[Determination of Histamine by a Rapid Fluorescamine-UPLC Method].

We have developed a rapid analysis method for determination of histamine using ultra-performance liquid chromatography and fluorescamine derivatization. Histamine was extracted from foods with trichloroacetic acid after homogenization. The supernatant obtained by centrifugation was filtered and derivatized without solid-phase extraction. The mobile phase was 10 mM phosphate-acetonitrile (75 : 25) without ion-pairing. Recovery tests of histamine from fishes, seasoning and fish products gave acceptable recovery (95.8-117.7%). This method should be useful for rapid evaluation of food poisoning outbreaks.

MZB1 in borderline resectable pancreatic cancer resected after neoadjuvant chemoradiotherapy.

BACKGROUND: A high accumulation of CD8+ tumor-infiltrating lymphocytes (TILs) induced by neoadjuvant chemoradiotherapy (NACRT) is associated with a favorable prognosis in patients with pancreatic ductal adenocarcinoma (PDAC). However, the correlation between a high accumulation of CD8+ TILs and a favorable prognosis has yet to be fully clarified. The aim of this study was to determine predictive markers of a high accumulation of CD8+ TILs, with a favorable prognosis, using proteomic analysis. MATERIALS AND METHODS: We studied 72 resected borderline resectable PDAC patients treated with NACRT between April 2009 and March 2014. Three matched pairs of high CD8+ TIL patients with a favorable prognosis and low CD8+ TIL patients with a poor prognosis were selected. Shotgun proteomics of the stroma and cancerous lesion was performed using formalin-fixed, paraffin-embedded tissue. Validation of the identified proteins was performed using immunohistochemical staining. Relationships between the identified proteins and TILs and clinical outcomes were assessed. RESULTS: Marginal zone B- and B1-cell-specific protein (MZB1) was detected in the tumor stroma. MZB1 expression was positively correlated with a high accumulation of CD8+ TILs. High stromal MZB1 expression also correlated with disease-free and overall survival. In a subgroup analysis of CD8+ expression, there was a significant association between stromal MZB1 expression and disease-free and overall survival in the high CD8+ TIL group. CONCLUSIONS: MZB1 is a potential marker of a high accumulation of CD8+ TILs in borderline resectable PDACs resected after NACRT. Combination of CD8+ TILs with MZB1 may be a new biomarker of resected cases after NACRT.

Identification of Tyrosine-Phosphorylated Proteins Upregulated during Epithelial-Mesenchymal Transition Induced with TGF-ß.

The epithelial-to-mesenchymal transition (EMT) is a unique process for the phenotypic changes of tumor cells characterized by a transition from polarized rigid epithelial cells to migrant mesenchymal cells, thus conferring the ability of tumor invasion and metastasis. A major challenge in the treatment of lung adenocarcinoma is to identify early stage patients at a high risk of recurrence or metastasis, thereby permitting the best therapeutic strategy and prognosis. In this study, we used a transforming growth factor-ß (TGF-ß)-induced EMT model to quantitatively identify protein tyrosine phosphorylation during the course of EMT in relation to malignant characteristics of lung adenocarcinoma cells. We performed relative quantitation analysis of tyrosine-phosphorylated peptides in TGF-ß-treated and -untreated lung adenocarcinoma cells and identified tyrosine-phosphorylated proteins that were upregulated in TGF-ß-treated cells. These include tensin-1 (TNS1) phosphorylated on Y1404, hepatocyte growth factor receptor (c-Met) phosphorylated on Y1234, and NT-3 growth factor receptor (TrkC) phosphorylated on Y516. We also found that these protein phosphorylation profiles were specifically observed in tissue samples of patients with poor prognostic lung adenocarcinoma. Tyrosine phosphorylations of these proteins represent possible candidates of prognostic prediction markers for lung adenocarcinoma.

Proteomic analysis of proteins related to prognosis of lung adenocarcinoma.

We attempted to identify prognosis-related proteins expressed in early resection lung adenocarcinomas that had higher metastatic potential. Early resection of lung adenocarcinoma tissues were collected from patients who experienced recurrence within 5 years after surgery; these patients are defined here as the poor prognosis group. From these samples, we prepared frozen tissue sections and then isolated cancerous areas by laser capture microdissection to allow extraction of cancer tissue-derived soluble proteins. Shotgun LC-MS/MS analysis detected and identified a total of 875 proteins in these cancer tissues. Relative quantitative analysis revealed that 17 proteins were preferentially expressed in the poor prognosis group relative to the good prognosis group, which consisted of patients who did not exhibit recurrence. Among them, 14-3-3 beta/alpha and calnexin were reported to be potentially involved in tumor recurrence and the malignant properties of lung cancer. Here immunological analyses confirmed disease-associated expression of these proteins. In a cell-culture model using A549, targeted depletion of either 14-3-3 beta/alpha or calnexin reduced proliferation, invasion, and migration, suggesting that both proteins are involved in determining the malignant properties of lung cancer that contribute to poor prognosis.

