Search

In this study, we conducted a collaborative study on the classification between silicone oil droplets and protein particles detected using the flow imaging (FI) method toward proposing a standardized classifier/model. We compared four approaches, including a classification filter composed of particle characteristic parameters, principal component analysis, decision tree, and convolutional neural network in the performance of the developed classifier/model. Finally, the points to be considered were summarized for measurement using the FI method, and for establishing the classifier/model using machine learning to differentiate silicone oil droplets and protein particles.

A broad-based interlaboratory study of glycosylation profiles of a reference and modified IgG antibody involving 103 reports from 76 laboratories., Graphical Abstract Highlights A broad-based interlaboratory study of the glycosylation of a reference antibody: NISTmAb. 103 reports were received from 76 diverse laboratories worldwide. Analysis involved two samples, the NISTmAb and an enzymatically modified sample, enabling within-lab separation of random and systematic errors using the “Youden two-sample” method. Consensus values were derived and similar performance across all experimental methods was noted., Glycosylation is a topic of intense current interest in the development of biopharmaceuticals because it is related to drug safety and efficacy. This work describes results of an interlaboratory study on the glycosylation of the Primary Sample (PS) of NISTmAb, a monoclonal antibody reference material. Seventy-six laboratories from industry, university, research, government, and hospital sectors in Europe, North America, Asia, and Australia submitted a total of 103 reports on glycan distributions. The principal objective of this study was to report and compare results for the full range of analytical methods presently used in the glycosylation analysis of mAbs. Therefore, participation was unrestricted, with laboratories choosing their own measurement techniques. Protein glycosylation was determined in various ways, including at the level of intact mAb, protein fragments, glycopeptides, or released glycans, using a wide variety of methods for derivatization, separation, identification, and quantification. Consequently, the diversity of results was enormous, with the number of glycan compositions identified by each laboratory ranging from 4 to 48. In total, one hundred sixteen glycan compositions were reported, of which 57 compositions could be assigned consensus abundance values. These consensus medians provide community-derived values for NISTmAb PS. Agreement with the consensus medians did not depend on the specific method or laboratory type. The study provides a view of the current state-of-the-art for biologic glycosylation measurement and suggests a clear need for harmonization of glycosylation analysis methods.

The evaluation of subvisible particles, including protein aggregates, in therapeutic protein products has been of great interest for both pharmaceutical manufacturers and regulatory agencies. To date, the flow imaging (FI) method has emerged as a powerful tool instead of light obscuration (LO) due to the fact that (1) protein aggregates contain highly transparent particles and thereby escape detection by LO and (2) FI provides detailed morphological characteristics of subvisible particles. However, the FI method has not yet been standardized nor listed in any compendium. In an attempt to assess the applicability of the standardization of the FI method, we conducted a collaborative study using FI and LO instruments in a Japanese biopharmaceutical consortium. Three types of subvisible particle preparations were shared across 12 laboratories and analyzed for their sizes and counts. The results were compared between the methods (FI and LO), inter-laboratories, and inter-instruments (Micro Flow Imaging and FlowCam). We clarified the marked difference between the detectability of FI and LO when counting highly transparent protein aggregates in the preparations. Although FlowCam provided a relatively higher number of particles compared with MFI, consistent results were obtained using the instrument from the same manufacturer in all 3 samples.

Insoluble particulate matter test for injections in pharmacopoeia is mandatory for parenteral drug products. In this test using light obscuration, four measurements of at least 5-mL are required. Since therapeutic protein injections of low dosage volumes are getting more popular, reduction of test volumes is desired. In this collaborative study, the impact of lower measurement volume on the accuracy and precision of particle count was evaluated using 2, 5, 10, and 25-μm polystyrene count standards for the validity of test with reduced sample volumes. Good accuracy (3000 particles/mL ± 10%) was obtained at all measurement volumes, and the inter-run variability (RSD) was the same levels between 5 and 1 mL. Although the inter-run variability increased at 0.2 mL, it was below 5%. These results indicated that light obscuration method can be used with 5 mL–0.2 mL, and that it is feasible for monitoring particles ≥2 μm.

As reported in the previous commentary (Ishii-Watabe et al., J Pharm Sci 2017), the Japanese biopharmaceutical research group is promoting collaborative multilaboratory studies to evaluate and standardize new methodologies for biopharmaceutical characterization and quality control. We have conducted the studies and held 2 annual meetings in 2018 and 2019. At the 2018 meeting, Dr. Rukman DeSilva of the U.S. Food and Drug Administration and Dr. Srivalli Telikepalli of the National Institute of Standards and Technology participated as guest speakers. At the 2019 meeting, we invited Prof. John Carpenter of the University of Colorado, Prof. Gerhard Winter and Prof. Wolfgang Friess of Ludwig Maximilian University of Munich, and Dr. Tim Menzen of Coriolis Pharma Research, as guest commentators. In both meetings, the main research topic was strategies for the characterization and control of protein aggregates/subvisible particles in drug products. Specifically, the use of the light obscuration method for insoluble particulate matter testing with reduced injection volumes, and a comparison of analytical performance between flow imaging and light obscuration were discussed. Other topics addressed included host cell protein analysis, bioassay, and quality control strategies. In this commentary, the recent achievements of the research group, meeting discussions, and future perspectives are summarized.

The N-glycan moiety of IgG-Fc has a significant impact on multifaceted properties of antibodies such as in their effector function, structure, and stability. Numerous studies have been devoted to understanding its biological effect since the exact composition of the Fc N-glycan modulates the magnitude of effector functions such as the antibody-dependent cell mediated cytotoxicity (ADCC), and the complement-dependent cytotoxicity (CDC). To date, systematic analyses of the properties and influence of glycan variants have been of great interest. Understanding the principles on how N-glycosylation modulates those properties is important for the molecular design, manufacturing, process optimization, and quality control of therapeutic antibodies. In this study, we have separated a model therapeutic antibody into three fractions according to the composition of the N-glycan by using a novel FcγRIIIa chromatography column. Notably, Fc galactosylation was a major factor influencing the affinity of IgG-Fc to the FcγRIIIa immobilized on the column. Each antibody fraction was employed for structural, biological, and physicochemical analysis, illustrating the mechanism by which galactose modulates the affinity to FcγRIIIa. In addition, we discuss the benefits of the FcγRIIIa chromatography column to assess the heterogeneity of the N-glycan.

