Release rate is a key variable affecting the therapeutic effectiveness of liposomal fasudil for the treatment of cerebral ischemia/reperfusion injury.

Liposomal fasudil as a treatment for cerebral ischemia/reperfusion (I/R) injury has been demonstrated to be effective in animal models due to the high accumulation of liposomes in damaged brain tissue. However, it is still unclear what effect drug release rate has on the treatment of I/R injury, where pathology progresses dramatically in a short time. In the present study, we assessed four formulations of liposomal fasudil. The results of an in vitro drug release assay showed that the release properties of fasudil were changed by varying the lipid composition and internal phase of the liposomes. Based on these results, differences in the transition of fasudil plasma concentration were monitored after the administration of each type of liposomal fasudil in normal rats. A pharmacokinetic study showed that higher levels of drug retention in liposomal fasudil resulted in higher fasudil plasma concentration. Finally, treatment of I/R injury model rats with liposomal fasudil revealed that a mid-level release rate of fasudil from liposomes resulted in the greatest therapeutic effect among the formulations. In conclusion, these results demonstrate that an optimized drug release rate from liposomes enhances the therapeutic effect of fasudil for the treatment of cerebral I/R injury.

Tumour-promoting activity of polycyclic aromatic hydrocarbons and their oxygenated or nitrated derivatives.

Various types of polycyclic aromatic compounds (PACs) in diesel exhaust particles are thought to contribute to carcinogenesis in mammals. Although the carcinogenicity, mutagenicity and tumour-initiating activity of these compounds have been evaluated, their tumour-promoting activity is unclear. In the present study, to determine the tumour-inducing activity of PACs, including previously known mutagenic compounds in atmospheric environments, a transformation assay for promoting activity mediated by the release of contact inhibition was conducted for six polycyclic aromatic hydrocarbons (PAHs), seven oxygenated PAHs (oxy-PAHs) and seven nitrated PAHs (nitro-PAHs) using mouse embryonic fibroblast cells transfected with the v-Ha-ras gene (Bhas 42 cells). Of these, two PAHs [benzo[k]fluoranthene (B[k]FA) and benzo[b]fluoranthene (B[b]FA)], one oxy-PAH [6H-benzo[cd]pyren-6-one (BPO)] and two nitro-PAHs (3-nitro-7H-benz[de]anthracen-7-one and 6-nitrochrysene) were found to exhibit particularly powerful tumour-promoting activity (≥10 foci following exposure to <100nM). In addition, clear mRNA expression of CYP1A1, which is associated with aryl hydrocarbon receptor (AhR)-mediated activation, was observed following the exposure of cells to two PAHs (B[k]FA and B[b]FA) and three oxy-PAHs (1,2-naphthoquinone, 11H-benzo[b]fluoren-11-one and BPO). Further, an HO-1 antioxidant response activation was observed following exposure to B[k]FA, B[b]FA and BPO, suggesting that the induction of tumour-promoting activity in these compounds is correlated with the dysfunction of signal transduction via AhR-mediated responses and/or oxidative stress responses.

Glycolytic enzyme GAPDH promotes peroxide stress signaling through multistep phosphorelay to a MAPK cascade.

Phosphorelay signaling of environmental stimuli by two-component systems is prevailing in bacteria and also utilized by fungi and plants. In the fission yeast Schizosaccharomyces pombe, peroxide stress signals are transmitted from the Mak2/3 sensor kinases to the Mpr1 histidine-containing phosphotransfer (HPt) protein and finally to the Mcs4 response regulator, which activates a MAP kinase cascade. Here we show that, unexpectedly, the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) physically associates with the Mcs4 response regulator and stress-responsive MAP kinase kinase kinases (MAPKKKs). In response to H2O2 stress, Cys-152 of the Tdh1 GAPDH is transiently oxidized, which enhances the association of Tdh1 with Mcs4. Furthermore, Tdh1 is essential for the interaction between the Mpr1 HPt protein and the Mcs4 response regulator and thus for phosphorelay signaling. These results demonstrate that the glycolytic enzyme GAPDH plays an essential role in the phosphorelay signaling, where its redox-sensitive cysteine residue may provide additional input signals.

Properties and functions of lactosylceramide from mouse neutrophils.