The phosphorylation of HIV-1 Gag by atypical protein kinase C facilitates viral infectivity by promoting Vpr incorporation into virions.

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) Gag is the main structural protein that mediates the assembly and release of virus-like particles (VLPs) from an infected cell membrane. The Gag C-terminal p6 domain contains short sequence motifs that facilitate virus release from the plasma membrane and mediate incorporation of the viral Vpr protein. Gag p6 has also been found to be phosphorylated during HIV-1 infection and this event may affect virus replication. However, the kinase that directs the phosphorylation of Gag p6 toward virus replication remains to be identified. In our present study, we identified this kinase using a proteomic approach and further delineate its role in HIV-1 replication. RESULTS: A proteomic approach was designed to systematically identify human protein kinases that potently interact with HIV-1 Gag and successfully identified 22 candidates. Among this panel, atypical protein kinase C (aPKC) was found to phosphorylate HIV-1 Gag p6. Subsequent LC-MS/MS and immunoblotting analysis with a phospho-specific antibody confirmed both in vitro and in vivo that aPKC phosphorylates HIV-1 Gag at Ser487. Computer-assisted structural modeling and a subsequent cell-based assay revealed that this phosphorylation event is necessary for the interaction between Gag and Vpr and results in the incorporation of Vpr into virions. Moreover, the inhibition of aPKC activity reduced the Vpr levels in virions and impaired HIV-1 infectivity of human primary macrophages. CONCLUSION: Our current results indicate for the first time that HIV-1 Gag phosphorylation on Ser487 is mediated by aPKC and that this kinase may regulate the incorporation of Vpr into HIV-1 virions and thereby supports virus infectivity. Furthermore, aPKC inhibition efficiently suppresses HIV-1 infectivity in macrophages. aPKC may therefore be an intriguing therapeutic target for HIV-1 infection.

Epidemiological characteristics of norovirus associated with sporadic gastroenteritis among children from the 2006/2007 to 2011/2012 season in Nara Prefecture, Japan.

The present study aimed to describe the epidemiological characteristics of norovirus (NoV) associated with sporadic gastroenteritis in regional populations of Nara Prefecture, Japan, from the 2006/2007 to 2011/2012 epidemic season. Fecal specimens of sporadic gastroenteritis collected between September 2006 and August 2012 in Nara Prefecture were examined for the presence of NoV by reverse transcription-polymerase chain reaction. The NoV genotype was determined by nucleotide sequence analysis. In total, 274 NoVs associated with sporadic gastroenteritis were identified. We detected 10 different NoV genotypes: GI/3, GI/4, GI/8, GII/2, GII/3, GII/4, GII/6, GII/7, GII/12, and GII/13. A high NoV detection rate of 35.9% was identified in 1-year-old children. Among the 274 NoV isolates, 142 were obtained from males and 131 were obtained from females (the source of one was unknown). The most prevalent genotype was GII/4, accounting for 117 of the 192 different NoVs identified by sequencing. More epidemiological data will be required to determine the epidemiological characteristics of NoVs in other areas of Japan.

N-Terminal methylation of proteasome subunit Rpt1 in yeast.

The 26S proteasome is a multicatalytic protease complex that degrades ubiquitinated proteins in eukaryotic cells. It consists of a proteolytic core (the 20S proteasome) as well as regulatory particles, which contain six ATPase (Rpt) subunits involved in unfolding and translocation of substrates to the catalytic chamber of the 20S proteasome. In this study, we used MS to analyze the N-terminal modifications of the yeast Rpt1 subunit, which contains the N-terminal recognition sequence for N-methyltransferase. Our results revealed that following the removal of the initiation Met residue of yeast Rpt1, the N-terminal Pro residue is either unmodified, mono-methylated, or di-methylated, and that this N-methylation has not been conserved throughout evolution. In order to gain a better understanding of the possible function(s) of the Pro-Lys (PK) sequence at positions 3 and 4 of yeast Rpt1, we generated mutant strains expressing an Rpt1 allele that lacks this sequence. The absence of the PK sequence abolished N-methylation, decreased cell growth, and increased sensitivity to stress. Our data suggest that N-methylation of Rpt1 and/or its PK sequence might be important in cell growth or stress tolerance in yeast.

