Sphingomyelin synthase 2 loss suppresses steatosis but exacerbates fibrosis in the liver of mice fed with choline-deficient, L-amino acid-defined, high-fat diet.

Sphingomyelin synthase 2 (SMS2) regulates sphingomyelin synthesis and contributes to obesity and hepatic steatosis. Here, we investigated the effect of SMS2 deficiency on liver fibrosis in mice fed with choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) or injected with carbon tetrachloride (CCl4), respectively. SMS2 deficiency suppressed hepatic steatosis, but exacerbated fibrosis induced by CDAHFD feeding. Sphingosine 1-phosphate (S1P), which is a key lipid mediator induces fibrosis in various organs, was increased in the liver of mice fed with CDAHFD. The increase of S1P became prominent by SMS2 deficiency. Meanwhile, SMS2 deficiency had no impact on CCl4-induced liver injury, fibrosis and S1P levels. Our findings demonstrated that SMS2 deficiency suppresses steatosis but worsens fibrosis in the liver in a specific condition with CDAHFD feeding.

Quantifying Protein-Specific N-Glycome Profiles by Focused Protein and Immunoprecipitation Glycomics.

Serum N-glycans have been reported to be potential diagnostic and therapeutic biomarkers for many diseases and conditions, such as inflammation, fibrosis, and cancer progression. We previously described the focused protein glycomic analysis (FPG) from gel-separated serum proteins. With this methodology, we sought novel glycan biomarkers for nonalcoholic steatohepatitis (NASH) and successfully identified some N-glycans that were significantly elevated in NASH patients compared to nonalcoholic fatty liver patients. Among them, trisialylated monofucosylated triantennary glycan (A3F) of alpha-1 antitrypsin showed the most dynamic change. For rapid identification of N-glycans on the focused proteins, we constructed a simplified method called immunoprecipitation glycomics (IPG), where the target proteins were immunoprecipitated with affinity beads and subsequently subjected to glycomic analysis by MALDI-TOF MS. Focusing on alpha-1 antitrypsin and ceruloplasmin as the target proteins, we compared the values of N-glycans determined by FPG and IPG. The quantified values of each N-glycan by these two methods showed a statistically significant correlation, indicating that high throughput and quantitative N-glycomics of targeted proteins can be achieved by the simplified IPG method. Thus, an analytical strategy combining FPG and IPG can be adapted to general biomarker discovery and validation in appropriate disease areas.

Identification of unique glycoisoforms of vitamin D-binding protein and haptoglobin as biomarker candidates in hepatocarcinogenesis of STAM mice.

Hepatocellular carcinoma (HCC) is the major subtype of primary liver cancer, and is typically diagnosed late in its course. Considering the limitations and the reluctance of patients to undergo a liver biopsy, a reliable, noninvasive diagnostic marker that predicts and assesses the treatment and prognosis of HCC is needed. With recent technological advances of mass spectrometry, glycomics is gathering momentum and holds substantial potential to discover new glycan markers in cancer research. Here, to discover specific glycan markers for the early diagnosis of HCC, we analyzed the glycan profiles of gel-separated serum proteins of progressive liver disease model mice. By focused protein glycomics of 12 gel-separated glycoproteins using sera from the mouse models, we revealed the entire profile of glycans in each major serum protein. We found that the levels of trisialylated triantennary glycans of haptoglobin and vitamin D-binding protein increased significantly as the disease progressed, while the alteration in these protein levels were modest. Furthermore, these glycan increases were not observed in age-matched control mice. In conclusion, our approach has identified specific glycan marker candidates for the early diagnosis of HCC.

FMISO accumulation in tumor is dependent on glutathione conjugation capacity in addition to hypoxic state.

