Pancreatic cancer is among the most lethal malignancies worldwide. This study aimed to identify a novel prognostic biomarker, facilitating treatment selection, using mass spectrometry (MS)-based proteomic analysis with formalin-fixed paraffin-embedded (FFPE) tissue.

The two main molecular species of sialic acid existing in nature are N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). Neu5Ac is abundant in mammalian brains and plays crucial roles in many neural functions. In contrast, Neu5Gc is present only at a trace level in vertebrate brains. The brain-specific suppression of Neu5Gc synthesis, which is a common feature in mammals, suggests that Neu5Gc has toxicity against brain functions. However, in vivo kinetics of Neu5Gc in the whole body, especially in the brain, has not been studied in sufficient detail. To determine the in vivo kinetics of Neu5Gc, 14C-Neu5Gc was enzymatically synthesized and injected into rat tail veins. Although most of 14C-Neu5Gc was excreted in urine, a small amount of 14C-Neu5Gc was detected in the brain. Brain autoradiography indicated that 14C-Neu5Gc was accumulated predominantly in the hippocampus. 14C-Neu5Gc transferred into the brain was incorporated into gangliosides including GM1, GD1a, GD1b, GT1b and GQ1b. Reduction of 14C-Neu5Gc after intracerebroventricular infusion was slower than that of 14C-Neu5Ac in the brain and hippocampus. The results suggest that Neu5Gc is transferred from blood into the brain across the blood brain barrier and accumulates in the brain more preferentially than does Neu5Ac.

Autoimmune autonomic ganglionopathy (AAG) is a rare acquired channelopathy that is characterized by pandysautonomia, in which autoantibodies to ganglionic nicotinic acetylcholine receptors (gAChR) may play a central role. Radioimmunoprecipitation (RIP) assays have been used for the sensitive detection of autoantibodies to gAChR in the serum of patients with AAG. Here, we developed luciferase immunoprecipitation systems (LIPS) to diagnose AAG based on IgGs to both the α3 and β4 gAChR subunits in patient serum. We reviewed the serological and clinical data of 50 Japanese patients who were diagnosed with AAG. With the LIPS testing, we detected anti-α3 and -β4 gAChR antibodies in 48% (24/50) of the patients. A gradual mode of onset was more common in the seropositive group than in the seronegative group. Patients with AAG frequently have orthostatic hypotension and upper and lower gastrointestinal tract symptoms, with or without anti-gAChR. The occurrence of autonomic symptoms was not significantly different between the seropositive and seronegative group, with the exception of achalasia in three patients from the seropositive group. In addition, we found a significant overrepresentation of autoimmune diseases in the seropositive group and endocrinological abnormalities as an occasional complication of AAG. Our results demonstrated that the LIPS assay was a useful novel tool for detecting autoantibodies against gAChR in patients with AAG.

Androgen receptor (AR) signaling is essential for the development of prostate cancer. Here, we report that runt-related transcription factor (RUNX1) could be a key molecule for the androgen-dependence of prostate cancer. We found RUNX1 is a target of AR and regulated positively by androgen. Our RUNX1 ChIP-seq analysis indicated that RUNX1 is recruited to AR binding sites by interacting with AR. In androgen-dependent cancer, loss of RUNX1 impairs AR-dependent transcription and cell growth. The RUNX1 promoter is bound by enhancer of zeste homolog 2 (EZH2) and is negatively regulated by histone H3 lysine 27 (K27) trimethylation. Repression of RUNX1 is important for the growth promotion ability of EZH2 in AR-independent cells. In clinical prostate cancer samples, the RUNX1 expression level is negatively associated with EZH2 and that RUNX1 loss correlated with poor prognosis. These results indicated the significance of RUNX1 for androgen-dependency and that loss of RUNX1 could be a key step for the progression of prostate cancer.

