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Postmenopausal women experience bone loss and weight gain. To date, crosstalk between estrogen receptor signals and nuclear factor-κB (NF-κB) has been reported, and estrogen depletion enhances bone resorption by osteoclasts via NF-κB activation. However, it is unclear when and in which tissues NF-κB is activated after menopause, and how NF-κB acts as a common signaling molecule for postmenopausal weight gain and bone loss. Therefore, we examined the role of NF-κB in bone and energy metabolism following menopause. NF-κB reporter mice, which can be used to measure NF-κB activation in vivo, were ovariectomized (OVX) and the luminescence intensity after OVX increased in the metaphyses of the long bones and perigonadal white adipose tissue, but not in the other tissues. OVX was performed on wild-type (WT) and p65 mutant knock-in (S534A) mice, whose mutation enhances the transcriptional activity of NF-κB. Weight gain with worsening glucose tolerance was significant in S534A mice after OVX compared with those of WT mice. The bone density of the sham group in WT or S534A mice did not change, whereas in the S534A-OVX group it significantly decreased due to the suppression of bone formation and increase in bone marrow adipocytes. Disulfiram, an anti-alcoholic drug, suppressed OVX-induced activation of NF-κB in the metaphyses of long bones and white adipose tissue (WAT), as well as weight gain and bone loss. Overall, the activation of NF-κB in the metaphyses of long bones and WAT after OVX regulates post-OVX weight gain and bone loss., Competing Interests: Declaration of competing interest The authors have declared no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Bloom dynamics of K. mikimotoi during summer 2015 in the Yatsushiro Sea, Japan, which caused fish mortality was studied using field survey data and satellite data. The bloom initially formed in the western area, subsequently appeared in the southern area, and finally expanded to the central area. The field-survey detected the horizontal displacement of the bloom which was also assessed by satellite data. Acoustic backscattering intensity of the current meter captured the modulation of the diurnal vertical migration of K. mikimotoi. After the modulation, K. mikimotoi distributed at a shallower depth in the nighttime than the period prior to the modulation. Factors affecting the modulation are suggested to be the continuous low nutrient conditions. Synchronization between the shallowed distribution during the nighttime and the wind driven surface northeastward current enabled a sudden horizontal transport toward the central area. Satellite and acoustic backscattering data are beneficial subsidiary tools for detecting blooms., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Kazuhiro Aoki reports financial support was provided by Fisheries Agency of Japan. Kazuhiro Aoki reports financial support was provided by Japan Society for the Promotion of Science., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Extracellular signal-regulated kinase (ERK) has been recognized as a critical regulator in various physiological and pathological processes. Extensive research has elucidated the signaling mechanisms governing ERK activation via biochemical regulations with upstream molecules, particularly receptor tyrosine kinases (RTKs). However, recent advances have highlighted the role of mechanical forces in activating the RTK-ERK signaling pathways, thereby opening new avenues of research into mechanochemical interplay in multicellular tissues. Here, we review the force-induced ERK activation in cells and propose possible mechanosensing mechanisms underlying the mechanoresponsive ERK activation. We conclude that mechanical forces are not merely passive factors shaping cells and tissues but also active regulators of cellular signaling pathways controlling collective cell behaviors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Platelet glycoprotein (GP) Ibα is the major ligand-binding subunit of the GPIb-IX-V complex that binds von Willebrand factor. GPIbα is heavily glycosylated, and its glycans have been proposed to play key roles in platelet clearance, von Willebrand factor binding, and as target antigens in immune thrombocytopenia syndromes. Despite its importance in platelet biology, the glycosylation profile of GPIbα is not well characterized., Objectives: The aim of this study was to comprehensively analyze GPIbα amino acid sites of glycosylation (glycosites) and glycan structures., Methods: GPIbα ectodomain that was recombinantly expressed or that was purified from human platelets was analyzed by Western blot, mass spectrometry glycomics, and mass spectrometry glycopeptide analysis to define glycosites and the structures of the attached glycans., Results: We identified a diverse repertoire of N- and O-glycans, including sialoglycans, Tn antigen, T antigen, and ABO(H) blood group antigens. In the analysis of the recombinant protein, we identified 62 unique O-glycosites. In the analysis of the endogenous protein purified from platelets, we identified 48 unique O-glycosites and 1 N-glycosite. The GPIbα mucin domain is densely O-glycosylated. Glycosites are also located within the macroglycopeptide domain and mechanosensory domain., Conclusions: This comprehensive analysis of GPIbα glycosylation lays the foundation for further studies to determine the functional and structural roles of GPIbα glycans., Competing Interests: Declaration of competing interests M.A.H. received consulting fees from Dova Pharmaceuticals, Janssen Pharmaceuticals, and Sonder Capital. C.R.B. is a co-founder and scientific advisory board member of Lycia Therapeutics, Palleon Pharmaceuticals, Enable Bioscience, Redwood Biosciences (a subsidiary of Catalent), OliLux Bio, Grace Science LLC, and InterVenn Biosciences. S.A.M. is a consultant for InterVenn Biosciences and Arkuda Therapeutics. S.A.M. and C.R.B. are inventors on a Stanford patent related to the use of mucinases for glycoproteomic analysis. The remaining co-authors have no conflicts of interest to disclose., (Copyright © 2023 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