Typical crystallizable fragment (Fc) glycans attached to the CH2 domain in therapeutic monoclonal antibodies (mAbs) are core-fucosylated and asialo-biantennary complex-type glycans, e.g., G2F (full galactosylation), G1aF (terminal galactosylation on the Man α1-6 arm), G1bF (terminal galactosylation on the Man α1-3 arm), and G0F (non-galactosylation). Terminal galactose (Gal) residues of Fc-glycans are known to influence effector functions such as antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity (CDC), but the impact of the G1F isomers (G1aF and G1bF) on the effector functions has not been reported. Here, we prepared four types of glycoengineered anti-CD20 mAbs bearing homogeneous G2F, G1aF, G1bF, or G0F (G2F mAb, G1aF mAb, G1bF mAb, or G0F mAb, respectively), and evaluated their biological activities. Interestingly, G1aF mAb showed higher C1q- and FcγR-binding activities, CDC activity, and FcγR-activation property than G1bF mAb. The activities of G1aF mAb and G1bF mAb were at the same level as G2F mAb and G0F mAb, respectively. Hydrogen–deuterium exchange/mass spectrometry analysis of dynamic structures of mAbs revealed the greater involvement of the terminal Gal residue on the Man α1-6 arm in the structural stability of the CH2 domain. Considering that mAbs interact with FcγR and C1q via their hinge proximal region in the CH2 domain, the structural stabilization of the CH2 domain by the terminal Gal residue on the Man α1-6 arm of Fc-glycans may be important for the effector functions of mAbs. To our knowledge, this is the first report showing the impact of G1F isomers on the effector functions and dynamic structure of mAbs. Abbreviations: ABC, ammonium bicarbonate solution; ACN, acetonitrile; ADCC, antibody-dependent cell-mediated cytotoxicity; C1q, complement component 1q; CDC, complement-dependent cytotoxicity; CQA, critical quality attribute; Endo, endo-β-N-acetylglucosaminidase; FA, formic acid; Fc, crystallizable fragment; FcγR, Fcγ receptors; Fuc, fucose; Gal, galactose; GlcNAc, N-acetylglucosamine; GST, glutathione S-transferase; HER2, human epidermal growth factor receptor 2; HDX, hydrogen–deuterium exchange; HILIC, hydrophilic interaction liquid chromatography; HLB-SPE, hydrophilic-lipophilic balance–solid-phase extraction; HPLC, high-performance liquid chromatography; mAb, monoclonal antibody; Man, mannose; MS, mass spectrometry; PBS, phosphate-buffered saline; SGP, hen egg yolk sialylglycopeptides.

In response to the successful use of monoclonal antibodies (mAbs) in the treatment of various diseases, systems for expressing recombinant mAbs using transgenic animals or plants have been widely developed. The silkworm (Bombyx mori) is a highly domesticated insect that has recently been used for the production of recombinant proteins. Because of their cost-effective breeding and relatively easy production scale-up, transgenic silkworms show great promise as a novel production system for mAbs. In this study, we established a transgenic silkworm stably expressing a human-mouse chimeric anti-CD20 mAb having the same amino acid sequence as rituximab, and compared its characteristics with rituximab produced by Chinese hamster ovary (CHO) cells (MabThera®). The anti-CD20 mAb produced in the transgenic silkworm showed a similar antigen-binding property, but stronger antibody-dependent cell-mediated cytotoxicity (ADCC) and weaker complement-dependent cytotoxicity (CDC) compared to MabThera. Post-translational modification analysis was performed by peptide mapping using liquid chromatography/mass spectrometry. There was a significant difference in the N-glycosylation profile between the CHO− and the silkworm-derived mAbs, but not in other post-translational modifications including oxidation and deamidation. The mass spectra of the N-glycosylated peptide revealed that the observed biological properties were attributable to the characteristic N-glycan structures of the anti-CD20 mAbs produced in the transgenic silkworms, i.e., the lack of the core-fucose and galactose at the non-reducing terminal. These results suggest that the transgenic silkworm may be a promising expression system for the tumor-targeting mAbs with higher ADCC activity.

The research and development of next-generation innovative medicines is a prominent interest of both the government and industries in Japan. On June 29, 2017, a kickoff meeting of a new research group focused on the quality issues of biopharmaceuticals was held in Tokyo with Prof. John Carpenter as an invited guest. The group's research focuses mainly on the evaluation and control of protein aggregates/subvisible particles in drug products, but the research topics also include glycan analysis, host-cell protein evaluation, bioassay validation, and analytical quality by design. The purpose of the group's activities is to resolve the critical and fundamental quality issues important to pharmaceutical companies through the collaboration of industries, academia, and regulatory agencies. In this commentary, our current plan to address these issues and the discussion at the kickoff meeting are described.

RATIONALE Glycan heterogeneity on recombinant human erythropoietin (rEPO) product is considered to be one of the critical quality attributes, and similarity tests of glycan heterogeneities are required in the manufacturing process changes and developments of biosimilars. A method for differentiating highly complex and diverse glycosylations is needed to evaluate comparability and biosimilarity among rEPO batches and products manufactured by different processes. METHODS The glycan heterogeneities of nine rEPO products (four innovator products and five biosimilar products) were distinguished by multivariate analysis (MVA) using the peak area ratios of each glycan to the total peak area of glycans in mass spectra obtained by liquid chromatography/mass spectrometry (LC/MS) of N-glycans from rEPOs. RESULTS Principal component analysis (PCA) using glycan profiles obtained by LC/MS proved to be a useful method for differentiating glycan heterogeneities among nine rEPOs. Using PC values as indices, we were able to visualize and digitalize the glycan heterogeneities of each rEPO. The characteristic glycans of each rEPO were also successfully identified by orthogonal partial least-squares discrimination analysis (OPLS-DA), an MVA method, using the glycan profile data. CONCLUSIONS PCA values were useful for evaluating the relative differences among the glycan heterogeneities of rEPOs. The characteristic glycans that contributed to the differentiation were also successfully identified by OPLS-DA. PCA and OPLS-DA based on mass spectrometric data are applicable for distinguishing glycan heterogeneities, which are virtually indistinguishable on rEPO products. Copyright © 2014 John Wiley & Sons, Ltd.

The recent patent expirations of erythropoietin (EPO) have promoted the development of biosimilars. Two and one biosimilar EPO products were approved in 2007 in Europe and in 2010 in Japan, respectively. Glycosylation heterogeneity of EPO is very complex, and its pattern has a large impact on its in vivo activity. In this study, glycoform profilings of biosimilar and innovator EPO products were performed using LC/ESI-MS. Glycoforms of EPO were detected within the range of m/z 1700-3600 at the 10(+)-16(+) charge states. The charge-deconvoluted spectra showed complex glycoform mass profiles at 28,000-32,000 Da, and most of the observed peaks were assigned to the peptide (18,236 Da)+glycans with the compositions of NeuAc10-14Hexn+3HexNAcnFuc3 (n=16-26) with or without some O-acetylations (+42 Da) and attachment of NeuGc for NeuAc or oxidation (+16 Da). Analysis of de-N-glycosylated EPO showed the distributions of O-glycans of NeuAc1-2Hex1HexNAc1 and site occupancy. Each EPO product showed a characteristic glycoform profile with respect to sialylation, glycan size, O-acetylation of sialic acids and O-glycosylation. Analysis of darbepoetin suggested that glycans of darbepoetin were highly sialylated and O-acetylated. LC/ESI-MS was shown to be useful to evaluate the similarity of the glycoform profiles of EPO.