Apelin is an inotropic and cardioprotective peptide that exhibits beneficial effects through activation of the APJ receptor in the pathology of cardiovascular diseases. Apelin induces the expression of angiotensin-converting enzyme 2 (ACE2) in failing hearts, thereby improving heart function in an angiotensin 1⁻7-dependent manner. Whether apelin antagonizes the over-activation of the renin⁻angiotensin system in the heart remains elusive. In this study we show that the detrimental effects of angiotensin II (Ang II) were exacerbated in the hearts of aged apelin-gene-deficient mice. Ang II-mediated cardiac dysfunction and hypertrophy were augmented in apelin knockout mice. The loss of apelin increased the ratio of angiotensin-converting enzyme (ACE) to ACE2 expression in the Ang II-stressed hearts, and Ang II-induced cardiac fibrosis was markedly enhanced in apelin knockout mice. mRNA expression of pro-fibrotic genes, such as transforming growth-factor beta (TGF-β) signaling, were significantly upregulated in apelin knockout hearts. Consistently, treatment with the ACE-inhibitor Captopril decreased cardiac contractility in apelin knockout mice. In vitro, apelin ameliorated Ang II-induced TGF-β expression in primary cardiomyocytes, accompanied with reduced hypertrophy. These results provide direct evidence that endogenous apelin plays a crucial role in suppressing Ang II-induced cardiac dysfunction and pathological remodeling.

Infrared thermal imaging of brain temperature changes is useful for evaluating cortical activity and disease states, such as stroke. However, the changes depend on a balance between changes in heat generation from metabolism and in heat convection related to blood flow. To discriminate between these effects and gain a clearer understanding of neurovascular metabolic coupling, brain temperature imaging must be improved to measure temperature and blood flow simultaneously. We develop an imaging technique that shows a two-dimensional (2-D) distribution of absolute brain temperature and relative cerebral blood flow changes in anesthetized rats by combining infrared thermal imaging with laser speckle imaging. The changes in brain metabolism and cerebral blood flow are achieved using two different anesthetics (isoflurane and α-chloralose) to evaluate our system. Isoflurane increased cerebral blood flow but decreased metabolism, whereas α-chloralose decreased both parameters. This technique enables simultaneous visualization of brain surface changes in temperature and cerebral blood flow in the same regions. This imaging system will permit further study of neurovascular metabolic coupling in normal and diseased brains.

Cysteine persulfide and cysteine polysulfides are cysteine derivatives having sulfane sulfur atoms bound to cysteine thiol. Accumulating evidence has suggested that cysteine persulfides/polysulfides are abundant in prokaryotes and eukaryotes and play important roles in diverse biological processes such as antioxidant host defense and redox-dependent signal transduction. Here, we show that enhancement of cellular polysulfides by using polysulfide donors developed in this study resulted in marked inhibition of lipopolysaccharide (LPS)-initiated macrophage activation. Polysulfide donor treatment strongly suppressed LPS-induced pro-inflammatory responses in macrophages by inhibiting Toll-like receptor 4 (TLR4) signaling. Other TLR signaling stimulants-including zymosan A-TLR2 and poly(I:C)-TLR3-were also significantly suppressed by polysulfur donor treatment. Administration of polysulfide donors protected mice from lethal endotoxin shock. These data indicate that cellular polysulfides negatively regulate TLR4-mediated pro-inflammatory signaling and hence constitute a potential target for inflammatory disorders.

Metastatic breast cancer is a highly lethal disease, and it is very important to evaluate the biomarkers associated with distant metastasis. However, molecular features of distant metastasis remain largely unknown in breast cancer. Estrogens play an important role in the progression of breast cancer and the majority of stage IV breast carcinomas express estrogen receptor (ER). Therefore, in this study, we examined molecular markers associated with distant metastasis in ER-positive breast carcinoma by microarray and immunohistochemistry. When we examined the gene expression profile of ER-positive stage IV breast carcinoma tissues (n = 7) comparing ER-positive stage I-III cases (n = 11) by microarray analysis, we newly identified OLFM4, LY6D and S100A7, which were closely associated with the distant metastasis. Subsequently, we performed immunohistochemistry for OLFM4, LY6D and S100A7 in 168 ER-positive breast carcinomas. OLFM4, LY6D and S100A7 immunoreactivities were significantly associated with stage, pathological T factor, distant metastasis and Ki67 status in the ER-positive breast carcinomas. Moreover, these immunoreactivities were significantly associated with a worse prognostic factor for distant metastasis-free and breast cancer-specific survival in ER-positive stage I-III breast cancer patients. However, when we performed immunohistochemistry for OLFM4, LY6D and S100A7 in 40 ER-negative breast carcinomas, these immunoreactivities were not generally associated with the clinicopathological factors examined, including distant metastasis and prognosis of patients, in this study. These results suggest that OLFM4, LY6D and S100A7 immunoreactivity are associated with an aggressive phenotype of ER-positive breast carcinoma, and these are potent markers for distant metastasis of ER-positive breast cancer patients.

