西村 基

Periodontal disease is a bacterial infection that destroys the gingiva and surrounding tissues of the oral cavity. Gingival crevicular fluid (GCF) is extracted from the gingival sulcus and pocket. Analysis of biochemical markers in GCF, which predict the progression of periodontal disease, may facilitate disease diagnosis. However, no useful GCF biochemical markers with high sensitivity for detecting periodontal disease have been identified. Thus, the search for biochemical markers of periodontal disease is of continued interest in experimental and clinical periodontal disease research. Using tandem mass tag (TMT) labeling, we analyzed GCF samples from healthy subjects and patients with periodontal disease, and identified a total of 619 GCF proteins based on proteomic analysis. Of these, we focused on two proteins, matrix metalloproteinase (MMP)-9 and neutrophil gelatinase-associated lipocalin (LCN2), which are involved in the progression of periodontal disease. Western blot analysis revealed that the levels of MMP-9 and LCN2 were significantly higher in patients with periodontal disease than in healthy subjects. In addition, ELISA also detected significantly higher levels of LCN2 in patients with periodontal disease than in healthy subjects. Thus, LC-MS/MS analyses of GCF using TMT labeling led to the identification of LCN2, which may be a promising GCF biomarker for the detection of periodontal disease.

Background: Nonalcoholic fatty liver disease (NAFLD) encompasses a wide spectrum of diseases, ranging from simple steatosis to nonalcoholic steatohepatitis (NASH), which carries a significant risk of progression to cirrhosis and hepatocellular carcinoma. Since NASH is a progressive but reversible condition, it is desirable to distinguish NASH from simple steatosis, and to treat NASH patients at an early stage. To establish appropriate diagnosis and therapy, the pathological mechanisms of the disease should be elucidated; however, these have not been fully clarified for both NASH and simple steatosis. This study aims to reveal the differences between simple steatosis and NASH.Methods: This study used fatty liver Shionogi (FLS) mice as a NASH model, for comparison with dd Shionogi (DS) mice as a model of simple steatosis. Genome-wide gene expression analysis was performed using Affymetrix GeneChip Mouse Genome 430 2.0 Array, which contains 45101 probe sets for known and predicted genes. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunohistochemistry were used to investigate gene expression changes and protein localizations.Results: DNA microarray analysis of the liver transcriptomes and qRT-PCR of both types of mice revealed that LCN2, CXCL1 and CXCL9 mRNAs were overexpressed in FLS mouse livers. Immunohistochemistry showed that CXCL1 protein was mainly localized to steatotic hepatocytes. CXCL9 protein-expressing hepatocytes and sinusoidal endothelium were localized in some areas of inflammatory cell infiltration. Most interestingly, hepatocytes expressing LCN2, a kind of adipokine, were localized around almost all inflammatory cell clusters. Furthermore, there was a positive correlation between the number of LCN2-positive hepatocytes in the specimen and the number of inflammatory foci.Conclusions: Overexpression and distinct localization of LCN2, CXCL1 and CXCL9 in the liver of fatty liver Shionogi mice suggest significant roles of these proteins in the pathogenesis of NASH.

Purpose Clinical application of biomarker candidates discovered by proteomic analysis is challenging. The purpose of this study was to standardize preanalytical conditions for measurement of serum levels of fibrinogen alpha C-chain 5.9 kDa fragment (FIC 5.9) and to test the diagnostic value of this peptide for detection of early hepatic fibrosis in patients with hepatitis C virus (HCV)-related chronic hepatitis. Experimental design Serum FIC 5.9 levels were measured by a sandwich ELISA. Effects on the serum FIC 5.9 level of temperature, the time between venipuncture and serum separation, and the types of collection tubes used were examined. The diagnostic value of serum FIC 5.9 as an early indicator of hepatic fibrosis due to HCV was then assessed. Results FIC 5.9 was produced in a time- and temperature-dependent manner after venipuncture. Abnormal FIC 5.9 values were found in 89.5% of FI stage patients. Receiver operating characteristic analyses confirmed the superiority of FIC 5.9 over other conventional markers for early detection of fibrosis. Conclusions and clinical relevance The serum FIC 5.9 level may be an early indicator of hepatic fibrosis in HCV-related chronic liver diseases. This study provides an example of a pipeline from biomarker discovery by proteome analysis to assay optimization and preliminary clinical validation.