Rheumatoid arthritis (RA)-associated IgG antibodies such as anti-citrullinated protein antibodies (ACPAs) have diverse glycosylation variants; however, key sugar chains modulating the arthritogenic activity of IgG remain to be clarified. Here, we show that reduced sialylation is a common feature of RA-associated IgG in humans and in mouse models of arthritis. Genetically blocking sialylation in activated B cells results in exacerbation of joint inflammation in a collagen-induced arthritis (CIA) model. On the other hand, artificial sialylation of anti-type II collagen antibodies, including ACPAs, not only attenuates arthritogenic activity, but also suppresses the development of CIA in the antibody-infused mice, whereas sialylation of other IgG does not prevent CIA. Thus, our data demonstrate that sialylation levels control the arthritogenicity of RA-associated IgG, presenting a potential target for antigen-specific immunotherapy., Post-translational modifications, such as glycosylation and sialylation, are thought to confer disease modifying effects on autoimmune-associated antibodies, including anti-citrullinated protein antibodies in rheumatoid arthritis. Here the authors show that sialylation converts arthritogenic IgG into inhibitors of collagen-induced arthritis in mice.

The development of therapeutic antibodies has grown over the last several years. Most of the recombinant monoclonal antibodies (mAbs) produced by mammalian cells are glycoproteins. Glycosylation of the mAbs can be associated with effector functions, such as antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity, as well as immunogenicity and clearance. Thus, mAb glycan heterogeneity is a significant characteristic associated with the safety and efficacy of the products. Therefore, glycan heterogeneity should be evaluated during research and development (R&D) and during development of mAbs manufacturing processes to identify the process parameters that affect glycan heterogeneity and to enhance understanding of the manufacturing process. There is an increasing need for a rapid, easy, and automated evaluation method for glycan heterogeneity. Liquid chromatography/mass spectrometry (LC/MS) is a method that can be used to analyze glycoforms. LC/MS is marked by the ability to measure the oligosaccharide composition of each glycoform, whereas other general methods, such as capillary electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and ion-exchange chromatography, cannot. However, a laborious off-line purification of mAbs is required to evaluate glycan heterogeneities. In this study, we demonstrate the use of a rapid, easy, and automated evaluation system for mAb glycoforms by LC/MS. This LC/MS system uses a column-switching system equipped with 2 columns, a protein A affinity column and a reversed-phase column (desalting column). We devised 2 column-switching systems: one that targeted intact mAbs (system 1) and one that targeted the light and heavy chains of the mAbs (system 2). Our results show that the proposed systems are applicable as a tool to evaluate the glycoforms in several situations, including the research, development, and production processes of mAbs. Additionally, we hope that our systems are useful as process analytical technology (PAT) for molecular heterogeneities containing glycoforms of mAbs in implementation of quality by design (QbD).

Insulin analog products for subcutaneous injection are prepared as solutions in which insulin analog molecules exist in several oligomeric states. Oligomeric stability can affect their onset and duration of action and has been exploited in designing them. To investigate the oligomeric stability of insulin analog products having different pharmacokinetics, we performed hydrogen/deuterium exchange mass spectrometry (HDX/MS), which is a rapid method to analyze dynamic aspects of protein structures. Two rapid-acting analogs (lispro and glulisine) incorporated deuteriums more and faster than recombinant human insulin, whereas a long-acting analog (glargine) and two intermediate-acting preparations (protamine-containing formulations) incorporated them less and more slowly. Kinetic analysis revealed that the number of slowly exchanged hydrogens (D(s)) (k0.01 min(-1)) accounted for the difference in HDX reactivity among analogs. Furthermore, we found correlations between HDX kinetics and pharmacokinetics reported previously. Their maximum serum concentration (C(max)) was linearly correlated with D(s) (r=0.88) and the number of maximum exchangeable hydrogens (D(∞)) (r=0.89). The maximum drug concentration time (t(max)) was also correlated with reciprocals of D(s) and D(∞) (r=0.86 and r=0.96, respectively). Here we demonstrate the ability of HDX/MS to evaluate oligomeric stability of insulin analog products.

Saliva contains a large number of proteins that participate in the protection of oral tissue. Exosomes are small vesicles (30-100 nm in diameter) with an endosome-derived limiting membrane that are secreted by a diverse range of cell types. We have recently demonstrated that exosomes are present in human whole saliva. In this study, we found that whole saliva contained at least two types of exosomes (exosome I and exosome II) that are different in size and protein composition. Proteomic analysis revealed that both types of exosomes contained Alix, Tsg101 and Hsp70, all exosomal markers, immunoglobulin A and polymeric immunoglobulin receptor, whereas they had different protein compositions. Most of dipeptidyl peptidase IV known as CD26 in whole saliva, was present on the exosome II and metabolically active in cleaving chemokines (CXCL11 and CXCL12). Human whole saliva exosomes might participate in the catabolism of bioactive peptides and play a regulatory role in local immune defense in the oral cavity.

Certain glycan motifs in glycoproteins are involved in several biological events and diseases. To understand the roles of these motifs, a method is needed to identify the glycoproteins that carry them. We previously demonstrated that liquid chromatography-multiple-stage mass spectrometry (LC-MSn) allowed for differentiation of oligosaccharides attached to Lewis-motifs, such as Lewisx(Lex, Galbeta1-4(Fucalpha1-3)GlcNAc) from other glycans. We successfully discriminated Lex-conjugated oligosaccharides from other N-linked oligosaccharides derived from mouse kidney proteins by using Lewis-motif-distinctive ions, a deoxyhexose (dHex)+hexose (Hex)+N-acetylhexsosamine (HexNAc) fragment (m/z 512), and a Hex+HexNAc fragment (m/z 366). In the present study, we demonstrated that this method could be used to identify the Lex-conjugated glycoproteins. All proteins in the mouse kidney were digested into peptides, and the fucosylated glycopeptides were enriched by lectin-affinity chromatography. The resulting fucosylated glycopeptides were subjected to two different runs of LC-MSn using a Fourier- transform ion cyclotron resonance mass spectrometer (FTICR-MS) and an ion trap-type mass spectrometer. After the first run, we picked out product ion spectra of the expected Lex-conjugated glycopeptides based on the presence of Lewis-motif-distinctive ions and assigned a peptide+HexNAc or peptide+(dHex)HexNAc fragment in each spectrum. Then the fucosylated glycopeptides were subjected to a second run in which the peptide-related fragments were set as precursor ions. We successfully identified gamma-glutamyl transpeptidase 1 (gamma-GTP1), low-density lipoprotein receptor-related protein 2 (LRP2), and a cubilin precursor as Lex-conjugated glycoproteins by sequencing of 2-5 glycopeptides. In addition, it was deduced that cadherin 16, dipeptidase I, H-2 class I histocompatibility antigen, K-K alpha precursor (H2-Kk), and alanyl (membrane) aminopeptidase could be Lex-conjugated glycoproteins from the good agreement between the experimental and theoretical masses and fragment patterns. The results indicated that our method could be applicable for the identification and screening of glycoproteins carrying target glycan-motifs, such as Lewis epitopes.