Aromatase inhibitors (AI) are commonly used to treat postmenopausal estrogen-receptor (ER)-positive breast carcinoma. However, resistance to AI is sometimes acquired, and the molecular mechanisms underlying such resistance are largely unclear. Recent studies suggest that AI treatment increases androgen activity during estrogen deprivation in breast carcinoma, but the role of the androgen receptor (AR) in breast carcinoma is still a matter of controversy. The purpose of this study is to examine the potential correlation between the AR- and AI-resistant breast carcinoma. To this end, we performed immunohistochemical analysis of 21 pairs of primary breast carcinoma and corresponding AI-resistant recurrent tissue samples and established two stable variant cell lines from ER-positive T-47D breast carcinoma cell line as AI-resistance models and used them in in vitro experiments. Immunohistochemical analysis demonstrated that the expression of prostate-specific antigen (PSA) and Ki-67 were significantly higher and ER and progesterone receptor (PR) were lower in recurrent lesions compared to the corresponding primary lesions. Variant cell lines overexpressed AR and PSA and exhibited neither growth response to estrogen nor expression of ER. Androgen markedly induced the proliferation of these cell lines. In addition, the expression profile of androgen-induced genes was markedly different between variant and parental cell lines as determined by microarray analysis. These results suggest that in some cases of ER-positive breast carcinoma, tumor cells possibly change from ER-dependent to AR-dependent, rendering them resistant to AI. AR inhibitors may thus be effective in a selected group of patients.

Transcriptional GATA factors are known lineage selector genes and regulate a variety of biological processes including specification and differentiation of tissues. In the present study, we examined expression profiles of six GATA factor genes in invasive ductal carcinomas (IDC) of the breast using microarray analysis (n = 20) and found that GATA4 expression was closely correlated with recurrence in patients. Because the significance of GATA4 has remained largely unknown in breast carcinoma, we further immunolocalized GATA4 in ductal carcinoma in situ (DCIS) of the breast (n = 48) and IDC (n = 163). GATA4 immunoreactivity was detected in the nuclei of carcinoma cells and was positive in 27% of DCIS and 31% of IDC cases. GATA4 status was significantly associated with nuclear grade and van Nuys classification in DCIS and was positively associated with distant metastasis, histological grade and HER2 status, but negatively correlated with progesterone receptor labeling index in IDC. Subsequent multivariate analysis demonstrated that GATA4 status was an independent prognostic factor for both disease-free and breast cancer-specific survival of IDC patients. All of these results indicate that GATA4 plays important roles in the progression of breast carcinoma from an early stage and that immunohistochemical GATA4 status is considered a potent prognostic factor in human breast cancer patients.

Helicobacter cinaedi is the most common enterohepatic Helicobacter species that causes bacteremia in humans, but its pathogenicity is unclear. Here, we investigated the possible association of H. cinaedi with atherosclerosis in vivo and in vitro. We found that H. cinaedi infection significantly enhanced atherosclerosis in hyperlipidaemic mice. Aortic root lesions in infected mice showed increased accumulation of neutrophils and F4/80(+) foam cells, which was due, at least partly, to bacteria-mediated increased expression of proinflammatory genes. Although infection was asymptomatic, detection of cytolethal distending toxin RNA of H. cinaedi indicated aorta infection. H. cinaedi infection altered expression of cholesterol receptors and transporters in cultured macrophages and caused foam cell formation. Also, infection induced differentiation of THP-1 monocytes. These data provide the first evidence of a pathogenic role of H. cinaedi in atherosclerosis in experimental models, thereby justifying additional investigations of the possible role of enterohepatic Helicobacter spp. in atherosclerosis and cardiovascular disease.

Avian influenza A viruses (IAVs) and human 1918, 1957, and 1968 pandemic IAVs all have neuraminidases (NAs) that are stable at low pH sialidase activity, yet most human epidemic IAVs do not. We examined the pH stability of H5N1 highly pathogenic avian IAV (HPAI) NAs and identified amino acids responsible for conferring stability at low pH. We found that, unlike other avian viruses, most H5N1 IAVs isolated since 2003 had NAs that were unstable at low pH, similar to human epidemic IAVs. These H5N1 viruses are thus already human virus-like and, therefore, have the frequent infections of humans.