Mass spectrometric identification of glycosylphosphatidylinositol-anchored peptides.

Glycosylphosphatidylinositol (GPI) anchoring is a post-translational modification widely observed among eukaryotic membrane proteins. GPI anchors are attached to proteins via the carboxy-terminus in the outer leaflet of the cell membrane, where GPI-anchored proteins (GPI-APs) perform important functions as coreceptors and enzymes. Precursors of GPI-APs (Pre-GPI-APs) contain a C-terminal hydrophobic sequence that is involved in cleavage of the signal sequence from the protein and addition of the GPI anchor by the transamidase complex. In order to confirm that a given protein contains a GPI anchor, it is essential to identify the C-terminal peptide containing the GPI-anchor modification site (ω-site). Previously, efficient identification of GPI-anchored C-terminal peptides by mass spectrometry has been difficult, in part because of complex structure of the GPI-anchor moiety. We developed a method to experimentally identify GPI-APs and their ω-sites. In this method, a part of GPI-anchor moieties are removed from GPI-anchored peptides using phosphatidylinositol-specific phospholipase C (PI-PLC) and aqueous hydrogen fluoride (HF), and peptide sequence is then determined by mass spectrometry. Using this method, we successfully identified 10 GPI-APs and 12 ω-sites in the cultured ovarian adenocarcinoma cells, demonstrating that this method is useful for identifying efficiently GPI-APs.

A distinct role for Pin1 in the induction and maintenance of pluripotency.

The prominent characteristics of pluripotent stem cells are their unique capacity to self-renew and pluripotency. Although pluripotent stem cell proliferation is maintained by specific intracellular phosphorylation signaling events, it has not been well characterized how the resulting phosphorylated proteins are subsequently regulated. We here report that the peptidylprolyl isomerase Pin1 is indispensable for the self-renewal and maintenance of pluripotent stem cells via the regulation of phosphorylated Oct4 and other substrates. Pin1 expression was found to be up-regulated upon the induction of induced pluripotent stem (iPS) cells, and the forced expression of Pin1 with defined reprogramming factors was observed to further enhance the frequency of iPS cell generation. The inhibition of Pin1 activity significantly suppressed colony formation and induced the aberrant differentiation of human iPS cells as well as murine ES cells. We further found that Pin1 interacts with the phosphorylated Ser(12)-Pro motif of Oct4 and that this in turn facilitates the stability and transcriptional activity functions of Oct4. Our current findings thus uncover an atypical role for Pin1 as a putative regulator of the induction and maintenance of pluripotency via the control of phosphorylation signaling. These data suggest that the manipulation of Pin1 function could be a potential strategy for the stable induction and proliferation of human iPS cells.

[Clinical review of influenza (H1N1) 2009 detected in 2009-2010 and 2010-2011 flu season in Nara Prefecture].

This study is based on clinical information on 894 subjects diagnosed with influenza (H1N1) 2009 in Nara Prefecture from June 15, 2009, to March 4, 2010, and from July 9, 2010, to March 6, 2011. Clinical data for 2009-2010 and 2010-2011 was compared. Results showed that 43% of 2009-2010 subjects were 0-9 years old and 38% were 10-19 years old. They also showed that 25% of 2010-2011 0-9 years old, 20% 10-19 years old, 20% 20-29 years old and 16% 30-39 years old. Both seasons showed a high percentage of subjects 0-9 years old. Numbers of subjects aged 20-39 years old increased in 2010-2011. Results thus suggest that an age shift occurred in subjects, in Nara Prefecture. The most frequent symptom was fever, e.g., 38 degrees C, in 88%. Upper airway inflammation was seen in 68%, lower airway inflammation in 20% and gastroenteritis in 6%. Lower airway inflammation decreased from 20% in 2009-2010 to 7% in 2010-2011. Neuraminidase inhibitor was prescribed for 408 (46%) subjects, oseltamivir for 262 (63%), zanamivir for 120 (29%), peramivir for 10 (2.4%), and laninamivir for 12 (2.9%). Two neuraminidase inhibitors were prescribed for 11 subjects. Oseltamivir prescription rates were lower among subjects 10-19 years old, following guidelines for the use of antiinfluenza drugs.