Changes in the glycosylation of some serum proteins are associated with certain diseases. In this study, we performed simultaneous site-specific glycosylation analysis of abundant serum glycoproteins by LC/Qq-TOF MS of human serum tryptic digest, the albumin of which was depleted. The glycopeptide peaks on the chromatogram were basically assigned by database searching with modified peak-list text files of MS/MS spectra and then based on mass differences of glycan units from characterized glycopeptides. Glycopeptide of IgG, haptoglobin and ceruloplasmin were confirmed by means of a comparison of their retention times and m/z values with those obtained by LC/MS of commercially available glycoproteins. Mass spectrometric carbohydrate heterogeneity in the assigned glycopeptides was analyzed by an additional LC/MS. We successfully demonstrated site-specific glycosylation of 23 sites in abundant serum glycoproteins.

N-Glycolylneuraminic acid (NeuGc), an acidic nine-carbon sugar, is produced in several animals, such as cattle and mice. Since human cells cannot synthesize NeuGc, it is considered to be immunogenic in humans. Recently, NeuGc contamination was reported in human embryonic stem cells cultured with xenogeneic serum and cells, suggesting that possibly NeuGc may harm the efficacy and safety of cell therapy products. Sialic acids have been determined by derivatization with 1,2-diamino-4,5-methylenedioxybenzene (DMB) followed by liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS); however, the limited availability of cell therapy products requires more sensitive and specific methods for the quality test. Here we studied the use of nano-flow liquid chromatography/Fourier transformation ion cyclotron resonance mass spectrometry (nanoLC/FTMS) and nanoLC/MS/MS for NeuGc-specific determination at a low femtomole level. Using our method, we found NeuGc contamination of the human cell line (HL-60RG cells) cultured with human serum. Our method needs only 2.5x10(3) cells for one injection and would be applicable to the determination of NeuGc in cell therapy products.

We developed an efficient and convenient strategy for protein identification and glycosylation analysis of a small amount of unknown glycoprotein in a biological sample. The procedure involves isolation of proteins by electrophoresis and mass spectrometric peptide/glycopeptide mapping by LC/ion trap mass spectrometer. For the complete glycosylation analysis, proteins were extracted in intact form from the gel, and proteinase-digested glycoproteins were then subjected to LC/multistage tandem MS (MSn) incorporating a full mass scan, in-source collision-induced dissociation (CID), and data-dependent MSn. The glycopeptides were localized in the peptide/glycopeptide map by using oxonium ions such as HexNAc+ and NeuAc+, generated by in-source CID, and neutral loss by CID-MS/MS. We conducted the search analysis for the glycopeptide identification using search parameters containing a possible glycosylation at the Asn residue with N-acetylglucosamine (203 Da). We were able to identify the glycopeptides resulting from predictable digestion with proteinase. The glycopeptides caused by irregular cleavages were not identified by the database search analysis, but their elution positions were localized using oxonium ions produced by in-source CID, and neutral loss by the data-dependent MSn. Then, all glycopeptides could be identified based on the product ion spectra which were sorted from data-dependent CID-MSn spectra acquired around localized positions. Using this strategy, we successfully elucidated site-specific glycosylation of Thy-1, glycosylphosphatidylinositol (GPI)-anchored proteins glycosylated at Asn23, 74, and 98, and at Cys111. High-mannose-type, complex-type, and hybrid-type oligosaccharides were all found to be attached to Asn23, 74 and 98, and four GPI structures could be characterized. Our method is simple, rapid and useful for the characterization of unknown glycoproteins in a complex mixture of proteins.

The Lewis x structure [Le x , Galβ1-4(Fucα1-3)GlcNAc] motif is one of the tumor antigens and plays an important role in oncogenesis, development, cellular differentiation and adhesion. The detection of Le x -carbohydrates and their structural analysis are necessary to clarify the role of Le x in several biological events. Mass spectrometry has been preferably used for the structural analysis of carbohydrates. Especially, collision-induced dissociation (CID) tandem mass spectrometry (MS/MS), which causes a glycosidic bond cleavage, is used for carbohydrate sequencing. However, Le x cannot be identified by MS/MS due to the existence of the positional isomers, such as Lewis a [Galβ1-3(a1-4Fuc)GlcNAc]. In the present study, we demonstrate the specific detection of Le x -carbohydrates in a biological sample by using multiple-stage MS/MS (MS"). Using pyridylaminated oligosaccharides bearing Le x , we found that the Le x -motif yields a cross-ring fragment by the cleavage of a bond between C-3 and C-4 of GlcNAc in Gal(Fuc)GlcNAc. The Le x -specific cross-ring fragment ion at m/z 259 was effectively detected by sequential scans, consisting of a full MS 1 scan, data-dependent CID MS 2 scan, MS 3 of [Gal(Fuc)GlcNAc+Na] + at m/z 534, and MS 4 of [GalGlcNAc+Na] + at m/z 388. The sequential scan was applied to N-linked oligosaccharide profiling using a LC/ESI-MS n system equipped with a graphitized carbon column. We successfully detected the Le x -motif and elucidated the structures of several Le x and Lewis y [(Fucα1-2)Gal/β1-4(Fucα1-3)GlcNAc] oligosaccharides in the murine kidney used as a model tissue. Our method is expected to be a powerful tool for the specific detection of the Le x -motif, and structural elucidation of Le x -carbohydrates in biological samples. Copyright ( 2005 John Wiley & Sons, Ltd.

LC/MS/MSは, アミノ酸配列情報に加え, 糖鎖構造に関する情報についても得ることができ, 糖ペプチドの解析にも有用である. QqTOF-MSを用いたLC/MS/MSは, 糖ペプチドのピークを特定し, ペプチドを同定し, 結合糖鎖構造に関する情報も得ることができる. 本稿では, 糖ペプチドの解析例として, APF及び電気泳動で分離されたGPIアンカー型タンパク質の解析例を示す.

Acoustic microscope can detect microstructural features with high resolving power compared to other lower frequency acoustic techniques. Many non-destructive evaluations using acoustic microscope, several of them used in industrial fields, were investigated. However, acoustic measurement of curved surface structure was restricted to basic studies as conventional acoustic microscope system was designed for flat specimen. We have developed a modified acoustic microscope for curved surface specimen testing and have measured acoustic image of round bar specimen. In this study, using this system, velocities of round bar specimen during fatigue test are monitored using the V(z) curve method. Measurement errors depending on curved surface measurement were investigated and it was found that the acoustic velocity tends to increase as fatigue damage increases.