Estrogens were recently demonstrated to be synthesized in non-small cell lung carcinomas (NSCLCs) via aromatase activity and aromatase inhibitor (AI) did suppressed estrogen receptor (ER) positive NSCLC growth. However, other enzymes involved in intratumoral production and metabolism of estrogens, i.e. 17β-hydroxysteroid dehydrogenases (i.e. 17βHSD1 and 17βHSD2) and others have not been studied. Therefore, in this study, we examined the clinical/ biological significance of 17β-hydroxysteroid dehydrogenases in NSCLCs.

Influenza viruses (IFVs) recognize sialoglycans expressed on the host cell surface. To understand the mechanisms underlying tissue and host tropisms of IFV, it is essential to elucidate the molecular interaction of the virus with the host sialoglycan receptor. We established and applied a new monoclonal antibody, clone HYB4, which specifically recognizes the Neu5Acα2-3 determinant at the non-reducing terminal Gal residue of both glycoproteins and gangliosides to investigate the biochemical properties of IFV receptors in A549 cells. HYB4 significantly blocked virus binding to A549 cells in a dose-dependent manner. Virus overlay assay indicated that several glycoproteins with molecular masses of 80-120 kDa of A549 cells were commonly recognized by different subtypes of IFV, such as H1N1 and H3N2. H1N1 virus binding to the glycoproteins was diminished by pretreatment with either sialidase or PNGase F. On TLC-immunostaining experiments with HYB4, GM₃ ganglioside was only detected in A549 cells. Interestingly, this antibody bound to GM₃ gangliosides on TLC and plastic surfaces, but not on lipid bilayers. In comparison with the recognition of Maackia amurensis lectins, HYB4 exclusively recognized Neu5Acα2-3Galβ1-4GlcNAc residues expressed on glycoproteins. These results strongly suggest that N-linked sialoglycans with the Neu5Acα2-3 determinant on several glycoproteins are receptors for influenza virus in A549 cells.

Influenza A virus (IAV) generally causes caspase-dependent apoptosis based on caspase-3 activation, resulting in nuclear export of newly synthesized viral nucleoprotein (NP) and elevated virus replication. Sulfatide, a sulfated galactosylsphingolipid, enhances IAV replication through promoting newly synthesized viral NP export induced by association of sulfatide with hemagglutinin delivered to the cell surface. Here, we demonstrated that sulfatide is involved in caspase-3-independent apoptosis initiated by the PB1-F2 protein of IAV by using genetically sulfatide-produced cells and PB1-F2-deficient IAVs. Sulfatide-deficient COS7 cells showed no virus-induced apoptosis, whereas SulCOS1 cells, sulfatide-enriched COS7 cells that genetically expressed the two transferases required for sulfatide synthesis from ceramide, showed an increase in IAV replication and were susceptible to caspase-3-independent apoptosis. Additionally, PB1-F2-deficient IAVs, which were generated by using a plasmid-based reverse genetics system from a genetic background of A/WSN/33 (H1N1), demonstrated that PB1-F2 contributed to caspase-3-independent apoptosis in IAV-infected SulCOS1 cells. Our results show that sulfatide plays a critical role in efficient IAV propagation via caspase-3-independent apoptosis initiated by the PB1-F2 protein.

SERMs bind to both estrogen receptor (ER)alpha and beta, resulting in tissue dependent estrogen agonist or antagonist responses. Both raloxifene and tamoxifen are most frequently used SERMs and exert estrogen agonistic effects on human bone tissues, but the details of their possible direct effects on human bone cells have remained largely unknown. In our present study, we examined the comparative effects of raloxifene, tamoxifen, and native estrogen, estradiol on human osteoblast cell line, hFOB in vitro. Both the cell numbers and the ratio of the cells in S phase fraction were significantly increased by the treatment of raloxifene or tamoxifen as well as estradiol treatments in hFOB. Gene profile patterns following treatment with raloxifene, tamoxifen, and estradiol demonstrated similar patterns in a microarray/hierarchal clustering analysis. We also examined the expression levels of these genes detected by this analysis using quantitative RT-PCR. MAF gene was induced by raloxifene treatment alone. GAS6 gene was induced by raloxifene and tamoxifen as well as estradiol. An estrogen receptor blocker, ICI 18, 286, inhibited an increase of GAS6 gene expression but not the levels of MAF gene mRNA expression. Results of our present study demonstrated that raloxifene exerted direct protective effects on human osteoblasts in both estrogen receptor dependent and independent manners.