Here, we tested the hypothesis that tensile and compressive stresses generated in the alveolar bone proper regulate site-specific cellular and functional changes in osteoclasts and osteoblasts. Thirty-two 13-week-old male mice were randomly divided into four groups: two experimental groups with vertical loading obliquely from the palatal side to the buccal side of the maxillary molar (0.9 N) 30 min per day for 8 or 15 days and unloaded controls (n = 8). Calcein and alizarin were administered 8 and 2 days before euthanization, respectively, to detect the time of bone formation. Undecalcified sections parallel to the occlusal plane were prepared on the palatal root and the surrounding alveolar bone in the middle of the root length. The alveolar perimeter was divided into 12 equal regions for site analysis, and the bone histomorphometric parameters were obtained for each region. Data from in vivo microfocus computed tomography were used to construct animal-specific finite element models. 2D stress distribution images were overlain on histology images obtained from the same location. Significant differences in the total perimeter between groups and between loading and unloading in each region were statistically analyzed (α = 0.05). Osteoclast counts and the alizarin label ratio were significantly higher in the loaded group than in the unloaded group in regions where the maximum von Mises and principal tensile stresses were the highest along the perimeter. The label ratio of calcein was significantly lower in the 8-day loaded group than in the unloaded group, indicating that the calcein-labeled surface was resorbed by osteoclasts that appeared during the loading period. The effect of loading was mitigated by an increase in the maximum principal compressive stress. We conclude that bone resorption and bone formation are functions of site-specific tension and compression in the alveolar bone proper, confirming our hypothesis. This finding is critical for the advancement of diagnosis and treatment planning in clinical dentistry., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Purpose: This study investigated the relationship between vertical and horizontal jumping ability and change of direction (COD) to measure athletic performance in 51 elite male handball players., Scope: Countermovement jump (CMJ), peak power, and standing long jump (SLJ) were measured. Participants performed a 20-m sprint test (time measured at 5, 10, and 20 m) and a zigzag test (COD: 135°, 90°, and 45°). The COD deficit, an index of the time required for COD, was calculated. The correlations between CMJ height and zigzag test times were relatively large (at 135°, r =  - 0.607; at 90°, r =  - 0.594; at 45°, r =  - 0.613; p < 0.01), whereas those between CMJ height and COD deficit were moderate (at 135°, r =  - 0.399, p < 0.01; at 90°, r =  - 0.350, p < 0.05; at 45°, r =  - 0.323, p < 0.05). SLJ showed a negative moderate correlation with COD deficit (at 135°, r =  - 0.439, p < 0.01; at 90°, r =  - 0.469, p < 0.01; at 45°, r =  - 0.380, p < 0.01)., Conclusions: This study is the first to analyse SLJ ability and COD deficit parameters of handball players. We found that SLJ ability is moderately related to COD time and deficit; therefore, SLJ measurement may be a useful predictor of athletic performance., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The purpose of this study was to investigate the effect of load-induced local mechanical strain on bone cell activity of peri-implant bone in mice. Titanium implants were placed in the maxillae of 13-week-old male C57BL/6J mice and subjected to intermittent 0.15 N, 0.3 N, or 0.6 N loads for 30 min/day for 6 days. The animals were sacrificed 2 days after the final loading. Unloaded mice were used as controls. An animal-specific three-dimensional finite element model was constructed based on morphological data retrieved from in vivo microfocus computed tomography for each mouse to calculate the mechanical strain distribution. Strain distribution images were overlaid on corresponding histological images of the same site in the same animal. The buccal cervical region of the peri-implant bone was predetermined as the region of interest (ROI). Each ROI was divided by four strain intensity levels: 0-20 με, 20-60 με, 60-100 με, and ≥100 με, and the bone histomorphometric parameters were analyzed by the total area of each strain range for all loaded samples. The distance between the calcified front and calcein labeling as a parameter representing the mineral apposition rate was significantly greater in the areas with strain intensity ≥100 με than in the area with strain intensity <100 με, suggesting that the bone formation activity of osteoblasts was locally enhanced by a higher mechanical strain. However, the shrunken osteocytes and the empty osteocyte lacunae were significantly lower in the highest strain area, suggesting that osteoclastogenesis was more retarded in higher strain areas than in lower strain areas. The histomorphometric parameters were not affected geometrically in the unloaded animals, suggesting that the load-induced mechanical strain caused differences in the histomorphometric parameters. Our findings support the hypothesis that bone cell activity related to bone resorption and formation is local strain-dependent on implant loading., (Copyright © 2021. Published by Elsevier Ltd.)