Co- and post-translational modifications of the 26S proteasome in yeast.

The yeast (Saccharomyces cerevisiae) 26S proteasome consists of the 19S regulatory particle (19S RP) and 20S proteasome subunits. We detected comprehensively co- and post-translational modifications of these subunits using proteomic techniques. First, using MS/MS, we investigated the N-terminal modifications of three 19S RP subunits, Rpt1, Rpn13, and Rpn15, which had been unclear, and found that the N-terminus of Rpt1 is not modified, whereas that of Rpn13 and Rpn15 is acetylated. Second, we identified a total of 33 Ser/Thr phosphorylation sites in 15 subunits of the proteasome. The data obtained by us and other groups reveal that the 26S proteasome contains at least 88 phospho-amino acids including 63 pSer, 23 pThr, and 2 pTyr residues. Dephosphorylation treatment of the 19S RP with lambda phosphatase resulted in a 30% decrease in ATPase activity, demonstrating that phosphorylation is involved in the regulation of ATPase activity in the proteasome. Third, we tried to detect glycosylated subunits of the 26S proteasome. However, we identified neither N- and O-linked oligosaccharides nor O-linked beta-N-acetylglucosamine in the 19S RP and 20S proteasome subunits. To date, a total of 110 co- and post-translational modifications, including N(alpha)-acetylation, N(alpha)-myristoylation, and phosphorylation, in the yeast 26S proteasome have been identified.

The tumour suppressor APC promotes HIV-1 assembly via interaction with Gag precursor protein.

Diverse cellular proteins and RNAs are tightly regulated in their subcellular localization to exert their local function. Here we report that the tumour suppressor adenomatous polyposis coli protein (APC) directs the localization and assembly of human immunodeficiency virus (HIV)-1 Gag polyprotein at distinct membrane components to enable the efficient production and spread of infectious viral particles. A proteomic analysis and subsequent biomolecular interaction assay reveals that the carboxyl terminus of APC interacts with the matrix region of Gag. Ectopic expression of APC, but not its familial adenomatous polyposis-related truncation mutant, prominently enhances HIV-1 production. Conversely, the depletion of APC leads to a significant decrease in membrane targeting of viral components, resulting in the severe loss of production of infectious virions. Furthermore, APC promotes the directional assembly of viral components at virological synapses, thereby facilitating cell-to-cell viral transmission. These findings reveal an unexpected role of APC in the directional spread of HIV-1.

Identification of candidate diagnostic serum biomarkers for Kawasaki disease using proteomic analysis.

Kawasaki disease (KD) is a systemic vasculitis and childhood febrile disease that can lead to cardiovascular complications. The diagnosis of KD depends on its clinical features, and thus it is sometimes difficult to make a definitive diagnosis. In order to identify diagnostic serum biomarkers for KD, we explored serum KD-related proteins, which differentially expressed during the acute and recovery phases of two patients by mass spectrometry (MS). We identified a total of 1,879 proteins by MS-based proteomic analysis. The levels of three of these proteins, namely lipopolysaccharide-binding protein (LBP), leucine-rich alpha-2-glycoprotein (LRG1), and angiotensinogen (AGT), were higher in acute phase patients. In contrast, the level of retinol-binding protein 4 (RBP4) was decreased. To confirm the usefulness of these proteins as biomarkers, we analyzed a total of 270 samples, including those collected from 55 patients with acute phase KD, by using western blot analysis and microarray enzyme-linked immunosorbent assays (ELISAs). Over the course of this experiment, we determined that the expression level of these proteins changes specifically in the acute phase of KD, rather than the recovery phase of KD or other febrile illness. Thus, LRG1 could be used as biomarkers to facilitate KD diagnosis based on clinical features.

Development of a routine laboratory direct detection system of staphylococcal enterotoxin genes.

A novel direct detection system has been developed for eight staphylococcal enterotoxin (SE)-encoding genes (sea, seb, sec, sed, see, seg, seh and sei) in milk. Specific detection by real-time PCR was successful for all SE-encoding genes in the reference strains. Furthermore, a novel DNA-preparation method with good reproducibility [coefficients of variation 0.31, 0.99 and 1.21 % at 10(6), 10(4) and 10(2) c.f.u. (ml milk sample)(-1), respectively] was developed to overcome PCR inhibition in the milk samples. The combination of this DNA-preparation method and real-time PCR resulted in high sensitivity [between 1.1 x 10(2) and 1.0 x 10(4) c.f.u. (ml milk sample)(-1)] and allowed the completion of the entire procedure within 4 h. Results of an evaluation of this method for the detection of SE-encoding genes using known outbreak milk samples produced results showing good correspondence with the reversed passive latex agglutination assay. In addition, this newly developed system can be applied to clinical samples such as faeces and vomit. Consequently, the system should be useful in the routine direct detection of SE-encoding genes in food-borne-poisoning samples.