Lactosylceramide (LacCer), which is essential for many cellular processes, is highly expressed on the plasma membranes of human neutrophils and mediates innate immune functions. Less is known, however, about the properties and biological functions of LacCer in mouse neutrophils. This study therefore analyzed the properties of mouse neutrophil LacCer. LacCer was observed on the surface of these cells, with flow cytometry indicating that mouse neutrophil LacCer could be detected by the anti-LacCer mAb T5A7, but not by the anti-LacCer antibodies Huly-m13 and MEM-74. The molecular species of LacCer were nearly identical in mouse and human neutrophils, including C24:0 and C24:1 fatty acid chain-containing species, although the LacCer content in plasma membranes was ∼ 20-fold lower in mouse than in human neutrophils. Surface plasmon resonance analysis revealed that T5A7 bound to a lipid monolayer composed of LacCer, DOPC, cholesterol and sphingomyelin (molar ratio 0.1 : 10 : 10 : 1), whereas Huly-m13 did not. T5A7 induced neutrophil migration, which was abolished by inhibitors of Src-family kinases, PI-3 kinases, and trimeric G (o/i) proteins. T5A7 also inhibited phagocytosis of non-opsonized zymosans by neutrophils. Taken together, these findings suggest that in mouse neutrophils, (i) LacCer is expressed as LacCer-enriched microdomains in cell surface plasma membranes, (ii) these microdomains are recognized by T5A7 but not by other known anti-LacCer antibodies and (iii) LacCer is involved in cell migration and phagocytosis.

Inhibition of connective tissue growth factor ameliorates disease in a murine model of rheumatoid arthritis.

OBJECTIVE: We have shown that connective tissue growth factor (CTGF) plays an important role in the pathogenesis of rheumatoid arthritis (RA). This study was undertaken to evaluate the effects of blockade of the CTGF pathway on the development of collagen-induced arthritis (CIA) in mice. METHODS: Arthritis was induced in DBA/1J mice by immunization with a combination of type II collagen (CII) and Freund's complete adjuvant. We evaluated the development of arthritis in mice with CIA left untreated versus treated with neutralizing anti-CTGF monoclonal antibody (mAb). RESULTS: Inhibition of CTGF in mice treated with neutralizing anti-CTGF mAb significantly ameliorated arthritis compared to the untreated mice with CIA. Serum levels of matrix metalloproteinase 3 were reduced by anti-CTGF mAb treatment. Moreover, blockade of CTGF decreased interleukin-17 expression on purified CD4+ T lymphocytes. Although the expression of the retinoic acid receptor-related orphan receptor γt gene was not suppressed by anti-CTGF mAb treatment, that of interferon regulatory factor 4 (IRF-4) and IκBζ (Nfkbiz), which are other important molecules for the differentiation of Th17 cells, was suppressed. In addition, blockade of CTGF inhibited pathologic proliferation of T lymphocytes in response to CII restimulation in vitro. Moreover, aberrant osteoclastogenesis in mice with CIA was restored by anti-CTGF mAb treatment. CONCLUSION: Our findings indicate that blockade of CTGF prevents the progression of arthritis in mice with CIA. Anti-CTGF mAb treatment suppresses pathologic T cell function and restores aberrant osteoclastogenesis in mice with CIA. CTGF may become a new target for the treatment of RA.

Associations of Cardiometabolic Indices With Peptides Related to Hypertensive Disorders of Pregnancy in Adult Men.