The objective of this study was to evaluate the developmental toxicity of phthalic acid (PA), which is one of the metabolites of phthalic acid esters (PAEs). Pregnant rats were given PA at a dose of 0 (control), 1.25, 2.5, or 5.0% in the diet on day 7 through day 16 of pregnancy. Average daily intakes of PA were 1021 mg/kg for the 1.25% group, 1763 mg/kg for the 2.5% group, and 2981 mg/kg for the 5.0% group. Maternal toxicity occurred in the 2.5 and 5.0% groups as can be seen by significant decreases in the maternal body weight gain and food consumption during the administration period. No significant changes in maternal parameters were found in the 1.25% group. Neither deaths nor clinical signs of toxicity were noted in any groups. No significant changes induced by PA were detected in the incidence of postimplantation loss and number and sex ratio of live fetuses. Significant decreases in the weight of male fetuses and number of ossification center of the caudal vertebrae were found in the 5.0% group. Morphological examinations of fetuses revealed no evidence of teratogenesis. Thus it appears unlikely that PA may be responsible for the production of the developmental toxicity of PAEs.

The objective of this study was to characterize the adverse effects of triphenyltin chloride (TPTCl) during early pregnancy. Following successful mating, female rats were given TPTCl by gastric intubation at 3.1, 4.7, or 6.3 mg/kg on days 0 to 3 of pregnancy or at 6.3, 12.5, or 25.0 mg/kg on days 4 to 6 of pregnancy and were sacrificed on day 20 of pregnancy. In successfully mated females, TPTCl totally prevented implantation in a dose-dependent manner. The pregnancy rate was significantly decreased after administration of TPTCl on days 0 to 3 at 4.7 and 6.3 mg/kg and on days 4 to 6 at 12.5 and 25.0 mg/kg. In females having implantations, the numbers of implantations and live fetuses and the incidences of pre- and postimplantation loss and fetal malformations in the TPTCl-treated groups were comparable to the controls. It could be concluded that TPTCl during early pregnancy causes failure in implantation and TPTCl has greater antiimplantation effects when administered during earlier than later stages of blastogenesis.

Glycan heterogeneity on recombinant human erythropoietin (rEPO) product is considered to be one of the critical quality attributes, and similarity tests of glycan heterogeneities are required in the manufacturing process changes and developments of biosimilars. A method for differentiating highly complex and diverse glycosylations is needed to evaluate comparability and biosimilarity among rEPO batches and products manufactured by different processes.The glycan heterogeneities of nine rEPO products (four innovator products and five biosimilar products) were distinguished by multivariate analysis (MVA) using the peak area ratios of each glycan to the total peak area of glycans in mass spectra obtained by liquid chromatography/mass spectrometry (LC/MS) of N-glycans from rEPOs.Principal component analysis (PCA) using glycan profiles obtained by LC/MS proved to be a useful method for differentiating glycan heterogeneities among nine rEPOs. Using PC values as indices, we were able to visualize and digitalize the glycan heterogeneities of each rEPO. The characteristic glycans of each rEPO were also successfully identified by orthogonal partial least-squares discrimination analysis (OPLS-DA), an MVA method, using the glycan profile data.PCA values were useful for evaluating the relative differences among the glycan heterogeneities of rEPOs. The characteristic glycans that contributed to the differentiation were also successfully identified by OPLS-DA. PCA and OPLS-DA based on mass spectrometric data are applicable for distinguishing glycan heterogeneities, which are virtually indistinguishable on rEPO products.

The effect of tributyltin chloride (TBTC1) administered during early pregnancy on pregnancy maintenance was evaluated in rats. Inseminated females were orally administered TBTC1 at a dose of 0, 8.1, 12.2 or 16.3 mg/kg on day 0 through day 7 of pregnancy. Females were sacrificed on day 20 of pregnancy and pregnancy outcome was determined. Pregnancy failure, which was evidenced by absence of implantation sites, was found in 0 of the 10, in 2 of the 11, in 10 of the 14 and in 10 of the 13 females at 0, 8.1, 12.2 and 16.3 mg/kg, respectively. The rate of pregnancy failure was significantly higher in the 12.2 and 16.3 mg/kg groups than that in the control group. In females with successful pregnancy, the numbers of corpora lutea, implantations and post-implantation loss per litter were comparable across all groups. No increase in the incidence of malformed fetuses was found in any TBTC1-treated groups. Thus it appears that TBTCl causes pregnancy failure after administration during very early pregnancy.

Mono-n-benzyl phthalate (MBeP), one of the major metabolites of the plasticizer n-butyl benzyl phthalate, was evaluated for developmental toxicity in Wistar rats. Rats were given MBeP by gastric intubation at 0, 250, 313, 375, 438 or 500 mg/kg from day 7 through day 15 of pregnancy. Significant decreases in the maternal body weight gains and food consumption during administration period were observed at 313 mg/kg and above and at 250 mg/kg and above, respectively. Significant increase in the incidence of postimplantation loss per litter was found at 438 and 500 mg/kg. The incidences of fetuses with external malformations at 438 mg/kg, of fetuses with skeletal malformations at 313 mg/kg and above and of fetuses with internal malformations at 375 mg/kg and above were higher than those in the control group. Defects in the cervical and thoracic vertebrae, ribs and kidney were frequently observed.

Recently, we cloned cDNAs for the three mouse PGE receptor subtypes, EP1, EP2 and EP3, and the prostacyclin receptor, and established cells that stably express each receptor. We examined the selectivity of TEI-3356, an isocarbacyclin analogue, compared with other EP agonists, sulprostone and misoprostol, using Chinese hamster ovary cells expressing each cloned receptor. TEI-3356 selectively displaced the [3H]PGE2 binding to EP3-expressing cell membranes, but showed very low affinity for both EP1 and EP2. Although TEI-3356 is an isocarbacyclin analogue, it showed low affinity for the prostacyclin receptor. On the other hand, sulprostone strongly displaced the [3H]PGE2 binding to EP1 and EP3, but not to EP2. Misoprostol weakly bound to the three subtypes without selectivity. TEI-3356 decreased the forskolin-induced cAMP formation in a concentration-dependent manner in the EP3-expressing cells, the half-maximal concentration for the inhibition being similar to that of sulprostone but lower than that of PGE2. These results demonstrate that TEI-3356 is a potent and highly selective agonist for the EP3 receptor.