Relationship between phosphorylation of sperm-specific antigen and prognosis of lung adenocarcinoma.

Lung cancer is generally considered as a highly malignant cancer. A major challenge for the management of lung adenocarcinoma patients is to predict the clinical course of the disease after resection. We analyzed the different levels of phosphorylation of proteins in lung adenocarcinoma tissues between a poor prognosis (PP) group, in which six patients exhibited recurrence within five years after surgery, and a good prognosis (GP) group, in which seven patients did not exhibit recurrence within five years after surgery. We found that phosphorylation at Ser92 of the sperm-specific antigen 2 (SSFA2) [phospho-SSFA2(pS92)] was stimulated in the PP group. Using samples from a total of 46 patients, we investigated the utility of phospho-SSFA2(pS92) to discriminate patients of GP and PP groups, with multiple reaction monitoring (MRM) mass spectrometry. Consequently, we confirmed that the PP group had significantly elevated phospho-SSFA2(pS92) levels. Additionally, no expression of SSFA2 recognized in the normal lung tissues. From these results, we demonstrate that phospho-SSFA2 (pS92) is related to the prognosis of early resected lung adenocarcinomas. Therefore, we suggest that phosphorylation of this protein indicates its role as a potential biomarker and new therapeutic target. BIOLOGICAL SIGNIFICANCE: Lung adenocarcinoma patients often experience a high rate of recurrence after surgery. It is important to discover biomarkers for prognostic prediction and therapeutic targets for treatment of early-stage lung adenocarcinoma. In this study, using tissue samples obtained from patients with lung adenocarcinoma that had been stored for five years at -80°C, we identified 13 unique phosphorylated peptides, which were differentially expressed between poor and good prognosis groups. We confirmed that phosphorylation at Ser92 of the sperm-specific antigen 2 (SSFA2)[phospho-SSFA2 (pS92)], was related to poor prognosis. Our study demonstrates that prognostic prediction of early-stage lung adenocarcinoma is possible, and suggests new therapeutic targets for its treatment.

A Novel Role for Flotillin-Containing Lipid Rafts in Negative-Feedback Regulation of Thyroid-Specific Gene Expression by Thyroglobulin.

BACKGROUND: Thyroglobulin (Tg) stored in thyroid follicles regulates follicular function in thyroid hormone (TH) synthesis by suppressing thyroid-specific gene expression in a concentration-dependent manner. Thus, Tg is an intrinsic negative-feedback regulator that can restrain the effect of thyrotropin (TSH) in the follicle. However, the underlying mechanisms by which Tg exerts its prominent autoregulatory effect following recognition by thyrocytes remains unclear. METHODS: In order to identify potential proteins that recognize and interact with Tg, mass spectrometry was used to analyze immunoprecipitated Tg-bound proteins derived from Tg-treated rat thyroid FRTL-5 cells. RESULTS: Flotillin 1 and flotillin 2, two homologs that are integral membrane proteins in lipid rafts, were identified as novel Tg-binding proteins with high confidence. Further studies revealed that flotillins physically interact with endocytosed Tg, and together these proteins redistribute from the cell membrane to cytoplasmic vesicles. Treatment with the lipid raft disrupter methyl-ß-cyclodextrin abolished both the endocytosis and the negative-feedback effect of Tg on thyroid-specific gene expression. Meanwhile, siRNA-mediated knockdown of flotillin 1 or flotillin 2 also significantly inhibited Tg effects on gene expression. CONCLUSION: Together these results indicate that flotillin-containing lipid rafts are essential for follicular Tg to be recognized by thyrocytes and exert its negative-feedback effects in the thyroid.

Analysis of gene expression during staurosporine-induced neuronal differentiation of human prostate cancer cells.