OBJECTIVE: 18F-fluoromisonidazole (FMISO), a well-known PET imaging probe for diagnosis of hypoxia, is believed to accumulate in hypoxic cells via covalent binding with macromolecules after reduction of the nitro group. Previously, we showed the majority of 18F-FMISO was incorporated into low-molecular-weight metabolites in hypoxic tumors, and the glutathione conjugate of reduced FMISO (amino-FMISO-GS) distributed in the tumor hypoxic regions as revealed by imaging mass spectrometry (IMS). The present study was conducted to clarify whether FMISO is metabolized to amino-FMISO-GS within tumor cells and how amino-FMISO-GS contributes to FMISO accumulation in hypoxic cells. We also evaluated the relationship between FMISO accumulation and the glutathione conjugation-related factors in the cells. METHODS: Tumor cells (FaDu, LOVO, and T24) were treated with 18F-FMISO and incubated under normoxic or hypoxic conditions for 4 h. The FMISO metabolites were analyzed with LC-ESI-MS. Several glutathione conjugation-related factors of tumor cells were evaluated in vitro. FaDu tumor-bearing mice were intravenously injected with 18F-FMISO and the tumors were excised at 4 h post-injection. Autoradiography, IMS and histologic studies were performed. RESULTS: Amino-FMISO-GS was the main contributor to FMISO incorporated in hypoxic FaDu cells in vitro and in vivo. Total FMISO uptake levels and amino-FMISO-GS levels were highest in FaDu, followed by LOVO, and then T24 (total uptake: 0.851 ± 0.009 (FaDu), 0.617 ± 0.021 (LOVO) and 0.167 ± 0.006 (T24) % dose/mg protein; amino-FMISO-GS: 0.502 ± 0.035 (FaDu), 0.158 ± 0.013 (LOVO), and 0.007 ± 0.001 (T24) % dose/mg protein). The glutathione level of FaDu was significantly higher than those of LOVO and T24. The enzyme activity of glutathione-S-transferase catalyzing the glutathione conjugation reaction in FaDu was similar levels to that in LOVO, and was higher than that in T24. Quantitative RT-PCR analysis revealed that the expression levels of efflux transporters of the glutathione conjugate (multidrug resistance-associated protein 1) were lowest in FaDu, followed by LOVO, and then T24. CONCLUSIONS: FMISO accumulates in hypoxic cells through reductive metabolism followed by glutathione conjugation. We illustrated the possibility that increased production and decreased excretion of amino-FMISO-GS contribute to FMISO accumulation in tumor cells under hypoxic conditions.

Direct Involvement of Arachidonic Acid in the Development of Ear Edema via TRPV3.

Arachidonic acid (AA) plays a pivotal role in the development of edema via its oxidized metabolites derived from cyclooxygenase (COX) and lipoxygenase (LOX), and is recently recognized as an activator of TRPV3. However, it is not clear whether AA plays some TRPV3-mediated pathological roles in the development of edema. Pharmacological and histological studies using ICRTRPV3+/+ and ICRTRPV3-/- mice indicated that higher ear edema responses to topical application of AA were observed in ICRTRPV3+/+ mice compared with ICRTRPV3-/- mice. However, there was no difference in the ear edema response to 12-O-tetradecanoylphorbol 13-acetate, skin histology, and skin barrier function between these mouse strains. Furthermore, oxidized fatty acids from the lesional site were analyzed to elucidate the TRPV3-mediated pathological roles of AA, and the results revealed that there were no differences in the level of COX or LOX metabolites derived from AA between both mouse strains. We concluded that AA plays a role in the development of TRPV3-mediated ear edema and that this result may contribute to better understanding of the pathophysiological mechanisms involved in the development of a certain type of edema.

Discovery of novel cyclic peptide inhibitors of dengue virus NS2B-NS3 protease with antiviral activity.

NS2B-NS3 protease is an essential enzyme for the replication of dengue virus (DENV), which continues to be a serious threat to worldwide public health. We designed and synthesized a series of cyclic peptides mimicking the substrates of this enzyme, and assayed their activity against the DENV-2 NS2B-NS3 protease. The introduction of aromatic residues at the appropriate positions and conformational restriction generated the most promising cyclic peptide with an IC50 of 0.95µM against NS2B-NS3 protease. Cyclic peptides with proper positioning of additional arginines and aromatic residues exhibited antiviral activity against DENV. Furthermore, replacing the C-terminal amide bond of the polybasic amino acid sequence with an amino methylene moiety stabilized the cyclic peptides against hydrolysis by NS2B-NS3 protease, while maintaining their enzyme inhibitory activity and antiviral activity.

Generation of Novel Anti-MUC1 Monoclonal Antibodies with Designed Carbohydrate Specificities Using MUC1 Glycopeptide Library.