Background: Among Amish communities of North America, biallelic mutations of ST3GAL5 (c.694C > T) eliminate synthesis of GM3 and its derivative downstream a- and b-series gangliosides. Systemic ganglioside deficiency is associated with infantile onset psychomotor retardation, slow brain growth, intractable epilepsy, deafness, and cortical visual impairment. We developed a robust quantitative assay to simultaneously characterize glycan and ceramide moieties of plasma glycosphingolipids (GSLs) among ST3GAL5 c.694C > T homozygotes (n = 8), their heterozygous siblings (n = 24), and wild type control (n = 19) individuals., Methods: Following extraction and saponification of total plasma lipids, GSLs were purified on a tC18 cartridge column, permethylated, and subjected to nanospray ionization mass spectrometry utilizing neutral loss scanning and data-dependent acquisition. Plasma GSLs were quantified against appropriate synthetic standards., Results: Our method demonstrated linearity from 5 to 250 μl of plasma. Recovery of synthetic GSLs spiked into plasma was 99-104% with no matrix interference. Quantitative plasma GSL profiles discriminated among ST3GAL5 genotypes: GM3 and GD3 were undetectable in ST3GAL5 c.694C > T homozygotes, who had markedly elevated lactosylceramide (19.17 ± 4.20 nmol/ml) relative to heterozygous siblings (9.62 ± 2.46 nmol/ml) and wild type controls (6.55 ± 2.16 nmol/ml). Children with systemic ganglioside deficiency had a distinctive shift in ceramide composition toward higher mass species., Conclusions: Our quantitative glycolipidomics method discriminates among ST3GAL5 c.694C > T genotypes, can reveal subtle structural heterogeneity, and represents a useful new strategy to diagnose and monitor GSL disorders in humans., (© 2019 Published by Elsevier B.V. on behalf of The Association for Mass Spectrometry: Applications to the Clinical Lab (MSACL).)

We recently found that the membrane-bound receptor activator of NF-κB ligand (RANKL) on osteoblasts works as a receptor to stimulate osteoblast differentiation, however, the reason why the RANKL-binding molecules stimulate osteoblast differentiation has not been well clarified. Since the induction of cell-surface receptor clustering is known to lead to cell activation, we hypothesized that the induction of membrane-RANKL clustering on osteoblasts might stimulate osteoblast differentiation. Immunoblotting showed that the amount of RANKL on the membrane was increased by the RANKL-binding peptide OP3-4, but not by osteoprotegerin (OPG), the other RANKL-binding molecule, in Gfp-Rankl-transfected ST2 cells. Observation under a high-speed atomic force microscope (HS-AFM) revealed that RANKL molecules have the ability to form clusters. The induction of membrane-RANKL-OPG-Fc complex clustering by the addition of IgM in Gfp-Rankl-transfected ST2 cells could enhance the expression of early markers of osteoblast differentiation to the same extent as OP3-4, while OPG-Fc alone could not. These results suggest that the clustering-formation of membrane-RANKL on osteoblasts could stimulate early osteoblast differentiation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

T antigen (Galβ1-3GalNAcα1-Ser/Thr) is an evolutionary-conserved mucin-type core 1 glycan structure in animals synthesized by core 1 β1,3-galactosyltransferase 1 (C1GalT1). Previous studies showed that T antigen produced by Drosophila C1GalT1 (dC1GalT1) was expressed in various tissues and dC1GalT1 loss in larvae led to various defects, including decreased number of circulating hemocytes, hyper-differentiation of hematopoietic stem cells in lymph glands, malformation of the central nervous system, mislocalization of neuromuscular junction (NMJ) boutons, and ultrastructural abnormalities in NMJs and muscle cells. Although glucuronylated T antigen (GlcAβ1-3Galβ1-3GalNAcα1-Ser/Thr) has been identified in Drosophila, the physiological function of this structure has not yet been clarified. In this study, for the first time, we unraveled