Postmortem Identification of Genetic Variations Associated with Sudden Unexpected Death in Young People.

Sudden unexpected death in the young (SUDY) is a traumatic occurrence for their family; however, information on the genetic variations associated with the condition is currently lacking. It is important to carry out postmortem genetic analyses in cases of sudden death to provide information for relatives and to allow appropriate genetic counselling and clinical follow-up. This study aimed to investigate the genetic variations associated with the occurrence of SUDY in Japan, using next-generation sequencing (NGS). The study included 18 cases of SUDY (16 males, 2 females; age 15-47 years) who underwent autopsy, including NGS DNA sequencing for molecular analysis. A total of 168 genes were selected from the sequencing panel and filtered, resulting in the identification of 60 variants in cardiac disease-related genes. Many of the cases had several of these genetic variants and some cases had a cardiac phenotype. The identification of genetic variants using NGS provides important information regarding the pathogenicity of sudden death.

Molecular autopsy for sudden death in Japan.

Japan has various death investigation systems; however, external examinations, postmortem computed tomography, macroscopic examinations, and microscopic examinations are performed regardless of the system used. These examinations can reveal morphological abnormalities, whereas the cause of death in cases with non-morphological abnormalities can be detected through additional examinations. Molecular autopsy and postmortem genetic analyses are important additional examinations. They are capable of detecting inherited arrhythmias or inherited metabolic diseases, which are representative non-morphological disorders that cause sudden death, especially in infants and young people. In this review, we introduce molecular autopsy reports from Japan and describe our experience with representative cases. The relationships between drug-related deaths and genetic variants are also reviewed. Based on the presented information, molecular autopsy is expected to be used as routine examinations in death investigations because they can provide information to save new lives.

Screening for Novel Type 2 Ryanodine Receptor Inhibitors by Endoplasmic Reticulum Ca2+ Monitoring.

Type 2 ryanodine receptor (RyR2) is a Ca2+ release channel on the endoplasmic (ER)/sarcoplasmic reticulum that plays a central role in the excitation-contraction coupling in the heart. Hyperactivity of RyR2 has been linked to ventricular arrhythmias in patients with catecholaminergic polymorphic ventricular tachycardia and heart failure, where spontaneous Ca2+ release via hyperactivated RyR2 depolarizes diastolic membrane potential to induce triggered activity. In such cases, drugs that suppress RyR2 activity are expected to prevent the arrhythmias, but there is no clinically available RyR2 inhibitors at present. In this study, we searched for RyR2 inhibitors from a well-characterized compound library using a recently developed ER Ca2+-based assay, where the inhibition of RyR2 activity was detected by the increase in ER Ca2+ signals from R-CEPIA1er, a genetically encoded ER Ca2+ indicator, in RyR2-expressing HEK293 cells. By screening 1535 compounds in the library, we identified three compounds (chloroxylenol, methyl orsellinate, and riluzole) that greatly increased the ER Ca2+ signal. All of the three compounds suppressed spontaneous Ca2+ oscillations in RyR2-expressing HEK293 cells and correspondingly reduced the Ca2+-dependent [3H]ryanodine binding activity. In cardiomyocytes from RyR2-mutant mice, the three compounds effectively suppressed abnormal Ca2+ waves without substantial effects on the action-potential-induced Ca2+ transients. These results confirm that ER Ca2+-based screening is useful for identifying modulators of ER Ca2+ release channels and suggest that RyR2 inhibitors have potential to be developed as a new category of antiarrhythmic drugs. SIGNIFICANCE STATEMENT: We successfully identified three compounds having RyR2 inhibitory action from a well-characterized compound library using an endoplasmic reticulum Ca2+-based assay, and demonstrated that these compounds suppressed arrhythmogenic Ca2+ wave generation without substantially affecting physiological action-potential induced Ca2+ transients in cardiomyocytes. This study will facilitate the development of RyR2-specific inhibitors as a potential new class of drugs for life-threatening arrhythmias induced by hyperactivation of RyR2.

Circadian Clock in Arabidopsis thaliana Determines Flower Opening Time Early in the Morning and Dominantly Closes Early in the Afternoon.