Numerous anti-mucin 1 (anti-MUC1) antibodies that recognize O-glycan core structures have already been developed. However, most of them show low specificities toward O-glycan structures and/or low affinity toward a monovalent epitope. In this study, using an MUC1 glycopeptide library, we established two novel anti-MUC1 monoclonal antibodies (1B2 and 12D10) with designed carbohydrate specificities. Compared with previously reported anti-MUC1 antibodies, 1B2 and 12D10 showed quite different features regarding their specificities, affinities, and reactivity profiles to various cell lines. Both antibodies recognized specific O-glycan structures at the PDT*R motif (the asterisk represents an O-glycosylation site). 1B2 recognized O-glycans with an unsubstituted O-6 position of the GalNAc residue (Tn, T, and 23ST), whereas 12D10 recognized Neu5Ac at the same position (STn, 26ST, and dST). Neither of them bound to glycopeptides with core 2 O-glycans that have GlcNAc at the O-6 position of the GalNAc residue. Furthermore, 1B2 and 12D10 showed a strong binding to not only native MUC1 but also 20-mer glycopeptide with a monovalent epitope. These anti-MUC1 antibodies should thus become powerful tools for biological studies on MUC1 O-glycan structures. Furthermore, the strategy of using glycopeptide libraries should enable the development of novel antibodies with predesigned O-glycan specificities.

Decreases in 15-lipoxygenase metabolites in Olmsted syndrome model rats.

BACKGROUND: Olmsted syndrome (OS) is a congenital dermatosis characterized by palmoplantar keratoderma and periorificial keratotic plaque. TRPV3 (transient receptor potential vanilloid subtype 3) encodes a thermosensitive Ca2+ channel and is the causative gene of OS. However, the molecular mechanism that causes the pathological development of OS is unclear. OBJECTIVE: We aimed to investigate the molecular mechanisms underlying OS pathology from the perspective of lipid metabolism. METHODS: Comprehensive lipidomics and microarray analyses were conducted on tissue samples from a non-lesional skin area of OS model rats (Ht rats) and from wild type (WT) rats as the control. RESULTS: Infiltration of leukocytes such as eosinophils and neutrophils and an increase in the fibrotic region were detected in the unaffected skin area of Ht rats compared with the WT rats. Among about 600 lipid species examined, the levels of 15-lipoxygenase (LOX) metabolites, the precursors of anti-inflammatory and pro-resolving lipid mediators, and dihydroceramides decreased by ≥16-fold in Ht rats compared with WT rats. Consistent with the decreases in the 15-LOX metabolites, expression levels of the genes that encode the 15-LOXs, Alox15 and Alox15b, were largely reduced. Conversely, increased expression levels were detected of Il36b, Ccl20, Cxcl1, and Cxcl2, which encode cytokines/chemokines, and S100a8 and S100a9, which encode the Ca2+ binding proteins that are implicated in epidermal proliferation. CONCLUSION: The pro-inflammatory state in the unaffected skin of Ht rats caused by decreases in 15-LOX metabolites and increases in cytokines/chemokines may contribute to the pathogenesis of OS.

AM251 Suppresses Epithelial-Mesenchymal Transition of Renal Tubular Epithelial Cells.

Epithelial-mesenchymal transition (EMT) of renal tubular epithelial cells is one of the causative mechanisms of kidney fibrosis. In our study, we screened lipophilic compounds using a lipid library including approximately 200 lipids to identify those that suppressed EMT induced by a transforming growth factor (TGF)-ß1 stimulus. Initial screening was performed with the immortalized HK-2 renal tubule epithelial cell line. The most promising compounds were further tested in RPTEC primary renal tubule epithelial cells. We found that the synthetic lipid AM251 suppressed two hallmark events associated with EMT, the upregulation of collagen 1A1 (COL1A1) and downregulation of E-cadherin. Though AM251 is known to act as an antagonist for the cannabinoid receptor type 1 (CB1) and an agonist for the G protein-coupled receptor 55 (GRP55), the suppression of EMT by AM251 was not mediated through either receptor. Microarray analyses revealed that AM251 inhibited induction of several EMT transcription factors such as SNAIL1, which is the key inducer of EMT, and the AP-1 transcription factors FOSB and JUNB. Activation of SMAD2/3 and p38 mitogen-activated protein kinase (MAPK) was inhibited by AM251, with greater inhibition of the latter, indicating that AM251 acted upstream of SMAD/p38 MAPK in the TGF-ß signaling pathway. Our findings regarding the effects of AM251 on the TGF-ß signaling pathway may inform development of a novel therapeutic agent suppressing EMT, thus preventing kidney fibrosis.