BACKGROUND: Seven peptides with low molecular weights in blood have been identified as possible biomarkers of hypertensive disorders of pregnancy (HDP). A history of HDP is known to be associated with a high risk of cardiovascular disease in the later life of women with HDP. However, it remains to be determined whether HDP-related peptides are useful biomarkers of cardiovascular disease in the general population. The purpose of this study was to determine the relationships between these peptides and cardiometabolic risk in adult men. METHODS: We investigated the relationships between HDP-related peptides and two recent indices of cardiometabolic risk, hematometabolic index (HMI) and lipid accumulation product (LAP), in male workers aged 35 to 69 years. Concentrations of the HDP-related seven peptides with mass/charge ratios (m/z) of 2081 (P-2081), 2091 (P-2091), 2127 (P-2127), 2209 (P-2209), 2378 (P-2378), 2858 (P-2858), and 3156 (P-3156) were measured simultaneously by using a mass spectrometer. Standardized partial regression coefficients (ß) were obtained in multivariable linear regression analysis, and mean levels of the log-transformed HMI and LAP were compared in tertile groups of each peptide in the analysis of covariance with adjustment for age, habits of smoking and alcohol drinking, history of diabetes, and medication therapy for dyslipidemia. RESULTS: There was a significant positive correlation between the HMI and the serum level of P-2378 (ß = 0.310), a fragment of complement component 4, while a significant inverse correlation (ß = -0.389) was obtained between the LAP and the serum level of P-3156, a fragment of inter-α-trypsin inhibitor heavy chain H4. Other peptides (P-2081, P-2091, P-2127, P-2209, and P-2858) did not show significant correlations with the HMI or LAP. The log-transformed HMI tended to be higher with an increase in the tertile for P-2378. The mean level of log-transformed LAP in the first tertile group of P-3156 was significantly higher than those in the second and third tertile groups of P-3156. CONCLUSION: The HDP-related peptides with m/z of 2378 and m/z of 3156 were shown to be associated with the HMI and LAP, respectively, which are recent indices reflecting cardiometabolic risk. Therefore, the peptides with m/z of 2378 and m/z of 3156 were thought to be potential biomarkers for discrimination of cardiovascular risk in adult men. Further studies on the relationships between the peptides and cardiovascular risk factors in non-pregnant women are needed to confirm the findings of this study.

Clinical peptidomic analysis by a one-step direct transfer technology: its potential utility for monitoring of pathophysiological status in female reproductive system disorders.

To date, numerous studies have searched for candidate molecules or clinical examination methods as potential biomarkers for monitoring intractable diseases, such as carcinomas. Evidence accumulated over the past decade shows that many proteolytic peptides appear in human humoral fluids, including peripheral blood, in association with an individual's health condition. Although an analysis of the whole peptide (the 'peptidome') using mass spectrometry is thought to be one of the most powerful and promising experimental approaches, it has failed to identify biomarkers in the clinical blood samples, presumably due to the methodological limitations. In general, commonly used techniques for proteomic analysis of blood require the removal of large amounts of serum/plasma proteins prior to mass spectrometry analysis, and this step seems to have resulted in the overlooking of important biomarkers during the analytical process. Here, we provide a brief overview of a new quantitative peptidomic analysis by a one-step direct transfer technology without depletion of major blood proteins. Using this technology, we herein report experimental data on serum peptidomic analysis for patients with pregnancy-induced hypertension as a clinical model. In addition, we refer to the potential utility of this approach for the monitoring of pathophysiological status in female reproductive system disorders in general.

Peptides associated with hypertensive disorders of pregnancy as possible biomarkers for severity of lower extremity arterial disease.

BACKGROUND AND AIMS: Seven circulating peptides, consisting of 18-28 amino acids, were identified as possible biomarkers of hypertensive disorders of pregnancy (HDP) in our previous study. However, it is unknown whether these peptides are relevant to cardiovascular disease. The purpose of this study was to clarify the relationships between serum levels of these peptides and leg arterial blood flow in patients with lower extremity arterial disease (LEAD). METHODS: The subjects were 165 outpatients with LEAD. Patients with advanced LEAD (stages 5 and 6 of the Rutherford classification) were not included. Leg arterial blood flow was evaluated by ankle brachial pressure index (ABI) and % decrease in ABI after leg exercise induced by a leg loader or treadmill. Concentrations of the seven peptides with m/z 2081 (P-2081), 2091 (P-2091), 2127 (P-2127), 2209 (P-2209), 2378 (P-2378), 2858 (P-2858) and 3156 (P-3156) were measured simultaneously with a mass spectrometer. RESULTS: P-2081, P-2127 and P-2209 levels showed significant positive correlations with leg arterial blood flow, while P-2091, P-2378 and P-2858 levels showed significant inverse correlations with leg arterial blood flow. There was no significant correlation between P-3156 levels and leg arterial blood flow. The above positive and inverse associations between peptide levels and leg arterial blood flow were also found in logistic regression analysis using tertile groups divided by the concentrations of each peptide. CONCLUSIONS: Serum levels of six HDP-related peptides (P-2081, P-2091, P-2127, P-2209, P-2378 and P-2858) were associated with lower extremity arterial blood flow in patients with LEAD, and thus these peptides are possible biomarkers for severity of LEAD.