Background: Vitamin D testing is increasing worldwide. Although immunoassays are still widely used in Japan for the measurement of serum 25-hydroxyvitamin D (25OHD) as an indicator of vitamin D status, development of a simple and high-throughput MS-based method is still needed for routine use in clinical laboratories.Methods: We designed a method using a triple quadrupole mass spectrometer equipped with a two-step separation approach that used the Aria TIX-2 HPLC system in the selected reaction monitoring mode. Analytical performance of the system and effects of various preanalytical factors were tested.Results: High-throughput quantitative analysis of 25OHD3 and D2 at 15 samples/h was achieved using 25 mu l of serum/plasma. Intra- and inter-assay CVs for 25OHD3 were 5% and 7%, respectively. Limit of detection for 25OHD3 was 0.31 ng/ml. No significant effects were seen for clotting time, repeated freeze-thaw cycles, anti-coagulants and possible interfering substances. A good correlation (r(2) = 0.947) was found between the present system and the DiaSorin radioimmunoassay. Serum 25OHD3 levels in apparently healthy Japanese subjects were 25.5 +/- 9.8 ng/ml for men and 20.9 +/- 7.1 ng/ml for women.Conclusions: This high-throughput LC-MS/MS 25-OHD assay has the potential to be used as a routine clinical laboratory assay for assessing vitamin D status. (c) 2012 Elsevier B.V. All rights reserved.

For the discrimination of methicillin-resistant S. aureus (MRSA) from methicillin-susceptible S. aureus (MSSA) or coagulase-negative staphylococci (CNS), we developed a bioluminescent enzyme immunoassay (BLEIA) for detecting penicillin-binding protein 2 (PBP2) and penicillin-binding protein 2' (PBP2') using biotinylated firefly luciferase. The BLEIA was able to detect recombinant PBP2 at 50 pg/ml and recombinant PBP2' at 500 pg/ml. PBP2 and PBP2' present in the membranes of S. aureus were extracted by acid and detergent treatment. The method was able to detect PBP2 or PBP2' extracted from 10(6) colony forming units of S. aureus because of efficient extraction and the high sensitivity of luciferase. In a study of clinical isolates previously characterized as either MRSA or MSSA by antibiotic susceptibility testing, all 34 specimens identified as MRSA were both PBP2 and PBP2' positive. The 34 MSSA specimens were PBP2 positive and PBP2' negative. Moreover, the BLEIA could detect PBP2' extracted from four species of methicillin-resistant CNS, but not PBP2 extracted from four species of methicillin-resistant and methicillin-susceptible CNS. This result suggested that PBP2 could be a unique marker for discrimination of S. aureus from CNS. A BLEIA that is able to detect PBP2 and PBP2' may be useful in clinical diagnostics. (C) 2012 Elsevier B.V. All rights reserved.

Spinocerebellar ataxia type 31 (SCA31) is defined by the presence of an insertion mutation containing a TGGAA repeat within the intron of the brain-expressed, associated with NEDD4 (BEAN) gene. Detecting this mutation is conventionally done by southern blotting or DNA sequencing, but these methods are technically demanding and not easily implemented in clinical diagnosis. Here, we adapted repeat-primed PCR (RP-PCR) to develop a clinical genetic test for SCA31 using only the PCR process to detect the TGGAA repeat within the insertion mutation. Pentanucleotide RP-PCR and subsequent DNA fragment analysis demonstrated characteristic ladder peaks with a 5-bp periodicity, originating from the TGGAA repeat, in 100% of samples (n = 14) from SCA31 patients in whom the presence of the TGGAA repeat had been verified by DNA sequencing. No peaks were observed in a normal control and two non-SCA31 patients, in whom the TGGAA repeat was absent. This method is valuable for genetic diagnosis of SCA31 in clinical practice. Journal of Human Genetics (2012) 57, 807-808; doi:10.1038/jhg.2012.112; published online 20 September 2012