Quantitative analysis of total serum glycome in human and mouse.

Model mice are frequently used in drug discovery research. Knowledge of similarities and differences between the mouse and human glycomes is critical when model mice are used for the discovery of glycan-related biomarkers and targets for therapeutic intervention. Since few comparative glycomic studies between human and mouse have been conducted, we performed a comprehensive comparison of the major classes of glycans in human and mouse sera using mass spectrometric and liquid chromatographic analyses. Up to 131 serum glycans, including N-glycans, free oligosaccharides (fOSs), glycosaminoglycans, O-glycans, and glycosphingolipid (GSL)-glycans, were quantified. In both serum samples, N-glycans were the most abundant in the total serum glycome, while fOSs were the least abundant. As expected, the diversity of sialic acid (i.e. Neu5Ac vs. Neu5Gc) was the major species difference between human and mouse in terms of N- and O-glycosylation, while GSL-glycomic profiles were completely different, even when the sialic acid diversity was taken into consideration. Furthermore, total serum glycomics of STAM mouse were unveiled as an initial step to identify novel biomarkers of liver diseases, with which we could identify several glycans with expression significantly increased or decreased expression.

Imaging Mass Spectrometry Revealed the Accumulation Characteristics of the 2-Nitroimidazole-Based Agent "Pimonidazole" in Hypoxia.

Hypoxia, or low oxygen concentration, is a key factor promoting tumor progression and angiogenesis and resistance of cancer to radiotherapy and chemotherapy. 2-Nitroimidazole-based agents have been widely used in pathological and nuclear medicine examinations to detect hypoxic regions in tumors; in particular, pimonidazole is used for histochemical staining of hypoxic regions. It is considered to accumulate in hypoxic cells via covalent binding with macromolecules or by forming reductive metabolites after reduction of its nitro group. However, the detailed mechanism of its accumulation remains unknown. In this study, we investigated the accumulation mechanism of pimonidazole in hypoxic tumor tissues in a mouse model by mass spectrometric analyses including imaging mass spectrometry (IMS). Pimonidazole and its reductive metabolites were observed in the tumor tissues. However, their locations in the tumor sections were not similar to the positively stained areas in pimonidazole-immunohistochemistry, an area considered hypoxic. The glutathione conjugate of reduced pimonidazole, a low-molecular-weight metabolite of pimonidazole, was found in tumor tissues by LC-MS analysis, and our IMS study determined that the intratumor localization of the glutathione conjugate was consistent with the area positively immunostained for pimonidazole. We also found complementary localization of the glutathione conjugate and reduced glutathione (GSH), implying that formation of the glutathione conjugate occurred in the tumor tissue. These results suggest that in hypoxic tumor cells, pimonidazole is reduced at its nitro group, followed by conjugation with GSH.

Characterization of the role of sphingomyelin synthase 2 in glucose metabolism in whole-body and peripheral tissues in mice.

Sphingomyelin synthase 2 (SMS2) is a proposed potential therapeutic target for obesity and insulin resistance. However, the contributions of SMS2 to glucose metabolism in tissues and its possible therapeutic mechanisms remain unclear. Thus, to determine whole-body glucose utilization and the contributions of each insulin-targeted tissue to glucose uptake, we performed a glucose kinetics study, using the radiolabeled glucose analog (18)F-2-fluoro-2-deoxy-D-glucose ((18)F-FDG), in wild-type (WT) and SMS2 knockout (KO) mice. Insulin signaling was enhanced in the liver, white adipose tissue and skeletal muscle of SMS2 KO mice compared with those of WT mice. In addition, compared with in WT mice, blood clearance of (18)F-FDG was accelerated in SMS2 KO mice when they were fed either a normal or a high fat diet. (18)F-FDG uptake was also increased in insulin-targeted tissues such as skeletal muscle in the SMS2 KO mice. Whereas skeletal muscle sphingolipid content was not clearly affected, plasma levels of very long-chain fatty acid (VLCFA)-containing ceramides were markedly increased in SMS2 KO mice, compared with in WT mice. We also generated liver-conditional SMS2 KO mice and performed glucose and insulin tolerance tests on mice with a high fat diet. However, no significant effect was observed. Thus, our study provided evidence that genetic inhibition of SMS2 elevated glucose clearance through activation of glucose uptake into insulin-targeted tissues such as skeletal muscle by a mechanism independent of hepatic SMS2. Our findings further indicate that this occurs, at least in part, via indirect mechanisms such as elevation of VLCFA-containing ceramides.