DGKζ is involved in LPS-activated phagocytosis through IQGAP1/Rac1 pathway.

Diacylglycerol kinase (DGK) plays an important role in phosphoinositide signaling cascade by regulating the intracellular level of diacylglycerol and phosphatidic acid. The DGK family is involved in various pathophysiological responses that are mediated through unique binding partners in different tissues and cells. In this study, we identified a small GTPase effector protein, IQGAP1, as a novel DGKζ-associated complex protein. A bacterial endotoxin, lipopolysaccharide (LPS), facilitated the complex formation in macrophages. Both proteins co-localized at the edge and phagocytic cup of the cell. Furthermore, RNA interference-mediated knockdown of DGKζ or IQGAP1 impaired LPS-induced Rac1 activation. Primary macrophages derived from DGKζ(-/-) mice attenuated LPS-induced phagocytosis of bacteria. These results suggest that DGKζ is involved in IQGAP1/Rac1-mediated phagocytosis upon LPS stimulation in macrophages.

Regenerating gene (REG) 1 alpha promotes pannus progression in patients with rheumatoid arthritis.

INTRODUCTION: A protein analysis using mass spectrometry revealed the existence of serum proteins with significant quantitative changes after the administration of infliximab. Among these proteins, regenerating gene (REG) 1α appears to be related to the pathogenesis of rheumatoid arthritis (RA). Therefore, the present study was conducted to examine the mechanism of REG1α in RA disease progression. METHODS: Serum samples were collected from RA patients and normal healthy controls. REG1α expression was evaluated by ELISA, RT-PCR, and indirect immunofluorescence microscopy. The functions of REG1α on synovial fibroblasts with regard to apoptosis, receptor activator of NF-κB ligand (RANKL) expression, and cellar proliferation were evaluated using siRNA to inhibit the intrinsic REG1α mRNA expression. RESULTS: The serum concentrations of REG1α in RA patients were higher than in normal healthy controls. The high expression of REG1α was also observed in the synovial tissue of RA patients compared to those of osteoarthropathy patients. In addition, tumor necrosis factor-α (TNF-α) upregulated REG1α expression in the synovial fibroblasts cell line (MH7A). Inhibition of REG1α expression suppressed the induction of RANKL expression by TNF-α. Furthermore, exogenous recombinant REG1α protein inhibited apoptosis and promoted cell proliferation in MH7A cells. These effects were abolished in the REG1α-siRNA MH7A cells. CONCLUSION: The present data suggest that TNF-α induces aberrant REG1α expression and that REG1α plays an important role in aberrant cell proliferation and RANKL expression of synovial fibroblasts, ultimately resulting in pannus formation. Restoration of normal physiological REG1α expression may contribute to disease amelioration.

Quantitative peptidomic analysis by a newly developed one-step direct transfer technology without depletion of major blood proteins: its potential utility for monitoring of pathophysiological status in pregnancy-induced hypertension.

We have recently developed a new target plate (BLOTCHIP®) for MALDI-MS. An advantage of this procedure is that it does not require the lowering of protein concentrations in test samples prior to analysis. Accordingly, this new technology enables the detection of peptides present in blood samples, including those that would otherwise be adsorbed to abundant blood proteins and would thus escape detection. Using this technology, we analyzed the peripheral blood of patients with pregnancy-induced hypertension (PIH; the most common serious complication of pregnancy) to test a potential utility of the technology for monitoring of the pathophysiological status. In the present study, we found 23 characteristic peptides for PIH in the blood serum of pregnant women. Offline LC-MALDI MS/MS identified 7 of the 23 peptides as fragments derived from kininogen-1 (three peptides), fibrinogen-α, complement component C4-A/B, α-2-HS-glycoprotein and inter-α-trypsin inhibitor heavy chain H4. 2-D scatter plots with combinations of the peptides found in the present study can be grouped for pregnant women with/without PIH, which would be satisfactory reflected for their status. Additionally, the levels of most of these peptides found were significantly decreased by albumin/IgG depletion prior to BLOTCHIP® analysis in accordance with conventional proteomics procedures. These results indicated that BLOTCHIP® analysis can be applied for discovery study of PIH biomarker candidates.