Staurosporine induces neuronal differentiation and suppresses malignancy of human prostate cancer TSU-Pr1 cells. To investigate the mechanism underlying neuronal differentiation and suppression of malignancy, we used cDNA microarrays to examine gene expression profiles in TSU-Pr1 cells treated with staurosporine. mRNAs isolated from untreated and staurosporine-treated TSU-Pr1 cells were hybridised to microarrays consisting of approximately 9100 genes. Changes in gene expression were verified by Northern blot analysis. Staurosporine-responsive genes were involved in a variety of cellular functions including growth regulation, differentiation, replication, DNA repair, G2/M transition and inhibition of apoptosis. Interestingly, expression of genes associated with cell proliferation and malignancy, such as Cyr61 and CTGF, was reduced. Expression of CD73/NT5E, which is involved in neuronal differentiation, was increased. In the present study, we identified various staurosporine-responsive genes in TSU-Pr1 cells. Further studies of the roles of these genes may clarify the mechanisms underlying neuronal differentiation and inhibition of malignancy by staurosporine and identify better approaches for the prevention and treatment of prostate cancer.

Oseltamivir-resistant seasonal A(H1N1) and A(H1N1)pdm09 influenza viruses from the 2007/2008 to 2012/2013 season in Nara Prefecture, Japan.

We examined the incidence of oseltamivir-resistant seasonal A(H1N1) and A(H1N1)pdm09 influenza viruses from the 2007/2008 to 2012/2013 season in Nara Prefecture, Japan. To detect the oseltamivir resistance marker in neuraminidase (NA), 365 influenza viruses (60 seasonal A(H1N1) and 305 A(H1N1)pdm09) were sequenced. The H275Y mutation in the NA gene, which confers resistance to oseltamivir, was identified in 93.8% (30/32) of seasonal A(H1N1) viruses that were circulating during the 2008/2009 season. Moreover, the detection rate of oseltamivir-resistant A(H1N1)pdm09 viruses was 4.1% (3/74) and 2.8% (5/180) in the 2009/2010 and 2010/2011 season, respectively. Four cases of oseltamivir-resistant A(H1N1)pdm09 virus infection occurred in the same hematology ward during the 2010/2011 season. Our data show a low frequency of oseltamivir-resistant A(H1N1)pdm09 virus in Nara Prefecture but suggested the possibility of human-to-human transmission of this virus.

Wheat germ cell-free system-based production of hemagglutinin-neuraminidase glycoprotein of human parainfluenza virus type 3 for generation and characterization of monoclonal antibody.

Human parainfluenza virus 3 (HPIV3) commonly causes respiratory disorders in infants and young children. Monoclonal antibodies (MAbs) have been produced to several components of HPIV3 and commercially available. However, the utility of these antibodies for several immunological and proteomic assays for understanding the nature of HPIV3 infection remain to be characterized. Herein, we report the development and characterization of MAbs against hemagglutinin-neuraminidase (HN) of HPIV3. A recombinant full-length HPIV3-HN was successfully synthesized using the wheat-germ cell-free protein production system. After immunization and cell fusion, 36 mouse hybridomas producing MAbs to HPIV3-HN were established. The MAbs obtained were fully characterized using ELISA, immunoblotting, and immunofluorescent analyses. Of the MAbs tested, single clone was found to be applicable in both flow cytometry and immunoprecipitation procedures. By utilizing the antibody, we identified HPIV3-HN binding host proteins via immunoprecipitation-based mass spectrometry analysis. The newly-developed MAbs could thus be a valuable tool for the study of HPIV3 infection as well as the several diagnostic tests of this virus.

[Validation study of a method for multiresidue analysis of pesticides in cereals and pulses using supercritical fluid extraction].

Supercritical fluid extraction (SFE) was applied to extraction of pesticides from cereals and pulses. Residues were extracted from homogenized samples mixed with water-absorbent polymer and supercritical carbon dioxide in a stainless steel tube, followed by elution with acetonitrile. Co-extractives were removed by means of mini-column clean-up. Measurement was performed by GC-MS/MS. Calibration was achieved by preparing matrix-matched calibration standards to counteract matrix effects. With the Japanese method validation guideline for pesticide residues as a reference, the method was assessed in 5 agricultural products spiked with 334 pesticides at 0.01 and 0.1 µg/g. Compounds at each level were extracted from 2 samples on 5 separate days. The trueness of the method for 137 pesticides in all samples was 70-120%, and the repeatability and within-run reproducibility were also consistent with the guideline. The trueness of the method for the other 101 pesticides was in the range of 50-70%, though the repeatability and within-run reproducibility were satisfactory. This method is available as a multiresidue analysis method for cereals and pulses.