The various monosaccharide composition analysis methods were evaluated as monosaccharide test for glycoprotein-based pharmaceuticals. Neutral and amino sugars were released by hydrolysis with 4-7N trifluoroacetic acid. The monosaccharides were N-acetylated if necessary, and analyzed by high-performance liquid chromatography (HPLC) with fluorometric or UV detection after derivatization with 2-aminopyridine, ethyl 4-aminobenzoate, 2-aminobenzoic acid or 1-phenyl-3-methyl-5-pyrazolone, or high pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Sialic acids were released by mild acid hydrolysis or sialidase digestion, and analyzed by HPLC with fluorometric detection after derivatization with 1,2-diamino-4,5-methylenedioxybenzene, or HPAEC-PAD. These methods were verified for resolution, linearity, repeatability, and accuracy using a monosaccharide standard solution, a mixture of epoetin alfa and beta, and alteplase as models. It was confirmed that those methods were useful for ensuring the consistency of glycosylation. It is considered essential that the analytical conditions including desalting, selection of internal standards, release of monosaccharides, and gradient time course should be determined carefully to eliminate interference of sample matrix. Various HPLC-based monosaccharide analysis methods were evaluated as a carbohydrate test for glycoprotein pharmaceuticals by an inter-laboratory study.

N-Glycolylneuraminic acid (NeuGc), an acidic nine-carbon sugar, is produced in several animals, such as cattle and mice. Since human cells cannot synthesize NeuGc, it is considered to be immunogenic in humans. Recently, NeuGc contamination was reported in human embryonic stem cells cultured with xenogeneic serum and cells, suggesting that possibly NeuGc may harm the efficacy and safety of cell therapy products. Sialic acids have been determined by derivatization with 1,2-diamino-4,5-methylenedioxybenzene (DMB) followed by liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS); however, the limited availability of cell therapy products requires more sensitive and specific methods for the quality test. Here we studied the use of nano-flow liquid chromatography/Fourier transformation ion cyclotron resonance mass spectrometry (nanoLC/FTMS) and nanoLC/MS/MS for NeuGc-specific determination at a low femtomole level. Using our method, we found NeuGc contamination of the human cell line (HL-60RG cells) cultured with human serum. Our method needs only 2.5x10(3) cells for one injection and would be applicable to the determination of NeuGc in cell therapy products.

We have previously described the site-specific glycosylation analysis of rat brain Thy-1 by LC/multistage tandem mass spectrometry (MS(n)) using proteinase-digested Thy-1. In the present study, detailed structures of oligosaccharides released from Thy-1 were elucidated by mass spectrometric oligosaccharide profiling using LC/MS with a graphitized carbon column (GCC-LC/MS). First, using model oligosaccharides, we improved the oligosaccharide profiling by ion trap mass spectrometry (IT-MS) coupled with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). Sequential scanning of a full MS(1) scan with FT-ICR-MS followed by data-dependent MS(n) with IT-MS in positive ion mode, and a subsequent full MS(1) scan with FT-ICR-MS followed by data-dependent MS(n) with IT-MS in negative ion mode enabled the monosaccharide composition analysis as well as profiling and sequencing of both neutral and acidic oligosaccharides in a single analysis. The improved oligosaccharide profiling was applied to elucidation of N-linked oligosaccharides from Thy-1 isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was demonstrated that Thy-1 possesses a significant variety of N-linked oligosaccharides, including Lewis a/x, Lewis b/y, and disialylated structure as a partial structure. Our method could be applicable to analysis of a small abundance of glycoproteins, and could become a powerful tool for glycoproteomics.

Ceruloplasmin has ferroxidase activity and plays an essential role in iron metabolism. In this study, a site-specific glycosylation analysis of human ceruloplasmin (CP) was carried out using reversed-phase high-performance liquid chromatography with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). A tryptic digest of carboxymethylated CP was subjected to LC-ESI-MS/MS. Product ion spectra acquired data-dependently were used for both distinction of the glycopeptides from the peptides using the carbohydrate B-ions, such as m/z 204 (HexNAc) and m/z 366 (HexHexNAc), and identification of the peptide moiety of the glycopeptide based on the presence of the b- and y-series ions derived from the peptide. Oligosaccharide composition was deduced from the molecular weight calculated from the observed mass of the glycopeptide and theoretical mass of the peptide. Of the seven potential N-glycosylation sites, four (Asn119, Asn339, Asn378, and Asn743) were occupied by a sialylated biantennary or triantennary oligosaccharide with fucose residues (0, 1, or 2). A small amount of sialylated tetraantennary oligosaccharide was detected. Exoglycosidase digestion suggested that fucose residues were linked to reducing end GlcNAc in biantennary oligosaccharides and to reducing end and/or alpha1-3 to outer arms GlcNAc in triantennary oligosaccharides and that roughly one of the antennas in triantennary oligosaccharides was alpha2-3 sialylated and occasionally alpha1-3 fucosylated at GlcNAc.

We developed an efficient and convenient strategy for protein identification and glycosylation analysis of a small amount of unknown glycoprotein in a biological sample. The procedure involves isolation of proteins by electrophoresis and mass spectrometric peptide/glycopeptide mapping by LC/ion trap mass spectrometer. For the complete glycosylation analysis, proteins were extracted in intact form from the gel, and proteinase-digested glycoproteins were then subjected to LC/multistage tandem MS (MSn) incorporating a full mass scan, in-source collision-induced dissociation (CID), and data-dependent MSn. The glycopeptides were localized in the peptide/glycopeptide map by using oxonium ions such as HexNAc+ and NeuAc+, generated by in-source CID, and neutral loss by CID-MS/MS. We conducted the search analysis for the glycopeptide identification using search parameters containing a possible glycosylation at the Asn residue with N-acetylglucosamine (203 Da). We were able to identify the glycopeptides resulting from predictable digestion with proteinase. The glycopeptides caused by irregular cleavages were not identified by the database search analysis, but their elution positions were localized using oxonium ions produced by in-source CID, and neutral loss by the data-dependent MSn. Then, all glycopeptides could be identified based on the product ion spectra which were sorted from data-dependent CID-MSn spectra acquired around localized positions. Using this strategy, we successfully elucidated site-specific glycosylation of Thy-1, glycosylphosphatidylinositol (GPI)-anchored proteins glycosylated at Asn23, 74, and 98, and at Cys111. High-mannose-type, complex-type, and hybrid-type oligosaccharides were all found to be attached to Asn23, 74 and 98, and four GPI structures could be characterized. Our method is simple, rapid and useful for the characterization of unknown glycoproteins in a complex mixture of proteins.

