients with decompensated cirrhosis who experience treatment failure on 12-week sofosbuvir (SOF)/velpatasvir (VEL) therapy that is the only approved regimen for decompensated cirrhosis in Japan. We experienced a patient with decompensated cirrhosis who showed virologic relapse at post-treatment week 7 following 12-week SOF/VEL therapy. She had resistance-associated substitutions (RASs) against VEL before therapy but did not achieve new RASs against VEL or SOF after therapy. We considered rescue therapy following strong demand from her and her family. The drug adherence of therapy was 100%, and the treatment was well tolerated. Because we prioritized the safety and drug adherence of the regimen, we performed prolonged 24-week SOF/VEL therapy without ribavirin at her own expense with the approval of the ethics board in the hospital where the first author belongs. Fortunately, a sustained virologic response 24 was achieved without any adverse events. Hepatocellular carcinoma that had developed after 12-week SOF/VEL therapy recurred and was treated near the end of rescue therapy, but hepatic functional reserve improved. Although this was a single case report and was assumed to be very rare, the same regimen might be effective for treatment failure with 12-week SOF/VEL therapy.

Nrf2-mediated anti-oxidant effects contribute to suppression of non-alcoholic steatohepatitis-associated hepatocellular carcinoma in murine model.

The exact mechanisms of hepatocellular carcinoma development in non-alcoholic steatohepatitis remain unclear. In this study, we used a new class of high-fat diet, which could induce hepatocellular carcinoma development without the use of general chemical carcinogens or knockout mice. We investigated the correlation between hepatocellular carcinoma and oxidative stress/anti-oxidant effects after depletion of the gut microbiota by treatment with antibiotics. Mice fed with the steatohepatitis-inducing high-fat diet (STHD-01) for 41 weeks developed hepatocellular carcinoma. Antibiotic-treatment in mice fed with STHD-01 significantly depleted the gut microbiota and significantly ameliorated liver injury/histology. The tumor numbers of hepatocellular carcinoma were dramatically decreased by the antibiotics-treatment. We analyzed the factors involved in oxidative stress and anti-oxidant effects. Oxidative stress was elevated in mice fed with STHD-01, whereas some anti-oxidant factors were significantly elevated after antibiotics treatment. These results suggest that the gut microbiota is a key factor in improving oxidative stress induced by STHD-01 feeding.

Bile acid metabolism regulated by the gut microbiota promotes non-alcoholic steatohepatitis-associated hepatocellular carcinoma in mice.

Gut microbiota plays a significant role in the development of hepatocellular carcinoma (HCC) in non-alcoholic steatohepatitis (NASH). However, understanding of the precise mechanism of this process remains incomplete. A new class steatohepatitis-inducing high-fat diet (HFD), namely STHD-01, can promote the development of HCC without the administration of chemical carcinogens. Using this diet, we comprehensively analyzed changes in the gut microbiota and its metabolic functions during the development of HCC in NASH. Mice fed the STHD-01 developed NASH within 9 weeks. NASH further progressed into HCC by 41 weeks. Treatment with antibiotics significantly attenuated liver pathology and suppressed tumor development, indicating the critical role of the gut microbiota in tumor development in this model. Accumulation of cholesterol and bile acids in the liver and feces increased after feeding the mice with STHD-01. Treatment with antibiotics did not reverse these phenotypes. In contrast, accumulation of secondary bile acids was dramatically reduced after the treatment with antibiotics, suggesting the critical role of the gut microbiota in the conversion of primary bile acids to secondary bile acids. Secondary bile acids such as deoxycholic acid activated the mTOR, pathway in hepatocytes. Activation of mTOR was observed in the liver of mice fed STHD-01, and the activation was reduced when mice were treated with antibiotics. Collectively, bile acid metabolism by the gut microbiota promotes HCC development in STHD-01-induced NASH.

Gut microbiota-mediated generation of saturated fatty acids elicits inflammation in the liver in murine high-fat diet-induced steatohepatitis.