Parvulin (Par14), a peptidyl-prolyl cis-trans isomerase, is a novel rRNA processing factor that evolved in the metazoan lineage.

Although parvulin (Par14/eukaryotic parvulin homolog), a peptidyl-prolyl cis-trans isomerase, is found associated with the preribosomal ribonucleoprotein (pre-rRNP) complexes, its roles in ribosome biogenesis remain undetermined. In this study, we describe a comprehensive proteomics analysis of the Par14-associated pre-rRNP complexes using LC-MS/MS and a knockdown analysis of Par14. Together with our previous results, we finally identified 115 protein components of the complexes, including 39 ribosomal proteins and 54 potential trans-acting factors whose yeast homologs are found in the pre-rRNP complexes formed at various stages of ribosome biogenesis. We give evidence that, although Par14 exists in both the phosphorylated and unphosphorylated forms in the cell, only the latter form is associated with the pre-40 S and pre-60 S ribosomal complexes. We also show that Par14 co-localizes with the nucleolar protein B23 during the interphase and in the spindle apparatus during mitosis and that actinomycin D treatment results in the exclusion of Par14 from the nucleolus. Finally we demonstrate that knockdown of Par14 mRNA decelerates the processing of pre-rRNA to 18 and 28 S rRNAs. We propose that Par14 is a component of the pre-rRNA complexes and functions as an rRNA processing factor in ribosome biogenesis. As the amino acid sequence of Par14 including that in the amino-terminal pre-rRNP binding region is conserved only in metazoan homologs, we suggest that its roles in ribosome biogenesis have evolved in the metazoan lineage.

Associations of cardiovascular risk with circulating peptides related to hypertensive disorders of pregnancy.

We previously identified seven peptides in serum that are associated with hypertensive disorders of pregnancy (HDP). However, the significance of these peptides in the general population is unknown. The aim of this study was to clarify the relationships of HDP-associated peptides with hypertension and other cardiovascular risks in adult men. We investigated the relationships of peptide levels with cardiovascular risk factors, including adiposity, blood pressure, blood lipids and glycemic status, in men (mean age: 46.4 years) who were receiving annual health checkups at their workplace. The concentrations of the abovementioned seven peptides in serum were measured simultaneously using a mass spectrometer. Among the seven peptides, only a peptide with m/z 2091 (P-2091) derived from fibrinogen-α showed a significant correlation with diastolic blood pressure (Spearman's rank correlation coefficient [r], -0.446). Another peptide with m/z 2378 (P-2378) originating from complement component 4 showed a significant positive correlation with body mass index (r, 0.273) and a significant inverse correlation with HDL cholesterol (r, -0.336). In addition, a peptide with m/z 3156 (P-3156) derived from an inter-α-trypsin inhibitor showed significant inverse correlations with body mass index (r, -0.258) and triglycerides (r, -0.334). There was no significant correlation of the levels of any of the seven peptides with hemoglobin A1c. Among the seven peptides related to HDP, P-2091, P-2378 and P-3156 were inversely associated with diastolic blood pressure, HDL cholesterol and triglycerides, respectively. Therefore, these peptides are possible biomarkers for discriminating cardiovascular risk in a general population.

Hypertensive disorders of pregnancy: Strategy to develop clinical peptide biomarkers for more accurate evaluation of the pathophysiological status of this syndrome.

Hypertensive disorders of pregnancy (HDP) is the most common and widely known as serious complication of pregnancy. As this syndrome is a major leading cause of maternal, fetal, and neonatal morbidity/mortality worldwide, many studies have sought to identify candidate molecules as potential disease biomarkers (DBMs) for use in clinical examinations. Accumulating evidence over the past 2 decades that the many proteolytic peptides appear in human humoral fluids, including peripheral blood, in association with an individual's health condition. This review provides the potential utility of peptidomic analysis for monitoring for pathophysiological status in HDP, and presents an overview of current status of peptide quantification technology. Especially, the technical limitations of the methods used for DBM discovery in the blood are discussed.

Knockdown of DEAD-box RNA helicase DDX5 selectively attenuates serine 311 phosphorylation of NF-κB p65 subunit and expression level of anti-apoptotic factor Bcl-2.