Human apolipoprotein B100 (apoB100) has 19 potential N-glycosylation sites, and 16 asparagine residues were reported to be occupied by high-mannose type, hybrid type, and monoantennary and biantennary complex type oligosaccharides. In the present study, a site-specific glycosylation analysis of apoB100 was carried out using reversed-phase high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC/ESI MS/MS). ApoB100 was reduced, carboxymethylated, and then digested by trypsin or chymotrypsin. The complex mixture of peptides and glycopeptides was subjected to LC/ESI MS/MS, where product ion spectra of the molecular ions were acquired data-dependently. The glycopeptide ions were extracted and confirmed by the presence of carbohydrate-specific fragment ions, such as m/z 204 (HexNAc) and 366 (HexHexNAc), in the product ion spectra. The peptide moiety of glycopeptide was determined by the presence of the b- and y-series ions derived from its amino acid sequence in the product ion spectrum, and the oligosaccharide moiety was deduced from the calculated molecular mass of the oligosaccharide. The heterogeneity of carbohydrate structures at 17 glycosylation sites was determined using this methodology. Our data showed that Asn2212, not previously identified as a site of glycosylation, could be glycosylated. It was also revealed that Asn158, 1341, 1350, 3309, and 3331 were occupied by high-mannose type oligosaccharides, and Asn 956, 1496, 2212, 2752, 2955, 3074, 3197, 3438, 3868, 4210, and 4404 were predominantly occupied by mono- or disialylated oligosaccharides. Asn3384, the nearest N-glycosylation site to the LDL-receptor binding site (amino acids 3359-3369), was occupied by a variety of oligosaccharides, including high-mannose, hybrid, and complex types. These results are useful for understanding the structure of LDL particles and oligosaccharide function in LDL-receptor ligand binding.

In our previous study, dibutyltin dichloride (DBTCl) caused preimplantation embryonic loss and postimplantation embryonic loss in rats following administration at 7.6 mg/kg and above on Days 0–3 and at 3.8 mg/kg and above on Days 4–7 of pregnancy, respectively. This study was designed to assess the effects of DBTCl on uterine function as a cause of early embryonic loss using pseudopregnant rats. DBTCl was given orally to pseudopregnant rats at 3.8, 7.6 or 15.2 mg/kg on pseudopregnant day (PPD) 0–3 or on PPD 4–7. The decidual cell response was induced by bilateral uterine scratch on PPD 4. The uterine weight on PPD 9 served as an index of uterine decidualization. Uterine weight and serum progesterone levels on PPD 9 were significantly decreased after administration of DBTCl at 7.6 mg/kg and above on PPD 0–3 and PPD 4–7. DBTCl had no effect on the serum estradiol levels and number of corpora lutea. Administration of progesterone reversed the suppression of uterine decidualization in rats given DBTCl on PPD 0–3. It can be concluded that DBTCl suppresses the uterine decidual cell response and decreases progesterone levels, and these effects are responsible for early embryonic loss due to DBTCl exposure.

We previously showed that dibutyltin dichloride (DBTCl) at 7.6 mg/kg and higher on days 0–3 of pregnancy caused implantation failure and a decline in serum progesterone levels in rats and hypothesized that the decline is responsible for the implantation failure. This study was conducted to determine the protective effects of progesterone on the DBTCl-induced implantation failure in rats. Rats were given oral DBTCl at 0, 7.6, or 15.2 mg/kg on days 0–3 of pregnancy and/or subcutaneous progesterone at 2 mg/rat on days 0–8 of pregnancy. The reproductive outcome was determined on day 9 of pregnancy. No effects of administration of progesterone alone on the pregnancy rate and number of implantations were found. The pregnancy rate and number of implantations were significantly decreased after administration of DBTCl alone. The pregnancy rate and number of implantations were higher in the groups given DBTCl and progesterone than the groups given DBTCl alone. The present data indicate that progesterone protects, at least in part, against the DBTCl-induced implantation failure and support our hypothesis that the decline in progesterone levels is a primary mechanism for the implantation failure due to DBTCl.

This study was conducted to determine the low-dose effects of bisphenol A (BPA) in a rat two-generation reproduction study. Groups of 25 male and 25 female Crj: CD (SD) IGS rats were given BPA at 0.2, 2, 20, or 200 microg/kg/day by gastric intubation throughout the study beginning at the onset of a 10- and 2-week premating period, in F0 males and females, respectively, and continuing through the mating, gestation, and lactation periods, for two generations. There were adult (F0, F1, F2) and postnatal day (PND) 22 (F1, F2) necropsies: the oldest F2 males and females being killed at postnatal weeks 7 and 14, respectively. No compound-related clinical signs or effects on body weight or food consumption were observed in any generation. There were no compound-related changes in surface righting reflex, negative geotaxis reflex, mid-air righting reflex, pinna detachment, incisor eruption, eye opening, testes descent, preputial separation, or vaginal opening in F1 and F2 generations, or behavior in the open field or water filled multiple T-maze in the F1 generation. No test compound-related changes in estrous cyclicity, copulation index, fertility index, number of implantations, gestation length, litter size, pup weight, pup sex ratio, pup viability, or other functional reproductive measures were noted in any generation. A few significant changes in the anogenital distance (AGD) per cube root of body weight ratio were found at 0.2 and 20 microg/kg in F1 males, at 2, 20, and 200 microg/kg in F1 females, and at 20 and 200 microg/kg in F2 females. However, the changes in the AGD were consistently small (within 5% of control values), and no continuous changes in the AGD or AGD/cube root of body weight ratio were detected. There were no compound-related changes in epididymal sperm counts or motility in F0 and F1 males. No compound-related necropsy findings or effects on organ weight including the reproductive organs were found in any generation. Histopathologic examinations revealed no evidence of compound-related changes in any organs including the reproductive organs of both sexes. The data indicate that oral doses of BPA of between 0.2 and 200 microg/kg over 2 generations did not cause significant compound-related changes in reproductive or developmental parameters in rats.

The objective of this study was to determine the toxic effects of butyltin trichloride (BTCl) during early pregnancy. Following successful mating, female rats were given BTCl by gastric intubation at 0, 56, 226, or 903 mg/kg on days 0–3 or 4–7 of pregnancy. Female rats were sacrificed on day 20 of pregnancy and fetuses were examined for number, abnormality, mortality, and weight. The maternal body weight gain and food consumption during the administration period was significantly decreased after administration of BTCl at 903 mg/kg on days 0–3 or 4–7 of pregnancy. The pregnancy rate in the BTCl-treated groups was comparable to the control value, regardless of the days on which BTCl was given. The incidence of pre-implantation embryonic loss was not significantly affected after administration of BTCl on days 0–3 or 4–7. In females having implantations, the numbers of corpora lutea, implantations, and live fetuses and the incidences of pre- and postimplantation loss in the groups given BTCl on days 0–3 were comparable to the controls. Although a significant increase in the incidence of postimplantation loss was observed after administration of BTCl on days 4–7 at 56 mg/kg, this change was small and inconsistent across doses and seems unlikely to be toxicologically significant. A significant decrease in weight of female fetuses was found after administration of BTCl at 903 mg/kg on days 0–3 or 4–7. It could be concluded that BTCl treatment during early pregnancy is maternal and developmental toxic at 903 mg/kg.