The protein composition of gingival crevicular fluid (GCF) may reflect the pathophysiology of periodontal diseases. A standard GCF proteomic pattern of healthy individuals would serve as a reference to identify biomarkers of periodontal diseases by proteome analyses. However, protein profiles of GCF obtained from apparently healthy individuals have not been well explored. As a step toward detection of proteomic biomarkers for periodontal diseases, we applied both gel-based and gel-free methods to analyze GCF obtained from healthy subjects as compared with supragingival saliva. To ensure optimized protein extraction from GCF, a novel protocol was developed. The proteins in GCF were extracted with high yield by urea buffer combined with ultrafiltration and the intensity of spots with supragingival saliva and GCF was compared using agarose two-dimensional electrophoresis. Eight protein spots were found to be significantly more intense in GCF. They included superoxide dismutase 1 (SOD1), apolipoprotein A-I (ApoA-I), and dermcidin (DCD). Moreover, GCF proteins from healthy subjects were broken down into small peptide fragments and then analyzed directly by LC-MS/MS analysis. A total of 327 proteins including ApoA-I, SOD1, and DCD were identified in GCF. These results may serve as reference for future proteomic studies searching for GCF biomarkers of periodontal diseases.

Mastermind (Mam) is one of the elements of Notch signaling, a system that plays a pivotal role in metazoan development. Mam proteins form transcriptionally activating complexes with the intracellular domains of Notch, which are generated in response to the ligand-receptor interaction, and CSL DNA-binding proteins. In mammals, three structurally divergent Mam isoforms (MamL1, MamL2 and MamL3) have been identified. There have also been indications that Mam interacts functionally with various other transcription factors, including the p53 tumor suppressor, beta-catenin and NF-kappa B. We have demonstrated previously that disruption of MamL1 causes partial deficiency of Notch signaling in vivo. However, MamL1-deficient mice did not recapitulate total loss of Notch signaling, suggesting that other members could compensate for the loss or that Notch signaling could proceed in the absence of Mam in certain contexts. Here, we report the generation of lines of mice null for MamL3. Although MamL3-null mice showed no apparent abnormalities, mice null for both MamL1 and MamL3 died during the early organogenic period with classic pan-Notch defects. Furthermore, expression of the lunatic fringe gene, which is strictly controlled by Notch signaling in the posterior presomitic mesoderm, was undetectable in this tissue of the double-null embryos. Neither of the single-null embryos exhibited any of these phenotypes. These various roles of the three Mam proteins could be due to their differential physical characteristics and/or their spatiotemporal distributions. These results indicate that engagement of Mam is essential for Notch signaling, and that the three Mam isoforms have distinct roles in vivo.

Serum biomarkers currently available for gastric cancers are not sufficiently sensitive and specific.We used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS) to generate comparative peptide profiles of serum samples obtained from gastric cancer patients (n = 81) and age- and sex-matched healthy controls (n = 66).Because of initial screening and further validation, we found that the intensities of a 2209 m/z MS peak were increased in the preoperative sera obtained from gastric cancer patients, and we identified this peak, a 2209 Da peptide, as a high molecular weight (HMW) kininogen fragment. Receiver operating characteristic analyses showed that the area under the curve (AUC) for the 2209 Da peptide (AUC = 0.715) was greater than those for conventional tumor markers (carcinoembryonic antigen AUC = 0.593, carbohydrate antigen 19-9 AUC = 0.527) used for the detection of stage I gastric cancers. Inverse correlations were observed between the levels of intact HMW kininogen and the 2209 Da peptide, suggesting that the upregulation of some protease activities is responsible for the overproduction of a kininogen fragment in gastric cancer patients.Serum levels of the 2209 Da peptide identified in this study have a greater diagnostic ability than those of conventional tumor markers used for the early detection of gastric cancer.