NF-κB plays a key role in the transcriptional regulation of genes involved in immunity, inflammation, cell proliferation, and oncogenesis. The NF-κB activation process includes nuclear translocation, followed by association with basal transcription machinery. These steps are tightly regulated by posttranslational modification of the proteins involved in this pathway. We recently reported that NF-κB transactivation activity is enhanced by knockdown of diacylglycerol kinase ζ (DGKζ), which belongs to an enzyme family that phosphorylates lipidic second messenger diacylglycerol to phosphatidic acid. To investigate details of the regulatory mechanism exerted by DGKζ, we identified DEAD-box RNA helicase DDX5 as a novel DGKζ-interacting protein and examined functional role of DDX5 in NF-κB transactivation activity. Here we show that DDX5 knockdown exerts no significant effect on nuclear translocation, but specifically attenuates Ser311 phosphorylation of p65 subunit. Luciferase reporter assay reveals that the NF-κB transcriptional activity is repressed in DDX5-knockdown cells. Furthermore, we found that DDX5 knockdown selectively downregulates the expression level of Bcl-2 of the NF-κB-inducible anti-apoptotic factors upon TNF-α stimulation. Considering the evidence collectively, we can infer that DGKζ-interacting multi-protein complex modulates the NF-κB transactivation activity in a negative and positive manner under conditions in which the expression level of a component of the complex is altered.

Equatorin: identification and characterization of the epitope of the MN9 antibody in the mouse.

Equatorin (MN9 antigenic molecule) is a widely distributed acrosomal protein in mammalian sperm. During the acrosome reaction, some amount of equatorin translocates to the plasma membrane, covering the equatorial region. From the results of studies of both in vitro and in vivo fertilization inhibition using the MN9 antibody, equatorin has been suggested to be involved in fusion with the oolemma. In the present study, we cloned equatorin and, using mass spectrometry and carbohydrate staining, found it to be a highly glycosylated protein. Equatorin is a sperm-specific type 1 transmembrane protein, and glycosidase treatment and recombinant protein assays verified that it is an N,O-sialoglycoprotein. In addition, the gamete interaction-related domain recognized by the MN9 antibody is posttranslationally modified. The modified domain was identified near threonine 138, which was most likely to be O-glycosylated when analyzed by amino acid substitution, dephosphorylation, and O-glycosylation inhibitor assays. Immunogold electron microscopy localized the equatorin N-terminus, where the MN9 epitope is present, on the acrosomal membrane facing the acrosomal lumen. These biochemical properties and the localization of equatorin are important for further analysis of the translocation mechanism leading to gamete interaction.

Association of human DNA helicase RecQ5beta with RNA polymerase II and its possible role in transcription.

Although RecQ5beta is a ssDNA (single-stranded DNA)-stimulated ATPase and an ATP-dependent DNA helicase with strand-annealing activities, its cellular function remains to be explored. In the present paper, we used immunopurification and MS-based analyses to show that human DNA helicase RecQ5beta is associated with at least four RNAP II (RNA polymerase II) subunits. RecQ5beta was also present in complexes immunoprecipitated using three different antibodies against the large subunit of RNAP II, or in complexes immunoprecipitated using an anti-FLAG antibody against either FLAG-RNAP II 33 kDa subunit or FLAG-Pin1. Different regions of the non-helicase domain of the RecQ5beta molecule were associated with hypophosphorylated and hyperphosphorylated forms of the RNAP II large subunit independently of DNA and RNA. RecQ5beta was also found in nuclear chromatin fractions and associated with the coding regions of the LDL (low-density lipoprotein) receptor and beta-actin genes. Knockdown of the RecQ5beta transcript increased the transcription of those genes. The results of the present study suggest that RecQ5beta has suppressive roles in events associated with RNAP II-dependent transcription.

NOP132 is required for proper nucleolus localization of DEAD-box RNA helicase DDX47.