Many plant species exhibit diurnal flower opening and closing, which is an adaptation influenced by the lifestyle of pollinators and herbivores. However, it remains unclear how these temporal floral movements are modulated. To clarify the role of the circadian clock in flower movement, we examined temporal floral movements in Arabidopsis thaliana. Wild-type (accessions; Col-0, Ler-0 and Ws-4) flowers opened between 0.7 and 1.4 h in a 16-h light period and closed between 7.5 and 8.3 h in a diurnal light period. In the arrhythmic mutants pcl1-1 and prr975, the former flowers closed slowly and imperfectly and the latter ones never closed. Under continuous light conditions, new flowers emerged and opened within a 23-26 h window in the wild-type, but the flowers in pcl1-1 and prr975 developed straight petals, whose curvatures were extremely small. Anti-phasic circadian gene expression of CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), LATE ELONGATED HYPOCOTYLE (LHY) and TIMING OF CAB EXPRESSION 1 (TOC1) occurred in wild-type flowers, but non-rhythmic expression was observed in pcl1-1 and prr975 mutants. Focusing on excised petals, bioluminescence monitoring revealed rhythmic promoter activities of genes expressed (CCA1, LHY and PHYTOCLOCK 1/LUX ARRHYTHMO, PCL1/LUX) in the morning and evening. These results suggest that the clock induces flower opening redundantly with unknown light-sensing pathways. By contrast, flower closing is completely dependent on clock control. These findings will lead to further exploration of the molecular mechanisms and evolutionary diversity of timing in flower opening and closing.

Sudden Unexpected Death of Infantile Dilated Cardiomyopathy with JPH2 and PKD1 Gene Variants.

A Japanese girl with polycystic kidney disease (PKD) developed normally, but at 8 months of age, she was hospitalized for acute onset dyspnea. On the day after admission to hospital, her general condition suddenly became worse. An echocardiogram showed left ventricular dilatation with thin walls, severe mitral valve regurgitation, and a reduced ejection fraction. She died of acute cardiac failure 3 hours after the sudden change. Postmortem analysis with light microscopy showed disarray of cardiomyocytes without obvious infiltration of lymphocytes, and we diagnosed her heart failure as idiopathic dilated cardiomyopathy (DCM). Clinical exome sequencing showed compound heterozygous variants in JPH2 (p.T237A/p.I414L) and a heterozygous nonsense mutation in PKD1 (p.Q4193*). To date, several variants in the JPH2 gene have been reported to be pathogenic for adult-onset hypertrophic cardiomyopathy or DCM in an autosomal dominant manner and infantile-onset DCM in an autosomal recessive manner. Additionally, autosomal dominant polycystic kidney disease is a systemic disease associated with several extrarenal manifestations, such as cardiomyopathy. Here we report a sudden infant death case of DCM and discuss the genetic variants of DCM and PKD.

Receptor for advanced glycation end products (RAGE)-mediated cytotoxicity of 3-hydroxypyridinium derivatives.

Advanced glycation end products (AGEs) formed from glyceraldehyde (Gcer) and glycolaldehyde (Gcol) are involved in the pathogenesis of diabetic complications, via interactions with a receptor for AGEs (RAGE). In this study, we aimed to elucidate the RAGE-binding structure in Gcer and Gcol-derived AGEs and identify the minimal moiety recognized by RAGE. Among Gcer and Gcol-derived AGEs, GLAP (glyceraldehyde-derived pyridinium) and GA-pyridine elicited toxicity in PC12 neuronal cells. The toxic effects of GLAP and GA-pyridine were suppressed in the presence of anti-RAGE antibody or the soluble form of RAGE protein. Furthermore, the cytotoxicity test using GLAP analog compounds indicated that the 3-hydroxypyridinium (3-HP) structure is sufficient for RAGE-dependent toxicity. Surface plasmon resonance analysis showed that 3-HP derivatives directly interact with RAGE. These results indicate that GLAP and GA-pyridine are RAGE-binding epitopes, and that 3-HP, a common moiety of GLAP and GA-pyridine, is essential for the interaction with RAGE.

Thermithiobacillus plumbiphilus sp. nov., a sulfur-oxidizing bacterium isolated from lead sulfide.