4-tert-Octylphenol (OP) is an alkylphenol that is an intermediate in the production of alkylphenol ethoxylates. OP has been reported to be the most potent estrogenic alkylphenol in vitro. In the present study, the effects of OP on initiation and maintenance of pregnancy were investigated in rats. Inseminated female rats were orally given OP at 0, 15.6, 31.3, 62.5 and 125 mg/kg on day 0 through day 8 of pregnancy. Female rats were sacrificed on day 20 of pregnancy, and pregnancy outcome was determined. Decreases in body weight gain and food consumption on days 0–9 were found at 31.3 mg/kg and above, and at 15.6 mg/kg and above, respectively. The pregnancy rate was not adversely affected by OP administration during early pregnancy even at 125 mg/kg. The incidence of post-implantation loss per litter at 31.3 mg/kg and above was significantly higher than that in the control group. The body weights of live fetuses in the OP-treated groups were not significantly different from those in the control group. No increase in the incidence of fetuses with external malformations was found in any OP-treated group. We concluded that OP during early pregnancy caused post-implantation embryonic loss at doses that showed maternal toxicity.

In our previous studies, tributyltin chloride (TBTCl) at doses of 16.3 mg/kg and above caused implantation failure (preimplantation embryonic loss) and postimplantation embryonic loss in rats following administration on gestational day (GD) 0 through GD 3 and GD 4 through GD 7, respectively. This study was designed to assess the effects of TBTCl on uterine function as a cause of early embryonic loss in pseudopregnant rats. TBTCl was given orally to pseudopregnant rats at doses of 4.1, 8.1, 16.3 and 32.5 mg/kg on pseudopregnant day (PPD) 0 to PPD 3 or 8.1, 16.3, 32.5 and 65.1 mg/kg on PPD 4 to PPD 7. The decidual cell response was induced by bilateral scratch trauma on PPD 4. The uterine weight on PPD 9 served as an index of uterine decidualization. Uterine weight and serum progesterone levels on PPD 9 were significantly decreased after administration of TBTCl at doses of 16.3 mg/kg and above on PPD 0 to PPD 3 or PPD 4 to PPD 7. Administration of TBTCl at doses of 8.1 mg/kg and above on PPD 0 to 3 also significantly decreased serum progesterone levels on PPD 4. TBTCl had no effect on ovarian weight and number of corpora lutea. It can be concluded that TBTCl suppresses the uterine decidual cell response and decreases progesterone levels, and these effects are responsible for early embryonic loss due to TBTCl exposure.

The present study was conducted to evaluate the adverse effects of dibutyltin dichloride (DBTCl) on initiation and maintenance of pregnancy after maternal exposure during early pregnancy in rats. After successful mating, female rats were given DBTCl by gastric intubation on Days 0 to 3 or on Days 4 to 7 of pregnancy at 0, 3.8, 7.6, or 15.2 mg/kg. Food-restricted pregnant rats were given an amount of feed equal to the feed intake of female rats treated with DBTCl at 15.2 mg/kg on Days 0 to 3 or on Days 4 to 7 of pregnancy. Female rats were sacrificed on Day 20 of pregnancy and pregnancy outcome was determined. After administration of DBTCl on Days 0 to 3, the rate of nonpregnant females and the incidence of preimplantation embryonic loss in the 7.6 mg/kg group were significantly higher than those in the control group, and those in the 15.2 mg/kg group were significantly higher than those in the control and pair-fed groups. In females with implantations, the numbers of implantations and live fetuses and the incidence of postimplantation embryonic loss in the groups given DBTCl on Days 0 to 3 were not significantly different from those in the control group. The incidence of postimplantation embryonic loss in the groups given DBTCl on Days 4 to 7 at 7.6 and 15.2 mg/kg was significantly higher than that in the control and pair-fed groups. It can be concluded that DBTCl adversely affects initiation and maintenance of pregnancy when administered during early pregnancy and that the manifestations of the adverse effects of DBTCl vary with the gestational stage at the time of maternal exposure.

The objective of this study was to determine the susceptible day for the developmental toxicity of di-n-butyl phthalate (DBP). Pregnant rats were given a single dose of DBP by gastric intubation at 1500 mg kg−1 on one of days 6–16 of pregnancy. A significant increase in the incidence of postimplantation loss was found in pregnant rats given DBP on one of days 6–16, except for days 7 and 11. Significant increases in the incidences of fetuses with skeletal malformations, of fetuses with skeletal and internal malformations and of fetuses with external and skeletal malformations were noted after a single dosing of DBP on day 8, on day 9 and on day 15, respectively. Deformity of the cervical vertebrae was frequently observed after administration of DBP on day 8. Deformity of the cervical and thoracic vertebrae and ribs and dilatation of the renal pelvis were predominantly found in fetuses of dams treated with DBP on day 9. Cleft palate and fusion of the sternebrae were exclusively detected after administration of DBP on day 15. It could be concluded that the manifestation of deviant development induced by DBP varies with the developmental stage at the time of administration and that DBP induces two discrete responses from embryos to teratogenicity on days 8 and 9 and on day 15 of pregnancy. © 1997 John Wiley & Sons, Ltd.

The objective of this study was to characterize the developmental toxicity of mono- n -benzyl phthalate (MBeP), which is one of the major metabolites of n -butyl benzyl phthalate. Pregnant rats were given MBeP by gastric intubation at 250,375,500, or 625 mg/kg on days 7 to 9,10 to 12, or 13 to 15 of pregnancy. A significantly increased incidence of postimplantation loss was found at 500 mg/kg and above regardless of the days of administration. While administration of MBeP on days 7 to 9 or 13 to 15 at 375 mg/kg and above was significantly teratogenic, no evidence of teratogenicity was detected when MBeP was given on days 10 to 12. Deformity of the vertebral column and ribs and dilatation of the renal pelvis were frequently observed after administration on days 7 to 9. Cleft palate and fused sternebrae were exclusively found after administration on days 13 to 15. These findings indicate that the susceptibility and spectrum of the developmental toxicity of MBeP vary with the developmental stages at the time of administration.

Prostaglandin E receptor EP3D is coupled to stimulation and inhibition of adenylate cyclase and stimulation of phosphatidylinositol turnover. To examine the roles of the interaction of the carboxylic acid of an agonist and its putative binding site, the arginine residue in the seventh transmembrane domain of EP3D, in receptor-G protein coupling, we have mutated the arginine to the non-charged glutamine. TEI-3356, an EP3 agonist with a negatively charged the carboxylic acid, and TEI-4343, a non-charged methylester of TEI-3356, inhibited the forskolin-stimulated cAMP formation in the same concentration-dependent manner, but stimulation of basal cAMP formation and Ca2+ mobilization by TEI-4343 was much lower than that by TEI-3356. In the mutant receptor, both TEI-3356 and TEI-4343 showed the inhibition of forskolin-stimulated cAMP formation in the same profile, but did not stimulate basal cAMP formation or Ca2+ mobilization. These findings suggest that the interaction between the carboxylic acid of agonist and the arginine residue is important in signal transduction for adenylate cyclase stimulation and Ca2+ mobilization but not for adenylate cyclase inhibition.