BACKGROUND: The gut microbiota plays crucial roles in the development of non-alcoholic steatohepatitis (NASH). However, the precise mechanisms by which alterations of the gut microbiota and its metabolism contributing to the pathogenesis of NASH are not yet fully elucidated. METHODS: Mice were fed with a recently reported new class of high-fat diet (HFD), steatohepatitis-inducing HFD (STHD)-01 for 9 weeks. The composition of the gut microbiota was analyzed by T-RFLP. Luminal metabolome was analyzed using capillary electrophoresis and liquid chromatography time-of-flight mass spectrometry (CE- and LC-TOFMS). RESULTS: Mice fed the STHD-01 developed NASH-like pathology within a short period. Treatment with antibiotics prevented the development of NASH by STHD-01. The composition of the gut microbiota and its metabolic activities were markedly perturbed in the STHD-01-fed mice, and antibiotic administration normalized these changes. We identified that long-chain saturated fatty acid and n-6 fatty acid metabolic pathways were significantly altered by STHD-01. Of note, the changes in gut lipidome caused by STHD-01 were mediated by gut microbiota, as the depletion of the gut microbiota could reverse the perturbation of these metabolic pathways. A saturated long-chain fatty acid, palmitic acid, which accumulated in the STHD-01 group, activated liver macrophages and promoted TNF-α expression. CONCLUSIONS: Lipid metabolism by the gut microbiota, particularly the saturation of fatty acids, affects fat accumulation in the liver and subsequent liver inflammation in NASH.

microRNA-mediated resistance to hypoglycemia in the HepG2 human hepatoma cell line.

BACKGROUND: It is generally accepted that the energy resources of cancer cells rely on anaerobic metabolism or the glycolytic system, even if they have sufficient oxygen. This is known as the Warburg effect. The cells skillfully survive under hypoglycemic conditions when their circumstances change, which probably at least partly involves microRNA (miRNA)-mediated regulation. METHODS: To determine how cancer cells exploit miRNA-mediated epigenetic mechanisms to survive in hypoglycemic conditions, we used DNA microarray analysis to comprehensively and simultaneously compare the expression of miRNAs and mRNAs in the HepG2 human hepatoma cell line and in cultured normal human hepatocytes. RESULTS: The hypoglycemic condition decreased the expression of miRNA-17-5p and -20a-5p in hepatoma cells and consequently upregulated the expression of their target gene p21. These regulations were also confirmed by using antisense inhibitors of these miRNAs. In addition to this change, the hypoglycemic condition led to upregulated expression of heat shock proteins and increased resistance to caspase-3-induced apoptosis. However, we could not identify miRNA-mediated regulations, despite using comprehensive detection. Several interesting genes were also found to be upregulated in the hypoglycemic condition by the microarray analysis, probably because of responding to this cellular stress. CONCLUSION: These results suggest that cancer cells skillfully survive in hypoglycemic conditions, which frequently occur in malignancies, and that some of the gene regulation of this process is manipulated by miRNAs.

Silencing of microRNA-122 is an early event during hepatocarcinogenesis from non-alcoholic steatohepatitis.

Non-alcoholic steatohepatitis (NASH) has emerged as a common cause of chronic liver disease and virus-independent hepatocellular carcinoma (HCC) in patients with obesity, diabetes, and metabolic syndrome. To reveal the molecular mechanism underlying hepatocarcinogenesis from NASH, microRNA (miRNA) expression profiles were analyzed in STAM mice, a NASH-HCC animal model. MicroRNA expression was also examined in 42 clinical samples of HCC tissue. Histopathological images of the liver of STAM mice at the ages of 6, 8, 12, and 18 weeks showed findings compatible with fatty liver, NASH, liver cirrhosis (LC), and HCC, respectively. Expression of miR-122 in non-tumor LC at the age of 18 weeks was significantly lower than that in LC at the age of 12 weeks. Expression of miR-122 was further decreased in HCCs relative to non-tumor LC at the age of 18 weeks. Expression of miR-122 was also decreased in clinical samples of liver tissue showing macrovesicular steatosis and HCC, being consistent with the findings in the NASH model mice. DNA methylation analysis revealed that silencing of miR-122 was not mediated by DNA hypermethylation of the promoter region. These results suggest that silencing of miR-122 is an early event during hepatocarcinogenesis from NASH, and that miR-122 could be a novel molecular marker for evaluating the risk of HCC in patients with NASH.

An ectotherm homologue of human predicted gene NAT16 encodes histidine N-acetyltransferase responsible for Nα-acetylhistidine synthesis.