A novel sulfur oxidizer, strain wk12T, was isolated from an industrially synthesized lead (II) sulfide. The G+C content of the genomic DNA was around 58.5 mol%. The major components in the cellular fatty acid profile were summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), C16 : 0 and summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c). The strain oxidized lead sulfide, thiosulfate and tetrathionate as electron donors to support autotrophic growth. Cells of strain wk12T were motile, rod-shaped (0.5-1.0 × 0.7-2.2 µm), and Gram-stain-negative. For growth, the temperature range was 5-37 °C, and optimum growth was observed at 28-32 °C. The pH range for growth was 5.8-8.7, with optimum growth at pH 6.4-7.1. Optimum growth of the isolate was observed in medium without NaCl, and no growth was observed in the medium containing 0.5 M or more NaCl. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolate belongs to the class Acidithiobacillia. The closest relative with a validly published name was Thermithiobacillus tepidarius DSM 3134T, with a 16S rRNA gene sequence similarity of 96 %. On the basis of phylogenetic and phenotypic properties, strain wk12T represents a novel species of the genus Thermithiobacillus, for which the name Thermithiobacillus plumbiphilus sp. nov. is proposed. The type strain is wk12T ( = NBRC 111508T = DSM 101799T).

Cooperative function of PLDδ and PLDα1 in abscisic acid-induced stomatal closure in Arabidopsis.

Phospholipase D (PLD) is involved in responses to abiotic stress and abscisic acid (ABA) signaling. To investigate the roles of two Arabidopsis (Arabidopsis thaliana) PLDs, PLDα1 and PLDδ, in ABA signaling in guard cells, we analyzed ABA responses in guard cells using Arabidopsis wild type, pldα1 and pldδ single mutants, and a pldα1 pldδ double mutant. ABA-induced stomatal closure was suppressed in the pldα1 pldδ double mutant but not in the pld single mutants. The pldα1 and pldδ mutations reduced ABA-induced phosphatidic acid production in epidermal tissues. Expression of either PLDα1 or PLDδ complemented the double mutant stomatal phenotype. ABA-induced stomatal closure in both pldα1 and pldδ single mutants was inhibited by a PLD inhibitor (1-butanol ), suggesting that both PLDα1 and PLDδ function in ABA-induced stomatal closure. During ABA-induced stomatal closure, wild-type guard cells accumulate reactive oxygen species and nitric oxide and undergo cytosolic alkalization, but these changes are reduced in guard cells of the pldα1 pldδ double mutant. Inward-rectifying K(+) channel currents of guard cells were inhibited by ABA in the wild type but not in the pldα1 pldδ double mutant. ABA inhibited stomatal opening in the wild type and the pldδ mutant but not in the pldα1 mutant. In wild-type rosette leaves, ABA significantly increased PLDδ transcript levels but did not change PLDα1 transcript levels. Furthermore, the pldα1 and pldδ mutations mitigated ABA inhibition of seed germination. These results suggest that PLDα1 and PLDδ cooperate in ABA signaling in guard cells but that their functions do not completely overlap.

A potent and selective cis-amide inhibitor of ryanodine receptor 2 as a candidate for cardiac arrhythmia treatment.

Ryanodine receptor 2 (RyR2) is a Ca2+ release channel mainly located on the sarcoplasmic reticulum (SR) membrane of heart muscle cells and regulates the concentration of Ca2+ in the cytosol. RyR2 overactivation causes potentially lethal cardiac arrhythmias, but no specific inhibitor is yet available. Herein we developed the first highly potent and selective RyR2 inhibitor, TMDJ-035, containing 3,5-difluoro substituents on the A ring and a 4-fluoro substituent on the B ring, based on a comprehensive structure-activity relationship (SAR) study of tetrazole compound 1. The SAR study also showed that the amide conformation is critical for inhibitory potency. Single-crystal X-ray diffraction analysis and variable-temperature 1H NMR revealed that TMDJ-035 strongly favors cis-amide configuration, while the inactive analogue TMDJ-011 with a secondary amide takes trans-amide configuration. Examination of the selectivity among RyRs indicated that TMDJ-035 displayed high selectivity for RyR2. TMDJ-035 suppressed abnormal Ca2+ waves and transients in isolated cardiomyocytes from RyR2-mutated mice. It appears to be a promising candidate drug for treating cardiac arrhythmias due to RyR2 overactivation, as well as a tool for studying the mechanism and dynamics of RyR2 channel gating.

Safety and efficacy of epoprostenol therapy in pulmonary veno-occlusive disease and pulmonary capillary hemangiomatosis.