Previously, we described a novel nucleolar protein, NOP132, which interacts with the small GTP binding protein RRAG A. To elucidate the function of NOP132 in the nucleolus, we identified proteins that interact with NOP132 using mass spectrometric methods. NOP132 associated mainly with proteins involved in ribosome biogenesis and RNA metabolism, including the DEAD-box RNA helicase protein, DDX47, whose yeast homolog is Rrp3, which has roles in pre-rRNA processing. Immunoprecipitation of FLAG-tagged DDX47 co-precipitated rRNA precursors, as well as a number of proteins that are probably involved in ribosome biogenesis, implying that DDX47 plays a role in pre-rRNA processing. Introduction of NOP132 small interfering RNAs induced a ring-like localization of DDX47 in the nucleolus, suggesting that NOP132 is required for the appropriate localization of DDX47 within the nucleolus. We propose that NOP132 functions in the recruitment of pre-rRNA processing proteins, including DDX47, to the region where rRNA is transcribed within the nucleolus.

Quantitative measurement of a candidate serum biomarker peptide derived from α2-HS-glycoprotein, and a preliminary trial of multidimensional peptide analysis in females with pregnancy-induced hypertension.

Purpose We previously attempted to develop quantitative enzyme-linked immunosorbent assay (ELISA) systems for the PDA039/044/071 peptides, potential serum disease biomarkers (DBMs) of pregnancy-induced hypertension (PIH), primarily identified by a peptidomic approach (BLOTCHIP®-mass spectrometry (MS)). However, our methodology did not extend to PDA071 (cysteinyl α2-HS-glycoprotein341-367), due to difficulty to produce a specific antibody against the peptide. The aim of the present study was to establish an alternative PDA071 quantitation system using liquid chromatography-multiple reaction monitoring (LC-MRM)/MS, to explore the potential utility of PDA071 as a DBM for PIH. Methods We tested heat/acid denaturation methods in efforts to purify serum PDA071 and developed an LC-MRM/MS method allowing for specific quantitation thereof. We measured serum PDA071 concentrations, and these results were validated including by three-dimensional (3D) plotting against PDA039 (kininogen-1439-456)/044 (kininogen-1438-456) concentrations, followed by discriminant analysis. Results PDA071 was successfully extracted from serum using a heat denaturation method. Optimum conditions for quantitation via LC-MRM/MS were developed; the assayed serum PDA071 correlated well with the BLOTCHIP® assay values. Although the PDA071 alone did not significantly differ between patients and controls, 3D plotting of PDA039/044/071 peptide concentrations and construction of a Jackknife classification matrix were satisfactory in terms of PIH diagnostic precision. Conclusions Combination analysis using both PDA071 and PDA039/044 concentrations allowed PIH diagnostic accuracy to be attained, and our method will be valuable in future pathophysiological studies of hypertensive disorders of pregnancy.

Identification of an anti-sperm auto-monoclonal antibody (Ts4)-recognized molecule in the mouse sperm acrosomal region and its inhibitory effect on fertilization in vitro.

We previously established an anti-mouse sperm auto-monoclonal antibody, Ts4, which shows immunoreactivity against several kinds of glycoproteins in the acrosomal region of epididymal spermatozoa, testicular germ cells, and early embryo, via binding to an epitope containing a common N-linked oligosaccharide (OS) chain on the molecules. In mice, we have already demonstrated that the OS chain in the epitope for Ts4 is a fucosylated agalacto-complex-type biantennary glycan carrying bisecting N-acetylglucosamine. In the testis, one of the specific OS chain-conjugated molecules is TEX101, a germ cell-marker glycoprotein, which is expressed in spermatocytes, spermatids, and testicular spermatozoa, but not in epididymal spermatozoa. In this study, we identified a Ts4-reactive glycoprotein in mouse cauda epididymal sperm. An immunoprecipitation method together with liquid chromatography-tandem mass spectrometry showed that alpha-N-acetylglucosaminidase (Naglu; a degradation enzyme of heparan sulfate) is one of the glycoproteins recognized by Ts4 in the epididymal spermatozoa. Western blot and immunohistochemical analyses revealed that mouse Naglu exists in two forms (82 and 77kDa) and is expressed in the acrosomal region and the flagellum of cauda epididymal sperm. Of the two Naglu-forms expressed in sperm, Ts4 immunoreacted against only the 82-kDa form located on the acrosomal region. The Ts4 mAb and anti-Naglu pAb negatively affected mouse fertilization in vitro. In addition, Ts4 inhibited sperm acrosome reaction induced by heparan sulfate. The Ts4-recognized fucosylated agalactobiantennary complex-type glycan with bisecting N-acetylglucosamine and Naglu on cauda epididymal spermatozoa may play a role in the process of fertilization.