Imaging Mass Spectrometry Reveals Acyl-Chain- and Region-Specific Sphingolipid Metabolism in the Kidneys of Sphingomyelin Synthase 2-Deficient Mice.

Obesity was reported to cause kidney injury by excessive accumulation of sphingolipids such as sphingomyelin and ceramide. Sphingomyelin synthase 2 (SMS2) is an important enzyme for hepatic sphingolipid homeostasis and its dysfunction is considered to result in fatty liver disease. The expression of SMS2 is also high in the kidneys. However, the contribution of SMS2 on renal sphingolipid metabolism remains unclear. Imaging mass spectrometry is a powerful tool to visualize the distribution and provide quantitative data on lipids in tissue sections. Thus, in this study, we analyzed the effects of SMS2 deficiency on the distribution and concentration of sphingomyelins in the liver and kidneys of mice fed with a normal-diet or a high-fat-diet using imaging mass spectrometry and liquid chromatography/electrospray ionization-tandem mass spectrometry. Our study revealed that high-fat-diet increased C18-C22 sphingomyelins, but decreased C24-sphingomyelins, in the liver and kidneys of wild-type mice. By contrast, SMS2 deficiency decreased C18-C24 sphingomyelins in the liver. Although a similar trend was observed in the whole-kidneys, the effects were minor. Interestingly, imaging mass spectrometry revealed that sphingomyelin localization was specific to each acyl-chain length in the kidneys. Further, SMS2 deficiency mainly decreased C22-sphingomyelin in the renal medulla and C24-sphingomyelins in the renal cortex. Thus, imaging mass spectrometry can provide visual assessment of the contribution of SMS2 on acyl-chain- and region-specific sphingomyelin metabolism in the kidneys.

A hybrid strategy using global analysis of oxidized fatty acids and bioconversion by Bacillus circulans.

RATIONALE: Targeted oxidized fatty acid analysis has been widely used to understand the roles of fatty acids in the development of diseases. However, because of the extensive structural diversity of fatty acids, it is considered that unknown lipid metabolites will remain undetected. Here, to discover and identify unknown lipid metabolites in biological samples, a global analytical system and a method of synthesizing lipid standards were investigated. METHODS: Oxidized fatty acids in mouse lung tissues were extracted using mixed-mode spin columns. Separation was achieved via ultra-high-performance liquid chromatography, mass spectrometric (MS) analysis was conducted in full scan mode using a Q Exactive Plus instrument equipped with an electrospray ionization probe, and structure analysis was carried out by high-resolution data-dependent tandem mass spectrometry (dd-MS(2)). In addition, lipid standards, which are not commercially available, were synthesized by bioconversion using Bacillus circulans. RESULTS: Oxidized fatty acids in mouse lung tissues were analyzed by high-resolution accurate-mass analysis, and multiple unknown molecules were discovered and tentatively identified using high-resolution dd-MS(2). Among these molecules, 21-hydroxydocosahexaenoic acid (21-HDoHE) and 22-HDoHE, which are not commercially available, were synthesized by bioconversion. By comparing the exact masses, retention times, and characteristic fragment ions of the synthesized standards, 21-HDoHE and 22-HDoHE were definitively identified in the mouse lung tissue. CONCLUSIONS: Our strategy of global analysis and bioconversion can be used for the discovery and identification of unknown lipid molecules.

The accumulation mechanism of the hypoxia imaging probe "FMISO" by imaging mass spectrometry: possible involvement of low-molecular metabolites.