BACKGROUND: Pulmonary veno-occlusive disease (PVOD) and pulmonary capillary hemangiomatosis (PCH) are rare causes of pulmonary hypertension. There is no proven medical therapy to treat these diseases, and lung transplantation is thought to be the only cure. Administration of vasodilators including epoprostenol sometimes causes massive pulmonary edema and could be fatal in these patients. METHODS AND RESULTS: Eight patients were treated with epoprostenol for 387.3±116.3 days (range, 102-1,063 days), who were finally diagnosed with PVOD or PCH by pathological examination. The maximum dose of epoprostenol given was 55.3±10.7 ng·kg(-1)·min(-1) (range, 21.0-110.5 ng·kg(-1)·min(-1)). With careful management, epoprostenol therapy significantly improved the 6-min walk distance (97.5±39.2 to 329.4±34.6 m, P<0.001) and plasma brain natriuretic peptide levels (381.3±136.8 to 55.2±14.4 pg/ml, P<0.05). The cardiac index significantly increased from 2.1±0.1 to 2.9±0.3 L·min(-1)·m(-2) (P<0.05). However, pulmonary artery pressure and pulmonary vascular resistance were not significantly reduced. For 4 patients, epoprostenol therapy acted as a bridge to lung transplantation. For the other patients who had no chance to undergo lung transplantation, epoprostenol therapy was applied for 528.0±216.6 days and the maximum dose was 63.9±19.0 ng·kg(-1)·min(-1). CONCLUSIONS: This study data suggest that cautious application of epoprostenol can be considered as a therapeutic option in patients with PVOD and PCH.

PROS1 variant in sudden death case of pulmonary embolism caused by calcification in the inferior vena cava: The importance of postmortem genetic analysis.

A Japanese man in his 30s died suddenly. Postmortem computed tomography and autopsy revealed a pulmonary embolism from an organizing thrombus in the inferior vena cava as the cause of death. Genomic analysis of congenital thrombophilia-related genes (i.e., SERPINC1, PROC, PROS1, F2, F5, PLG, and MTHFR) revealed a heterozygous variant of PROS1 (p.A139V), which has been reported in patients with congenital protein S deficiency. After a genetic conference that included forensic pathologists, molecular scientists, genetic researchers, genetic clinicians, and clinical physicians, the results of the genetic analysis were explained to the family. Biochemical analyses of protein S (PS) activity and total PS antigen levels were performed with samples from the deceased's family and genetic analysis was not performed until clinical symptoms appear. Herein we demonstrate the importance of genetic background in cases of a sudden death due to pulmonary embolism.

I536T variant of RBM20 affects splicing of cardiac structural proteins that are causative for developing dilated cardiomyopathy.

RBM20 is one of the genes predisposing to dilated cardiomyopathy (DCM). Variants in the RS domain have been reported in many DCM patients, but the pathogenicity of variants within the RNA-recognition motif remains unknown. Two human patients with the I536T-RBM20 variant without an apparent DCM phenotype were identified in sudden death cohorts. A splicing reporter assay was performed, and an I538T knock-in mouse model (Rbm20I538T) was generated to determine the significance of this variant. The reporter assay demonstrated that the human I536T variant affected the TTN splicing pattern compared to wild-type. In the mouse experiments, Rbm20I538T mice showed different splicing patterns in Ttn, Ldb3, Camk2d, and Ryr2. The expressions of Casq1, Mybpc2, and Myot were upregulated in Rbm20I538T mice, but Rbm20I538T mice showed neither DCM nor cardiac dysfunction on histopathological examination and ultrasound echocardiography. The I536T-RBM20 (I538T-Rbm20) variant changes gene splicing and affects gene expression, but the splicing and expression changes in Ttn and Ca handling genes such as Casq1, Camk2d, and Ryr2 do not cause DCM morphology in the mouse model. KEY MESSAGES: • Two human patients with the I536T-RBM20 variant without a DCM phenotype were identified. • A splicing reporter assay demonstrated that the variant affected the TTN splicing. • Rbm20I538T mice showed neither DCM nor cardiac dysfunction. • Rbm20I538T mice showed different splicing patterns and the gene expressions.

Human protein factory for converting the transcriptome into an in vitro-expressed proteome,.

Appropriate resources and expression technology necessary for human proteomics on a whole-proteome scale are being developed. We prepared a foundation for simple and efficient production of human proteins using the versatile Gateway vector system. We generated 33,275 human Gateway entry clones for protein synthesis, developed mRNA expression protocols for them and improved the wheat germ cell-free protein synthesis system. We applied this protein expression system to the in vitro expression of 13,364 human proteins and assessed their biological activity in two functional categories. Of the 75 tested phosphatases, 58 (77%) showed biological activity. Several cytokines containing disulfide bonds were produced in an active form in a nonreducing wheat germ cell-free expression system. We also manufactured protein microarrays by direct printing of unpurified in vitro-synthesized proteins and demonstrated their utility. Our 'human protein factory' infrastructure includes the resources and expression technology for in vitro proteome research.