biological roles of glucuronylated T antigen. Our data show that in Drosophila, glucuronylation of T antigen is predominantly carried out by Drosophila β1,3-glucuronyltransferase-P (dGlcAT-P). We created dGlcAT-P null mutants and found that mutant larvae showed lower expression of glucuronylated T antigen on the muscles and at NMJs. Furthermore, mislocalization of NMJ boutons and a partial loss of the basement membrane components collagen IV (Col IV) and nidogen (Ndg) at the muscle 6/7 boundary were observed. Those two phenotypes were correlated and identical to previously described phenotypes in dC1GalT1 mutant larvae. In addition, dGlcAT-P null mutants exhibited fewer NMJ branches on muscles 6/7. Moreover, ultrastructural analysis revealed that basement membranes that lacked Col IV and Ndg were significantly deformed. We also found that the loss of dGlcAT-P expression caused ultrastructural defects in NMJ boutons. Finally, we showed a genetic interaction between dGlcAT-P and dC1GalT1. Therefore, these results demonstrate that glucuronylated core 1 glycans synthesized by dGlcAT-P are key modulators of NMJ bouton localization, basement membrane formation, and NMJ arborization on larval muscles., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

A massive bloom of the dinoflagellate Karenia mikimotoi appeared in 2014 in Imari Bay, Japan. Bloom dynamics and hydrographical conditions were examined by field survey. The bloom initially developed in the eastern area of Imari Bay, subsequently after rainfall during the neap tides, cell density exceeded over 10,000cellsml. Vertical distribution of K. mikimotoi was primarily controlled by the light intensity and secondarily by the water quality during the daytime. Almost all cell-density maxima occurred in depths with weak daytime light intensities of <300μmolm -2 s -1 . In some cases of weak light intensity, cell-density maxima occurred in depths with favorable hydrodynamic conditions for the growth. Spatially classified areas were identified by cluster analysis using the growth rate calculated from seawater temperature and salinity. This study quantitatively evaluated the environmental factors of the eastern area, where the bloom initially occurred, during the development of the bloom., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Adult T-cell leukemia/lymphoma (ATLL) is a distinct form of peripheral T-cell lymphoma with poor prognosis, which is caused by the human T-lymphotropic virus type 1 (HTLV-1). In contrast to the unequivocal importance of HTLV-1 infection in the pathogenesis of ATLL, the role of acquired mutations in HTLV-1 infected T cells has not been fully elucidated, with a handful of genes known to be recurrently mutated. In this study, we identified unique RHOA mutations in ATLL through whole genome sequencing of an index case, followed by deep sequencing of 203 ATLL samples. RHOA mutations showed distinct distribution and function from those found in other cancers. Involving 15% (30/203) of ATLL cases, RHOA mutations were widely distributed across the entire coding sequence but almost invariably located at the guanosine triphosphate (GTP)-binding pocket, with Cys16Arg being most frequently observed. Unexpectedly, depending on mutation types and positions, these RHOA mutants showed different or even opposite functional consequences in terms of GTP/guanosine diphosphate (GDP)-binding kinetics, regulation of actin fibers, and transcriptional activation. The Gly17Val mutant did not bind GTP/GDP and act as a dominant negative molecule, whereas other mutants (Cys16Arg and Ala161Pro) showed fast GTP/GDP cycling with enhanced transcriptional activation. These findings suggest that both loss- and gain-of-RHOA functions could be involved in ATLL leukemogenesis. In summary, our study not only provides a novel insight into the molecular pathogenesis of ATLL but also highlights a unique role of variegation of heterologous RHOA mutations in human cancers., (© 2016 by The American Society of Hematology.)