Hypothalamic gonadotropin-releasing hormone (GnRH) neurons originate in the olfactory placode and migrate to the forebrain during embryonic development. We found that GnRH neurons migrated in two different modes in the chick medial telencephalon: they initially underwent axophilic migration in association with a subset of olfactory fibers in a dorsocaudal direction. This was followed by ventrally directed tangential migration to the basal forebrain. Since many of the ventrally migrating GnRH neurons did not follow distinct fiber fascicles, it is proposed that diffusible guidance molecules played a role in this migratory process. A long-range diffusible factor, netrin 1, was expressed in the lower part of the commissural plate and the subpallial septum, but not along the axophilic migratory route of GnRH neurons. Failure of ventrally directed migration of GnRH neurons and their misrouting to the dorsomedial forebrain was induced by misexpression of netrin 1 in the dorsocaudal part of the septum near the top of the commissural plate, which is where the migration of GnRH neurons changed to a ventral direction. In such cases, a subset of olfactory fibers also extended, but close contact between aberrant fibers and misrouted GnRH neurons did not exist. A coculture experiment demonstrated that netrin 1 exerts an attractive effect on migrating GnRH neurons. These results provide evidence that netrin 1 acts as chemoattractant to migrating GnRH neurons at the dorsocaudal part of the septum and has the potential to regulate the ventral migration of GnRH neurons to the ventral septum and the preoptic area.

Survivin is one of the apoptosis inhibitor genes and is rarely expressed in adult -tissues. However, survivin expression has been detected in various human cancers and -correlations have been recognized between the level of expression of this gene in tumors and prognosis. In this study, we investigated the correlations between survivin mRNA expression in osteosarcoma tissues and clinicopathological parameters.

The estrogen receptor (ER) functions as a dimer and is involved in several different biological functions. However ER dimeric proteins have not been identified by in situ methodologies. Structured illumination microscopy (SIM) has been recently developed, which enabled the localization of protein and protein interaction. Therefore, in this study, we firstly demonstrated that ERs formed both homodimers and heterodimers in breast carcinoma cell lines using Nikon's SIM (N-SIM). ERα/α homodimers were detected in the nuclei of both ERα-positive MCF-7 and T-47D cells; 23.0% and 13.4% of ERα proteins formed ERα/α homodimers, respectively. ERα/β heterodimers were also detected in MCF-7 and T-47D. Approximately 6.6% of both ERα and ERβ1 proteins formed ERα/β1 heterodimers in MCF-7. In addition, 18.1% and 22.4% of ERα and ERβ proteins formed ERα/β2 heterodimers and ERα/β5 heterodimers in MCF-7, respectively. In addition, by using proximity ligation assay (PLA) in MCF-7, estradiol-induced ERα/α homodimers and ERα/β1 heterodimers were both detected after 15 to 45 min of treatment and at 15 min, respectively. The percentage of total ER proteins could also be determined using N-SIM. By using both methods, it has become possible to evaluate precise localization and ratio of ER dimers among different cell types.

Estrogen receptors promote target gene transcription when they form a dimer, in which two identical (homodimer) or different (heterodimer) proteins are bound to each other. In hormone-dependent cancers, hormone receptor dimerization plays pivotal roles, not only in the pathogenesis or development of the tumors, but also in the development of therapeutic resistance. Protein⁻protein interactions (PPIs), including dimerization and complex formation, have been also well-known to be required for proteins to exert their functions. The methods which could detect PPIs are genetic engineering (i.e., resonance energy transfer) and/or antibody technology (i.e., co-immunoprecipitation) using cultured cells. In addition, visualization of the target proteins in tissues can be performed using antigen⁻antibody reactions, as in immunohistochemistry. Furthermore, development of microscopic techniques (i.e., electron microscopy and confocal laser microscopy) has made it possible to visualize intracellular and/or intranuclear organelles. We have recently reported the visualization of estrogen receptor dimers in breast cancer tissues by using the in situ proximity ligation assay (PLA). PLA was developed along the lines of antibody technology development, and this assay has made it possible to visualize PPIs in archival tissue specimens. Localization of PPI in organelles has also become possible using super-resolution microscopes exceeding the resolution limit of conventional microscopes. Therefore, in this review, we summarize the methodologies used for studying PPIs in both cells and tissues, and review the recently reported studies on PPIs of hormones.