BACKGROUND: Nα-Acetylhistidine (NAH) is present in very high concentrations exclusively in the brain and lens of ectothermic vertebrates, including ray-finned fishes, amphibians and reptiles, and not in those of endothermic birds and mammals. Although NAH is known to be synthesized from l-His and acetyl-CoA by histidine N-acetyltransferase (HISAT; EC 2.3.1.33), the gene encoding HISAT has remained unknown for any organism. METHODS: HISAT was purified from the blue mackerel brain, and its partial amino acid sequences were analyzed using mass spectrometry and Edman degradation. Using the sequence information, the corresponding gene was cloned and sequenced. Recombinant proteins encoded by the fish gene and its human homologue were expressed in a cell-free translation system. RESULTS: HISAT was identified to be a protein encoded by a fish homologue of the human predicted gene NAT16 (N-acetyltransferase 16). HISAT is an unstable enzyme that is rapidly and irreversibly inactivated during preincubation at 37°C in the absence of acetyl-CoA. In fish brain, the HISAT gene is expressed as two splice variants containing an identical ORF but differing lengths of 5'-UTR. Both variants are expressed exclusively in the fish brain and lens. Interestingly, the recombinant human NAT16 protein, unlike the recombinant fish HISAT, has only trace enzyme activity for NAH synthesis. CONCLUSIONS: These results propose that the function of mammalian NAT16 has been altered from l-His acetylation (NAH synthesis) to another different biological role. GENERAL SIGNIFICANCE: The molecular identification of HISAT will allow progress in the understanding of the physiological function of NAH in ectothermic vertebrates.

Molecular cloning and biochemical characterization of two novel Neu3 sialidases, neu3a and neu3b, from medaka (Oryzias latipes).

Mammalian Neu3 sialidases are involved in various biological processes, such as cell death and differentiation, through desialylation of gangliosides. The enzymatic profile of Neu3 seems to be highly conserved from birds to mammals. In fish, the functional properties of Neu3 sialidase are not clearly understood, with the partial exception of the zebrafish form. To cast further light on the molecular evolution of Neu3 sialidase, we identified the encoding genes in the medaka Oryzias latipes and investigated the properties of the enzyme. PCR amplification using medaka brain cDNA allowed identification of two novel medaka Neu3 genes, neu3a and neu3b. The YRIP, VGPG motif and Asp-Box, characteristic of consensus motifs of sialidases, were well conserved in the both medaka Neu3 sialidases. When each gene was transfected into HEK293 to allow cell lysates for the use of enzymatic characterization, two Neu3 sialidases showed strict substrate specificity toward gangliosides, similar to mammalian Neu3. The optimal pH values were at pH 4.2 and pH 4.0, respectively, and neu3b in particular showed a broad optimum. Immunofluorescence assays indicated neu3a localization at plasma membranes, while neu3b was found in cytosol. The tissue distribution of two genes was then investigated by estimation of mRNA expression and sialidase activity, both being dominantly expressed in the brain. In neu3a gene-transfected neuroblastoma cells, the enzyme was found to positively regulate retinoic acid-induced differentiation with the elongation of axon length. On the other hand, neu3b did not affect neurite formation. These results and phylogenetic analysis suggested that the medaka neu3a is an evolutionally conserved sialidase with regard to enzymatic properties, whereas neu3b is likely to have originally evolved in medaka.

Purification and identification of two carnosine-cleaving enzymes, carnosine dipeptidase I and Xaa-methyl-His dipeptidase, from Japanese eel (Anguilla japonica).

Three enzymes, carnosine dipeptidase I (EC 3.4.13.20, CNDP1), carnosine dipeptidase II (EC 3.4.13.18, CNDP2), and Xaa-methyl-His dipeptidase (or anserinase: EC 3.4.13.5, ANSN), are known to be capable of catalyzing the hydrolysis of carnosine (ß-alanyl-l-histidine), in vertebrates. Here we report the purification and identification of two unidentified carnosine-cleaving enzymes from Japanese eel (Anguilla japonica). Two different dipeptidases were successfully purified to homogeneity from the skeletal muscle; one exhibited a broad substrate specificity, while the other a narrow specificity. N-terminal amino-acid sequencing, deglycosylation analysis, and genetic analysis clearly revealed that the former is a homodimer of glycosylated subunits, encoded by ANSN, and the latter is another homodimer of glycosylated subunits, encoded by CNDP1; that is, Xaa-methyl-His dipeptidase, and carnosine dipeptidase I respectively. This is the first report on the identification of carnosine dipeptidase I from a non-mammal. Database search revealed presence of a CNDP1 ortholog only from salmonid fishes, including Atlantic salmon and rainbow trout, but not from other ray-finned fish species, such as zebrafish, fugu, and medaka whose genomes have been completely sequenced. The mRNAs of CNDP1 and ANSN are strongly expressed in the liver of Japanese eel, compared with other tissues, while that of CNDP2 is widely distributed in all tissues tested.