Background: The liver secretes very-low-density lipoproteins (VLDLs) and plays a key role in lipid metabolism. Plasma total triglyceride (TG) level variations have been studied in patients with hepatitis C virus (HCV)-related chronic hepatitis (CH-C). However, the results of these studies are variable. A homogenous assay protocol was recently proposed to directly measure the TG content in VLDL (VLDL-TG) and VLDL remnants.Methodology/Principal Findings: Using the assay protocol, we determined serum VLDL-TG levels in 69 fasting patients with biopsy-proven HCV-related chronic liver disease and 50 healthy subjects. Patients were classified into stages F0-F4 using the 5-point Desmet scale. Serum total TG levels in patients with non-cirrhotic (F1-F3) CH-C did not demonstrate significant differences compared with healthy subjects, but serum VLDL-TG levels did demonstrate significant differences. Mean serum VLDL-TG levels tended to decrease with disease progression from F1 to F4 (cirrhosis). Compared with healthy subjects, serum non-VLDL-TG levels significantly increased in patients with stages F2 and F3 CH-C; however, we observed no significant difference in patients with liver cirrhosis. Furthermore, the serum VLDL-TG/non-VLDL-TG ratio, when taken, demonstrated a significant decrease in patients with CH-C from the mildest stage F1 onward.Conclusions/Significance: The decrease in serum VLDL-TG levels was attenuated by increase in non-VLDL-TG levels in patients with non-cirrhotic CH-C, resulting in comparable total TG levels. Results of previous studies though variable, were confirmed to have a logical basis. The decrease in the serum VLDL-TG/non-VLDL-TG ratio as early as stage F1 demonstrated TG metabolic alterations in early stages of CH-C for the first time. The involvement of TG metabolism in CH-C pathogenesis has been established in experimental animals, while conventional TG measurements are generally considered as poor indicators of CH-C progression in clinical practice. The serum VLDL-TG/non-VLDL-TG ratio, which focuses on TG metabolic alterations, may be an early indicator of CH-C.

It is well-known that there are significant "sex differences" in many laboratory tests. After puberty onset, sex-specific organs are well developed and clinical tests concerning their functions show obvious sex differences. On the other hand, sex differences are minimal or negligible in other test items representing functions of organs common in the both sexes. But, there are some exceptions. While in childhood, sex steroids secretions from sex-specific organs are inhibited in both sexes, after puberty onset, differences in the amount of secretion of sex steroids increase, resulting, either in a direct or indirect ways, in sex differences of the blood levels of many laboratory tests. The mechanisms of sex differences are not clear in many test items. Indeed, sex difference in height is not necessarily completely unraveled. Further studies on mechanisms of sex differences in laboratory tests will reveal mysterious differences between men and women.

We have constructed a high-quality and multi-applicable cDNA library specific for mouse pancreatic islets. This is the first pancreatic islet cDNA library created using a recombination-based method, which can readily be converted into other applications including yeast two-hybrid and mammalian expression libraries. Based on sequence data of the library, we constructed a sequence database specific for mouse pancreatic islets. Among the 8882 non-redundant clones, 5799 were classified into specific functional categories using a classification system designed by the Gene Ontology Consortium, 10% of which were "molecular function unknown" genes. We also developed cDNA microarray membranes with 8108 non-redundant clones. Analyses of expression profiles of three different cell lines and of MIN6 cells with or without overexpression of transcription factor NeuroD1 established the usefulness and applicability of our microarrays. The mouse pancreatic islet cDNA library, sequence database, set of clones, and microarrays developed in this study should be useful resources for studies of pancreatic islets and related diseases including diabetes mellitus.

We have been conducting a mouse cDNA project to predict protein-coding sequences of mouse homologues of human KIAA and FLJ genes since 2001. As an extension of these projects, we herein present the entire sequences of 500 mKIAA cDNA clones and 4 novel cDNA clones that were incidentally identified during this project. We have isolated cDNA clones from the size-fractionated mouse cDNA libraries derived from 7 tissues and 3 types of cultured cells. The average size of the 504 cDNA sequences reached 4.3 kb and that of the deduced amino acid sequences from these cDNAs was 807 amino acid residues. We assigned the integrity of CDSs from the comparison with the corresponding human KIAA cDNA sequences. The comparison of mouse and human sequences revealed that two different human KIAA cDNAs are derived from Single genes. Furthermore, 3 out of 4 proteins encoded in the novel cDNA clones showed moderate sequence, similarity with human KIAA proteins, thus we could obtain new members of KIAA protein families through our mouse cDNA projects.