(18)F-fluoromisonidazole (FMISO) has been widely used as a hypoxia imaging probe for diagnostic positron emission tomography (PET). FMISO is believed to accumulate in hypoxic cells via covalent binding with macromolecules after reduction of its nitro group. However, its detailed accumulation mechanism remains unknown. Therefore, we investigated the chemical forms of FMISO and their distributions in tumours using imaging mass spectrometry (IMS), which visualises spatial distribution of chemical compositions based on molecular masses in tissue sections. Our radiochemical analysis revealed that most of the radioactivity in tumours existed as low-molecular-weight compounds with unknown chemical formulas, unlike observations made with conventional views, suggesting that the radioactivity distribution primarily reflected that of these unknown substances. The IMS analysis indicated that FMISO and its reductive metabolites were nonspecifically distributed in the tumour in patterns not corresponding to the radioactivity distribution. Our IMS search found an unknown low-molecular-weight metabolite whose distribution pattern corresponded to that of both the radioactivity and the hypoxia marker pimonidazole. This metabolite was identified as the glutathione conjugate of amino-FMISO. We showed that the glutathione conjugate of amino-FMISO is involved in FMISO accumulation in hypoxic tumour tissues, in addition to the conventional mechanism of FMISO covalent binding to macromolecules.

Antisense peptide nucleic acid­peptide conjugates for functional analyses of genes in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is one of the most common and clinically important pathogens because of its resistance to a wide variety of antibiotics. A number of treatments of P. aeruginosa have been developed, but there is still no definitive one. Antisense drugs have a great potential to treat multidrug-resistant P. aeruginosa because this technology, in principle, can inhibit the expression of any essential genes. Nucleic Acid Ther.2012, 22, 323 reported that peptide nucleic acid (PNA) antisenses conjugated to the carrier peptide (RXR)4 and targeted to ftsZ and acpP (essential genes) had antibacterial activity in P. aeruginosa. However, growth inhibition was also found with peptide-PNA antisense conjugates of mismatched sequences (negative controls), and hence there remains a possibility for considerable enhancement of basal level activity due to the general toxicity. To assess the true potential of peptide-PNA conjugates, we measured sequence-dependent knockdown of the (RXR)4-PNA conjugates by using a scrambled sequence as a negative control. In addition, we evaluated (RXR)4-PNA antisenses against three other essential genes (lepB, lptD and mraY) and a non-essential gene (PA1303), and confirmed that multiple sequences targeting only the essential genes showed antimicrobial activity in P. aeruginosa PAO1 cells. We also conducted a rescue experiment and confirmed that the antimicrobial activity of anti-mraY antisenses was an on-target effect, not due to general toxicity. These findings indicate that the (RXR)4­PNA antisense should be a useful tool for target validation of a specific gene and could be a therapeutic platform capable of targeting a variety of genes in P. aeruginosa.

Histological analyses by matrix-assisted laser desorption/ionization-imaging mass spectrometry reveal differential localization of sphingomyelin molecular species regulated by particular ceramide synthase in mouse brains.

Sphingomyelin (SM) is synthesized by SM synthase (SMS) from ceramide (Cer). SM regulates signaling pathways and maintains organ structure. SM comprises a sphingoid base and differing lengths of acyl-chains, but the importance of its various forms and regulatory synthases is not known. It has been reported that Cer synthase (CerS) has restricted substrate specificity, whereas SMS has no specificity for different lengths of acyl-chains. We hypothesized that the distribution of each SM molecular species was regulated by expression of the CerS family. Thus, we compared the distribution of SM species and CerS mRNA expression using molecular imaging. Spatial distribution of each SM molecular species was investigated using ultra-high-resolution imaging mass spectrometry (IMS). IMS revealed that distribution of SM molecular species varied according to the lengths of acyl-chains found in each brain section. Furthermore, a combination study using in situ hybridization and IMS revealed the spatial expression of CerS1 to be associated with the localization of SM (d18:1/18:0) in cell body-rich gray matter, and CerS2 to be associated with SM (d18:1/24:1) in myelin-rich white matter. Our study is the first comparison of spatial distribution between SM molecular species and CerS isoforms, and revealed their distinct association in the brain. These observations were demonstrated by suppression of CerS2 using siRNA in HepG2 cells; that is, siRNA for CerS2 specifically decreased C22 very long-chain fatty acid (VLCFA)- and C24 VLCFA-containing SMs. Thus, histological analyses of SM species by IMS could be a useful approach to consider their molecular function and regulative mechanism.