Effects of tetracycline hydrochloride on measurements with the laser fluorescence device DIAGNOdent: in vitro and in vivo studies.

Dental materials that fluoresce affect the reading of the laser fluorescence device DIAGNOdent. Tetracycline hydrochloride (TCH) shows fluorescence and is retained in teeth. The aim of this study was to evaluate the effects of TCH on the DIAGNOdent reading. Filter-paper discs that contained various amounts of TCH were prepared (0.16-10 mg per disc). One-day-old newborn rats were subcutaneously injected with TCH for 29 days, and their mandibles were then removed. The DIAGNOdent values (D-V) of the discs and first molars of the rats were measured before and after they were subjected to ultraviolet irradiation (UV). The D-V of discs containing TCH increased depending on the amount of TCH. The D-Vs of discs with lower amounts of TCH (0.16-1.25 mg) were approximately 10-15, and these values increased to 30-40 under UV. In addition, the D-Vs of molars after UV were twofold greater than those before UV. These results suggest that TCH might affect the readings obtained by DIAGNOdent.

Tea polyphenols inhibit rat osteoclast formation and differentiation.

Matrix metalloproteinases (MMPs) play an important role in degeneration of the matrix associated with bone and cartilage. Regulation of osteoclast activity is essential in the treatment of bone disease, including osteoporosis and rheumatoid arthritis. Polyphenols in green tea, particularly epigallocatechin-3-gallate (EGCG), inhibit MMPs expression and activity. However, the effects of the black tea polyphenol, theaflavin-3,3'-digallate (TFDG), on osteoclast and MMP activity are unknown. Therefore, we examined whether TFDG and EGCG affect MMP activity and osteoclast formation and differentiation in vitro. TFDG or EGCG (10 and 100 µM) was added to cultures of rat osteoclast precursors cells and mature osteoclasts. Numbers of multinucleated osteoclasts and actin rings decreased in polyphenol-treated cultures relative to control cultures. MMP-2 and MMP-9 activities were lower in TFDG- and EGCG-treated rat osteoclast precursor cells than in control cultures. MMP-9 mRNA levels declined significantly in TFDG-treated osteoclasts in comparison to control osteoclasts. TFDG and EGCG inhibited the formation and differentiation of osteoclasts via inhibition of MMPs. TFDG may suppress actin ring formation more effectively than EGCG. Thus, TFDG and EGCG may be suitable agents or lead compounds for the treatment of bone resorption diseases.

Tea Polyphenols Inhibit Rat Osteoclast Formation and Differentiation.

Matrix metalloproteinases (MMPs) play an important role in degeneration of the matrix associated with bone and cartilage. Regulation of osteoclast activity is essential in the treatment of bone disease, including osteoporosis and rheumatoid arthritis. Polyphenols in green tea, particularly epigallocatechin-3-gallate (EGCG), inhibit MMPs expression and activity. However, the effects of the black tea polyphenol, theaflavin-3,3'-digallate (TFDG), on osteoclast and MMP activity are unknown. Therefore, we examined whether TFDG and EGCG affect MMP activity and osteoclast formation and differentiation in vitro. TFDG or EGCG (10 and 100 µM) was added to cultures of rat osteoclast precursors cells and mature osteoclasts. Numbers of multinucleated osteoclasts and actin rings decreased in polyphenol-treated cultures relative to control cultures. MMP-2 and MMP-9 activities were lower in TFDG- and EGCG-treated rat osteoclast precursor cells than in control cultures. MMP-9 mRNA levels declined significantly in TFDG-treated osteoclasts in comparison to control osteoclasts. TFDG and EGCG inhibited the formation and differentiation of osteoclasts via inhibition of MMPs. TFDG may suppress actin ring formation more effectively than EGCG. Thus, TFDG and EGCG may be suitable agents or lead compounds for the treatment of bone resorption diseases.

Purification and identification of a novel primitive secretory enzyme catalyzing the hydrolysis of imidazole-related dipeptides in the jawless vertebrate Lethenteron reissneri.