Transforming growth factor-beta (TGF-beta) has been implicated in the development of diabetic glomerulopathy. In order to evaluate a role of Smad3, one of the major signaling molecules downstream of TGF-beta, in the pathogenesis of diabetic glomerulopathy, Smad3-null mice were made diabetic with streptozotocin injection and analyzed 4 weeks after induction of diabetes. Electron microscopy revealed that the thickness of glomerular basement membrane (GBM) in wild-type diabetic mice was significantly higher than that in non-diabetic mice, whereas no appreciable GBM thickening was found in Smad3-null diabetic mice. Urinary albumin excretion was dramatically increased in wild-type diabetic mice, whereas Smad3-null diabetic mice did not show any overt albuminuria. Northern blotting revealed that mRNA levels of fibronectin and alpha3 chain of type IV collagen (alpha3Col4) in renal cortex of wild-type diabetic mice were approximately twice as much as those of non-diabetic mice, whereas their mRNA levels were not increased in Smad3-null diabetic mice. Real-time polymerase chain reaction (PCR) also confirmed diabetes-induced upregulation of fibronectin and alpha3Col4 in glomeruli of wild-type mice. Glomerular expression of TGF-beta1, as assessed by real-time PCR, was enhanced to a similar degree in wild-type and smad3-null diabetic mice, indicating that the observed differences between wild-type and Smad3-null mice are not attributable to difference in the expression of TGF-beta1. These data clearly demonstrate a critical role of Smad3 in the early phase of diabetic glomerulopathy. This may be due at least partly to the present findings that diabetes-induced upregulation of fibronectin and alpha3Col4 is dependent on Smad3 function. (C) 2003 Elsevier Science (USA). All rights reserved.

ATP-sensitive potassium (K-ATP) channels play important roles in many cellular functions such as hormone secretion and excitability of muscles and neurons. Classical ATP-sensitive potassium (K-ATP) channels are heteromultimeric membrane proteins comprising the pore-forming Kir6.2 subunits and the sulfonylurea receptor subunits (SUR1 or SUR2), The molecular mechanism by which hormones and neurotransmitters modulate K-ATP channels via protein kinase A (PKA) is poorly understood. We mutated the PKA consensus sequences of the human SUR1 and Kir6.2. subunits and tested their phosphorylation capacities in Xenopus oocyte homogenates and in intact cells. We identified the sites responsible for PKA phosphorylation in the C-terminus of Kir6.2 (S372) and SUR1 (S1571), Kir6.2 can be phosphorylated at its PKA phosphorylation site in intact cells after G-protein (Gs)-coupled receptor or direct PKA stimulation. While the phosphorylation of Kir6.2 increases channel activity, the phosphorylation of SUR1 contributes to the basal channel properties by decreasing burst duration, interburst interval and open probability, and also increasing the number of functional channels at the cell surface, Moreover, the effect of PKA could be mimicked by introducing negative charges in the PKA phosphorylation sites. These data demonstrate direct phosphorylation by PKA of the K-ATP channel, and may explain the mechanism by which Gs-coupled receptors stimulate channel activity. Importantly, they also describe a model of heteromultimeric ion channels in which there are functionally distinct roles of the phosphorylation of the different subunits.

A cDNA clone encoding angiopoietin-3 protein (Ang3), a novel member of the angiopoietin family, was identified. Ang3 cDNA was cloned from a human aorta cDNA library. Ang3 is a 503 amino acid protein having 45.1% and 44.7% identity with human angiopoietin-1 and human angiopoietin-2, respectively. Ang3 mRNA is expressed in lung and cultured human umbilical vein endothelial cells (HUVECs). Ang3 mRNA expression in HUVECs was slightly decreased by vascular endothelial cell growth factor treatment, suggesting that the regulation of Ang3 mRNA expression is different from that of Ang2.