Comparative analysis of human SRC-family kinase substrate specificity in vitro.

Src family kinases (SFKs) are the earliest known family of tyrosine kinases and are widely thought to play essential roles in cellular signal transduction. Although numerous functional analyses have been performed, no study has analyzed the specificity of all SFKs on an equal platform. To gain a better understanding of SFK phosphorylation, we designed a high-throughput in vitro kinase assay on the subproteome scale using surface plasmon resonance. We reacted each of the 11 human SFKs with 519 substrate proteins, and significant phosphorylation was detected in 33.6% (1921) of the total 5709 kinase-substrate combinations. A large number of novel phosphorylations were included among them. Many substrates were shown to be phosphorylated by multiple SFKs, which might reflect functional complementarity of SFKs. Clustering analysis of phosphorylation results grouped substrates into 10 categories, while the similarity of SFK catalytic specificity exhibited no significant correlation with that of amino acid sequences. In silico predictions of SRC-specific phosphorylation sites were not consistent with experimental results, implying some unknown SRC recognition modes. In an attempt to find biologically meaningful novel substrates, phosphorylation data were integrated with annotation data. The extensive in vitro data obtained in this study would provide valuable clues for further understanding SFK-mediated signal transduction.

Inhibitory effects of simvastatin on platelet-derived growth factor signaling in pulmonary artery smooth muscle cells from patients with idiopathic pulmonary arterial hypertension.

Idiopathic pulmonary arterial hypertension (IPAH) is a progressive disease characterized by inappropriate increase of pulmonary artery smooth muscle cells (PASMCs) leading to occlusion of pulmonary arterioles. Inhibition of platelet-derived growth factor (PDGF) signaling is starting to garner attention as a targeted therapy for IPAH. We assessed the inhibitory effects of simvastatin, a 3-hydroxy-3-methylglutanyl coenzyme A reductase inhibitor, on PDGF-induced proliferation and migration of PASMCs obtained from 6 patients with IPAH who underwent lung transplantation. PDGF stimulation caused a significantly higher growth rate of PASMCs from patients with IPAH than that of normal control PASMCs as assessed by (3)H-thymidine incorporation. Simvastatin (0.1 micromol/L) significantly inhibited PDGF-induced cell proliferation of PASMCs from patients with IPAH but did not inhibit proliferation of normal control cells at the same concentration. Western blot analysis revealed that simvastatin significantly increased the expression of cell cycle inhibitor p27. PDGF significantly increased the migration distance of IPAH-PASMCs compared with that of normal PASMCs, and simvastatin (1 micromol/L) significantly inhibited PDGF-induced migration. Immunofluorescence staining revealed that simvastatin (1 micromol/L) inhibited translocation of Rho A from the cytoplasm to membrane and disorganized actin fibers in PASMCs from patients with IPAH. In conclusion, simvastatin had inhibitory effects on inappropriate PDGF signaling in PASMCs from patients with IPAH.

Cytokine reducing effect of azelnidipine in human peripheral blood mononuclear cells.

Numerous clinical trials have shown that calcium channel blocker (CCB) therapy improves the clinical outcome in patients with cardiovascular diseases. Since the progression of several types of cardiovascular diseases is closely associated with inflammation, alleviation of inflammation may be one potential mechanism of those beneficial effects of CCB therapy. We examined whether a new CCB (azelnidipine) could influence the inflammatory response of human peripheral blood mononuclear cells (PBMCs), which are recruited to inflammatory lesions and modulate inflammation. We investigated whether azelnidipine affected intracellular signaling and cytokine production by phytohemagglutinin (PHA)-stimulated human PBMCs in vitro. PBMCs were obtained from 10 healthy volunteers and stimulated with PHA. Then relative intracellular calcium ion concentration ([Ca(2+)](i)) was assessed by fluorescence microscopy, and the production of monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-alpha (TNF-alpha) were measured by enzyme-linked immunosorbent assay. Stimulation with PHA significantly raised [Ca(2+)](i) and enhanced the production of MCP-1 and TNF-alpha by human PBMCs. Azelnidipine significantly diminished the PHA-induced rise of [Ca(2+)](i), and the production of MCP-1 and TNF-alpha. These findings indicate that azelnidipine might have an anti-inflammatory influence on human PBMCs, although the mechanisms and the difference from other CCBs still remain unclear and further exploration should be required.