In 2010, a massive bloom of the raphidophycean flagellate Chattonella occurred in the Ariake Sea and Tachibana Bay. Bloom dynamics and hydrographical conditions were examined by field survey. The development and decline of the bloom occurred three times in Tachibana Bay. First and third bloom developments synchronized with precipitation, and the second bloom developed in synchronization with a salinity decrease which occurred in relation to an increase of river discharge from the Chikugo River which takes several days to flow from the Ariake Sea. These results imply that the bloom was transported with the low salinity water from the Ariake Sea to Tachibana Bay. During blooms along the northern coast of Shimabara Peninsula, the predominant phytoplankton species changed from Chattonella to Skeletonema. Low salinity water intrusion induced an interregional difference of the Chattonella and Skeletonema bloom spatially-differentiated by the salinity in the Ariake Sea and Tachibana Bay., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Mosquitoes are important vectors of parasitic and viral diseases with Anopheles gambiae transmitting malaria and Aedes aegypti spreading yellow and Dengue fevers. Using two different approaches (solid-phase extraction and reversed-phase or hydrophilic interaction HPLC fractionation followed by MALDI-TOF MS or permethylation followed by NSI-MS), we examined the N-glycans of both A. gambiae and A. aegypti larvae and demonstrate the presence of a range of paucimannosidic glycans as well as bi- and tri-antennary glycans, some of which are modified with fucose or with sulphate or glucuronic acid residues; the latter anionic modifications were also found on N-glycans of larvae from another dipteran species (Drosophila melanogaster). The sulphate groups are attached primarily to core α-mannose residues (especially the α1,6-linked mannose), whereas the glucuronic acid residues are linked to non-reducing β1,3-galactose. Also, O-glycans were found to possess glucuronic acid and sulphate as well as phosphoethanolamine modifications. The presence of sulphated and glucuronylated N-glycans is a novel feature in dipteran glycomes; these structures have the potential to act as additional anionic glycan ligands involved in parasite interactions with the vector host., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Introduction: The objectives of this study were to investigate the effect of phytic acid, inositol hexakisphosphate (IP6), as a final rinse on the surface of instrumented root canals and smear-layered flat dentin surfaces treated with sodium hypochlorite (NaOCl) and to evaluate its effect on the viability and alkaline phosphatase activity of osteoblast-like cells (MC3T3-E1)., Methods: The universally accepted chelating agent EDTA was used as the control in all conducted experiments. Root canals of human canines were instrumented with rotary files and irrigated with 5% NaOCl, followed by a final rinse of 17% EDTA (1 minute), 1% IP6 (1 minute or 30 seconds), or distilled water. NaOCl-treated flat coronal dentin surfaces were also treated with 17% EDTA (1 minute), 1% IP6 (1 minute or 30 seconds), or distilled water. The presence or absence of smear layer was evaluated with scanning electron microscopy. Cell viability and alkaline phosphatase assays were performed to evaluate the effect of IP6 and EDTA on cultured MC3T3-E1 cells., Results: The results demonstrated the ability of IP6 to remove the smear layer from instrumented root canals and flat coronal dentin surfaces. When compared with EDTA, IP6 was less cytotoxic and did not affect the differentiation of MC3T3-E1 cells., Conclusions: IP6 shows the potential to be an effective and biocompatible chelating agent., (Copyright © 2015 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

The dynamics of river plume in relation to harmful blooms of the raphidophycean flagellate, Chattonella antiqua in summer 2008-2010 in the Yatsushiro Sea, Japan were studied using a hydrodynamic model and monitoring data. In the southern area, the bloom formed in the waters stratified by a halocline caused by the southward expansion of riverine water from the Kuma River after the bloom initially forming in the northern area. The timing of the southward riverine water advection can be explained by the balance between the wind stress term and the pressure gradient term calculated from the horizontal density difference between the northern and southern areas. The wind stress and pressure gradient terms were evaluated using the sea surface temperature, salinity, wind speed and direction at two stations. Real time monitoring or continuous observations in these areas will enable nowcasts of bloom expansion when a bloom develops in the northern area., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