Imidazole-related dipeptides, such as carnosine and anserine, occur widely in skeletal muscles of jawed vertebrates. All of the known enzymes that catalyze the hydrolysis of these dipeptides belong to the M20A metallopeptidase subfamily; two secretory enzymes, serum carnosinase (EC 3.4.13.20) and anserinase (EC 3.4.13.5), and one non-secretory enzyme, cytosolic nonspecific dipeptidase (EC 3.4.13.18). Here we report the enzymatic characterization and molecular identification of an unidentified enzyme, which catalyzes the hydrolysis of these dipeptides, from the skeletal muscle of Far Eastern brook lamprey (Lethenteron reissneri). A 60-kDa subunit protein of the enzyme was purified to near homogeneity. We cloned two M20A genes from the skeletal muscle of Far Eastern brook lamprey; one was a secretory-type gene encoding for the 60-kD protein, and another was a non-secretory-type gene presumably encoding for cytosolic nonspecific dipeptidase. Our findings indicate that the purified enzyme is a N-glycosylated secretory M20A dipeptidase distributed specifically in the jawless vertebrate group, and may be derived from a common ancestor gene between serum carnosinase and anserinase. We propose that this dipeptidase is a novel secretory M20A enzyme and is classified as neither serum carnosinase nor anserinase.

Homologue of mammalian apolipoprotein A-II in non-mammalian vertebrates.

Although apolipoprotein with molecular weight 14 kDa (apo-14 kDa) is associated with fish plasma high-density lipoproteins (HDLs), it remains to be determined whether apo-14 kDa is the homologue of mammalian apoA-II. We have obtained the full cDNA sequences that encode Japanese eel and rainbow trout apo-14 kDa. Homologues of Japanese eel apo-14 kDa sequence could be found in 14 fish species deposited in the DDBJ/EMBL/GenBank or TGI database. Fish apo-14 kDa lacks propeptide and contains more internal repeats than mammalian apoA-II. Nevertheless, phylogenetic analysis allowed fish apo-14 kDa to be the homologue of mammalian apoA-II. In addition, in silico cloning of the TGI, Ensembl, or NCBI database revealed apoA-IIs in dog, chicken, green anole lizard, and African clawed frog whose sequences had not so far been available, suggesting both apoA-I and apoA-II as fundamental constituents of vertebrate HDLs.

Occurrence of a novel acetylated amino acid, N(alpha)-acetylhistidine, in skeletal muscle of freshwater fish and other ectothermic vertebrates.

The occurrence of N(alpha)-acetylhistidine (NAH) in skeletal muscle of 91 species of freshwater fish and 9 species of other ectothermic vertebrates was investigated, with consideration of phylogenetic relationships. Of the 91 freshwater fish species examined, 13 species (7 cichlids, 5 anabantids, and 1 catfish) contained considerable amounts (>1 micromol/g) of NAH in their skeletal muscles. The highest level (10.37 micromol/g) of NAH was found in the tissue of Betta splendens (Siamese fighting fish). Moreover, the NAH contents in the tissues of Trichogaster trichopterus (three spot gourami), Kryptopterus bicirrhis (glass catfish), Oreochromis niloticus (Nile tilapia), Mikrogeophagus ramirezi (ram cichlid) and Parachromis managuensis (Guapote tigre) were 3.17-6.16 micromol/g. The skeletal muscle of amphibians (5 species) and reptiles (4 species) had a low level (<0.25 micromol/g) of NAH. The present findings clearly demonstrate NAH as the fifth imidazole-related compound, in addition to histidine, carnosine, anserine and ophidine (balenine), recognized as a major non-protein nitrogenous constituent in the skeletal muscle of vertebrate animals.

Pharmacological topics of bone metabolism: a novel bisphosphonate for the treatment of periodontitis.

It has been reported that the pharmacological characteristics of bisphosphonates vary depending on the side chain attached to the carbon atom of the P-C-P bond. TRK-530 is a novel synthetic bisphosphonate with an anti-oxidant methylthio-phenylthio side chain. This compound has been suggested to have both anti-inflammatory and anti-bone-resorbing effects. Such a compound could be effective for the treatment of diseases with excessive bone resorption accompanied by inflammation. We have been studying this compound as a potential therapeutic agent for periodontitis. To date, we have found that 1) TRK-530 inhibited osteoclastic bone resorption in animals and in bone organ culture, 2) both systemic and topical administration of TRK-530 prevented alveolar bone loss in animals with experimental periodontitis, 3) TRK-530 prevented prostaglandin E(2) synthesis by inhibiting the expression of cyclooxygenase (COX)-2 mRNA, and 4) TRK-530 inhibited the formation of dental calculus. The above results suggest that TRK-530 might be useful for the treatment of alveolar bone loss in periodontitis.