RNA sequencing reveals abnormal LDB3 splicing in sudden cardiac death.

The aim of this study is to determine the molecular mechanism of sudden death in a previously healthy patient. Clinical exome sequencing revealed I536T-RBM20 variant, which alters RNA splicing of TTN and is causative for dilated cardiomyopathy. Comprehensive RNA sequencing (RNA-seq) was also performed in the patient samples and the control samples. Splicing abnormality was compared in cardiac muscle and skeletal muscle. RNA-seq analysis of the cardiac and skeletal muscle showed abnormal splicing of LDB3, not of TTN. Exon 11 of LDB3 was abnormally included in the patient samples compared with the control samples. This abnormal LDB3 splicing pattern in skeletal muscle has been reported in myotonic dystrophy type 1 (DM1) patients. We, thus, confirmed that the patient had expanded CTG repeat in DMPK and the diagnosis was genetically DM1. This finding suggest that one of the molecular mechanisms of sudden cardiac death in this asymptomatic subclinical DM1 patient might be LDB3 abnormal splicing due to the CTG repeat in DMPK, rather than RBM20 variant. RNA-seq analysis is useful to determine the exact molecular diagnosis for sudden cardiac death.

Dominance of Sulfuritalea species in nitrate-depleted water of a stratified freshwater lake and arsenate respiration ability within the genus.

Facultative autotrophs of the genus Sulfuritalea within the class Betaproteobacteria have been predicted to be an important bacterial population in stratified freshwater lakes based on previous PCR-based studies. Here, we designed a new probe specific for the genus Sulfuritalea and performed catalysed reporter deposition-fluorescence in situ hybridisation to enumerate cells of Sulfuritalea species throughout the water column in a stratified freshwater lake. The cells stained with the Sulfuritalea-specific probe were detected in all hypoxic water samples collected in different seasons and years. Their abundance ranged from 1.4 × 104 to 2.1 × 105 cells ml-1 , corresponding to 0.5-5.5% of the total DAPI-stained cells and 2.3-15% of the total bacterial cells. A high abundance of Sulfuritalea species was recorded in hypoxic water samples without nitrate, which is the only known anaerobic electron acceptor for Sulfuritalea. Nitrate-independent anaerobic respiration was further investigated using a single cultured representative of this genus, and its growth via arsenate respiration was experimentally demonstrated. In conclusion, Sulfuritalea species were found to be a major component of the planktonic bacterial community in nitrate-depleted hypoxic water, where arsenate respiration is one of the possible energy metabolisms of Sulfuritalea.

Differential responses of normal human coronary artery endothelial cells against multiple cytokines comparatively assessed by gene expression profiles.

Endothelial cells play an important role in terms of biological functions by responding to a variety of stimuli in the blood. However, little is known about the molecular mechanism involved in rendering the variety in the cellular response. To investigate the variety of the cellular responses against exogenous stimuli at the gene expression level, we attempted to describe the cellular responses with comprehensive gene expression profiles, dissect them into multiple response patterns, and characterize the response patterns according to the information accumulated so far on the genes included in the patterns. We comparatively analyzed in parallel the gene expression profiles obtained with DNA microarrays from normal human coronary artery endothelial cells (HCAECs) stimulated with multiple cytokines, interleukin-1beta, tumor necrosis factor-alpha, interferon-beta, interferon-gamma, and oncostatin M, which are profoundly involved in various functional responses of endothelial cells. These analyses revealed that the cellular responses of HCAECs against these cytokines included at least 15 response patterns specific to a single cytokine or common to multiple cytokines. Moreover, we statistically extracted genes contained within the individual response patterns and characterized the response patterns with the genes referring to the previously accumulated findings including the biological process defined by the Gene Ontology Consortium (GO). Out of the 15 response patterns in which at least one gene was successfully extracted through the statistical approach, 11 response patterns were differentially characterized by representing the number of genes contained in individual criteria of the biological process in the GO only. The approach to dissect cellular responses into response patterns and to characterize the pattern at the gene expression level may contribute to the gaining of insight for untangling the diversity of cellular functions.