Development of an ultrasensitive immunoassay using affinity maturated antibodies for the measurement of rodent insulin.

The measurement of plasma insulin is important for clinical diagnosis of diabetes and for preclinical research of metabolic diseases, especially in rodent models used in drug discovery research for type 2 diabetes. Fasting immunoreactive insulin (F-IRI) concentrations are used to calculate the homeostasis model assessment ratio (HOMA-R), an index of insulin sensitivity. However, even the most sensitive commercially available enzyme-linked immunosorbent assay (ELISA) kits cannot measure the very low F-IRI concentrations in normal rats and mice. Therefore, we sought to develop a new rodent insulin ELISA with greater sensitivity for low F-IRI concentrations. Despite repeated efforts, high-affinity antibodies could not be generated by immunizing mice with mouse insulin (self-antigen). Therefore, we generated two weak monoclonal antibodies (13G4 and 26B2) that were affinity maturated and used to develop a highly sensitive ELISA. The measurement range of the sandwich ELISA with the affinity maturated antibodies (13G4m1 and 26B2m1) was 1.5 to 30,000 pg/ml, and its detection limit was at least 10 times lower than those of commercially available kits. In conclusion, we describe the development of a new ultrasensitive ELISA suitable for measuring very low plasma insulin concentrations in rodents. This ELISA might be very useful in drug discovery research in diabetes.

Identification of potential prognostic markers for knee osteoarthritis by serum proteomic analysis.

BACKGROUND: As osteoarthritis (OA) is a highly heterogeneous disease in terms of progression, establishment of prognostic biomarkers would be highly beneficial for treatment. The present study was performed to identify novel biomarkers capable of predicting the progression of knee OA. METHODS: A total of 69 plasma samples (OA patients undergoing radiographic progression, n = 25; nonprogression, n = 33; healthy donors, n = 11) were analyzed by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS), and ion peaks of interest were identified by liquid chromatography and matrix-assisted laser desorption/ionization (MALDI)-TOF MS. The identities of these proteins were further validated by immunoprecipitation combined with SELDI-TOF MS analysis. RESULTS: SELDI-TOF MS analysis indicated that the intensities of 3 ion peaks differed significantly between progressors and nonprogressors. Subsequent analyses indicated that these peaks corresponded to apolipoprotein C-I, C-III, and an N-terminal truncated form of transthyretin, respectively. The identities of these proteins were confirmed by the loss of ion peaks in SELDI-TOF MS spectra by immunoprecipitation using specific antibodies for the respective proteins. CONCLUSIONS: Three potential biomarkers were identified whose serum levels differed significantly between OA progressors and nonprogressors. These biomarkers are expected to be prognostic biomarkers for knee OA and to facilitate the development of novel disease-modifying treatments for OA.

Development of a neutralizing antibody specific for the active form of matrix metalloproteinase-13.

Matrix metalloproteinase-13 (MMP-13) is important in the pathology of osteoarthritis (OA). Although MMP-13 is considered a therapeutic target for OA, it is unclear how MMP-13 activity is regulated by the system that comprises various proteinases and their inhibitors. MMP-13 neutralizing antibodies could be a useful tool for investigating the involvement of MMP-13 in cartilage degeneration in OA-affected joints because antibodies possess high affinity and specificity compared with low-molecular weight chemical compounds. On the basis of three-dimensional structure and amino acid sequence information on MMP-13, we selected an appropriate antigen peptide and generated a neutralizing antibody by immunizing mice with the antigen. The most significant property of monoclonal antibody 14D10 was the specific binding to the active form of MMP-13, but not to the latent form, or other MMPs. With this property, active MMP-13 was measured selectively by an enzyme-linked immunosorbet assay. Furthermore, 14D10 suppressed the cleavage of type II collagen in human articular chondrocyte cultures, and 14D10 is thought to inhibit MMP-13 activity effectively. Thus, the highly selective MMP-13 neutralizing antibody (14D10) might be a useful tool for investigating the mechanism of type II collagen degradation in arthritic pathology.