To explore the mechanism of bone destruction associated with oral cancer, we identified factors that stimulate osteoclastic bone resorption in oral squamous cell carcinoma. Two clonal cell lines, HSC3-C13 and HSC3-C17, were isolated from the maternal oral cancer cell line, HSC3. The conditioned medium from HSC3-C13 cells showed the highest induction of Rankl expression in the mouse stromal cell lines ST2 and UAMS-32 as compared to that in maternal HSC3 cells and HSC3-C17 cells, which showed similar activity. The conditioned medium from HSC3-C13 cells significantly increased the number of osteoclasts in a co-culture with mouse bone marrow cells and UAMS-32 cells. Xenograft tumors generated from these clonal cell lines into the periosteal region of the parietal bone in athymic mice showed that HSC3-C13 cells caused extensive bone destruction and a significant increase in osteoclast numbers as compared to HSC3-C17 cells. Gene expression was compared between HSC3-C13 and HSC3-C17 cells by using microarray analysis, which showed that CXCL2 gene was highly expressed in HSC3-C13 cells as compared to HSC3-C17 cells. Immunohistochemical staining revealed the localization of CXCL2 in human oral squamous cell carcinomas. The increase in osteoclast numbers induced by the HSC3-C13-conditioned medium was dose-dependently inhibited by addition of anti-human CXCL2-neutralizing antibody in a co-culture system. Recombinant CXCL2 increased the expression of Rankl in UAMS-32 cells. These results indicate that CXCL2 is involved in bone destruction induced by oral cancer. This is the first report showing the role of CXCL2 in cancer-associated bone destruction., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Selectins on activated vascular endothelium mediate inflammation by binding to complementary carbohydrates on circulating neutrophils. The human neutrophil receptor for E-selectin has not been established. We report here that sialylated glycosphingolipids with 5 N-acetyllactosamine (LacNAc, Galbeta1-4GlcNAcbeta1-3) repeats and 2 to 3 fucose residues are major functional E-selectin receptors on human neutrophils. Glycolipids were extracted from 10(10) normal peripheral blood human neutrophils. Individual glycolipid species were resolved by chromatography, adsorbed as model membrane monolayers and selectin-mediated cell tethering and rolling under fluid shear was quantified as a function of glycolipid density. E-selectin-expressing cells tethered and rolled on selected glycolipids, whereas P-selectin-expressing cells failed to interact. Quantitatively minor terminally sialylated glycosphingolipids with 5 to 6 LacNAc repeats and 2 to 3 fucose residues were highly potent E-selectin receptors, constituting more than 60% of the E-selectin-binding activity in the extract. These glycolipids are expressed on human blood neutrophils at densities exceeding those required to support E-selectin-mediated tethering and rolling. Blocking glycosphingolipid biosynthesis in cultured human neutrophils diminished E-selectin, but not P-selectin, adhesion. The data support the conclusion that on human neutrophils the glycosphingolipid NeuAcalpha2-3Galbeta1-4GlcNAcbeta1-3[Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3](2)[Galbeta1-4GlcNAcbeta1-3](2)Galbeta1-4GlcbetaCer (and closely related structures) are functional E-selectin receptors.

Receptor activator of NF-kappaB (RANK) and its ligand (RANKL) are pivotal regulators of osteoclast differentiation. RANK and RANKL also mediate T cell/dendritic cell (DC) interaction. Previous study has shown that RANK/RANKL interaction induces prolonged DC survival and antigen presentation. In the present study, we have newly established a hybridoma which produces neutralizing anti-RANKL monoclonal antibody (IK22-5). By treating collagen-induced arthritis (CIA) mice with IK22-5, we have investigated the role of RANKL in the pathogenesis of CIA. Although IK22-5 had no effect on immune responses or inflammation, it ameliorated bone loss at the site of inflammation. Histological analyses revealed that osteoclast formation was impaired at the site of joint inflammation in IK22-5-treated CIA mice. These results suggest the utility of anti-RANKL mAb for the prevention of osteoporosis associated with joint inflammation in RA.

For chemical characterization of glycosphingolipids, it is necessary to determine the chemical compositions of three constituents, i.e., sugars, fatty acids, and sphingoids. A new rapid analytical method is described using a one-pot reaction in a household microwave oven, producing sugars, fatty acids, and especially sphingoids free of by-products, from a single aliquot of a biological sample. Glycosphingolipids were hydrolyzed by microwave exposure with 0.1 M NaOH/CH(3)OH for 2 min followed by 1 M HCl/CH(3)OH for 45 s. The alkaline methanolysis step produced intermediate lysoglycosphingolipids virtually free of by-products such as the O-methyl ethers usually seen. The fatty acid methyl esters were extracted with n-hexane, and other reaction products were dried, taken up in aqueous alkaline methanol, and shaken with chloroform. Sphingoids partitioned into the organic phase under these conditions, whereas the sugar portion that partitioned into the aqueous phase was re-N-acetylated and remethanolyzed for 30 s by microwave exposure. Analysis of the profiles of glycosphingolipid constituents obtained using the microwave oven method showed that they were quantitatively and qualitatively comparable to those obtained by time-consuming conventional methods, which require reaction for several hours. Analysis of the three constituents, including analysis by gas chromatography, may be obtained within 1 day using the method described here.