Degradation of vitamin B12 in dietary supplements.

Beverages and solid dietary supplements rich in various added vitamins and minerals have recently become available. It seems reasonable to consider that the intake of these foods is convenient for easy ingestion of nutrients, but problems caused by blending different nutrients in high concentrations have arisen. We focused on vitamin B12 (B12) among vitamins and determined the B12 contents of beverages and solid dietary supplements purchased from a retail shop. The B12 contents of three of five beverages were less than stated on the labels. On the other hand, certain beverages unexpectedly contained much more B12 than stated on the labels. In these beverages the amount of B12 decreased rapidly with time, whereas B12 content was lower than stated on the label in only one of four solid dietary supplements. The content of B12 was affected by storage time, light exposure, temperature and vitamin C. From experimental analysis with a competitive binding assay method employing a ACS Chemiluminescent B12 kit, examining differential binding by intrinsic factors and spectral analysis of B12, it was determined that some of the B12 might have been converted into B12 analogues or small degradation products by multinutrient interaction during storage.

Hemolymph patterns of free amino acids in the brine shrimp Artemia franciscana after three days starvation at different salinities.

The hemolymph pattern of free amino acids was examined in the brine shrimp, Artemia franciscana (Great Salt Lake origin). After one-month acclimation to 35 or 60 ppt salinity at 27 degrees C, the animals were transferred to 10, 35 or 60 ppt salinities to continue acclimation for 3 days without feeding at 27 degrees C. The osmolarity of one of the new media was raised by glycerol addition. In the hemolymph, 8 amino acids such as taurine, alanine, threonine, serine, lysine, glycine, arginine and leucine, comprised approximately 70% of the total content of free amino acids. This pattern suggested internal proteolysis due to starvation at high temperature. The total content of free amino acids significantly increased at 10 and 60 ppt salinities in comparison to 35 ppt. The hemolymph patterns from the 10 ppt and glycerol-added media showed a singularly high peak of taurine or alanine.

Fructooligosaccharides consumption inhibits the loss of iron from the incisor enamel surface in gastrectomized rat.

We examined the effects of fructooligosaccharides (FOS) on the reduction in the incisor iron content in gastrectomized rat. Twenty-eight 5- wk-old male Sprague-Dawley rats were divided into two groups: sham operated (bSH) and gastrectomized (bGX). After 4 wk, each group was divided into two subgroups according to the presence or absence of 7.5% FOS in the synthetic diet (SH, SH+FOS, GX, and GX+FOS). At 10 wk after surgery, the maxilla was prepared to examine the iron content of the incisor enamel surface at four points. These points corresponded to the iron content at 6, 7, 8, and 10 wk, respectively. Blood was collected to determine serum iron levels at 4 and 10 wk. The serum iron level significantly decreased at 4 and 10 wk in the GX group. At 10 wk, the level in the GX+FOS group significantly increased but did not reach that in the SH group. The iron content of the enamel surface time-dependently increased and no significant differences were seen between SH and GX+FOS at 8 and 10 wk. These results suggest that FOS consumption impaired the loss of enamel content following gastrectomy, and this effect preceded the effect on the serum iron level.

Current topics in pharmacological research on bone metabolism: inhibitory effects of bisphosphonates on the differentiation and activity of osteoclasts.

Despite the extensive use of bisphosphonates (BPs) in the treatment of metabolic bone diseases associated with increased osteoclastic bone resorption, the precise mechanism of their action on bone metabolism is still unclear. To clarify at which stages of osteoclast differentiation and activation that BPs influence, we examined the osteoclasts generated from mononuclear precursors and osteoclasts in the calvaria by laser scanning confocal microscopy. The studies showed that BPs inhibit lipopolysaccharide- or parathyroid hormone-induced osteoclast differentiation, fusion, attachment, actin ring formation, and activation and that both beta3 integrin and osteopontin have an important role in cytoskeletal rearrangements associated with cell attachment and resorption in osteoclasts.