We have developed a highly sensitive microarray protein chip, ProteoChip, coated with ProLinker, novel calixcrown derivatives with a bifunctional coupling property that permits efficient immobilization of capture proteins on solid matrixes and makes high-throughput analysis of protein-protein interactions possible. The analysis of quartz crystal microbalance showed that both monoclonal antibody (mAb) and antigen (Ag) bound to the gold film of the sensor surface coated with ProLinker B and that it is useful for studies of Ab-Ag interactions. ProteoChip, aminated glass slide coated with ProLinker A, was also demonstrated to be useful for preparation of high-density array spots by using a microarrayer and for analysis of analyte Ags either by direct or sandwich methods of fluorescence immunoassay. The detection sensitivity of ProteoChip was as low as 1-10 femtogram/mL of analyte protein, useful for detection of tumor markers. ProteoChip was also useful for studies of direct protein-protein interactions as demonstrated by analysis of integrin-extracellular matrix protein interaction. These experimental results suggest that ProteoChip is a powerful tool for development of chip-based lead screening microarrays to monitor protein-protein interactions (i.e. drug target) as well as for biomarker assays which require high detection sensitivity.

The sustained expansion of a tumor mass requires new blood vessel formation to provide rapidly proliferating tumor cells with an adequate supply of oxygen and nutrients. Hypoxia-inducible factor-1 (HIF-1) plays an essential role in tumor angiogenesis and growth by regulating the transcription of genes in response to hypoxic stress. This study was designed to investigate the effects of melatonin on tumor growth and angiogenesis, as well as the mechanism underlying the antitumor activities of melatonin. In this study, we show that the administration of melatonin inhibits tumor growth and blocks tumor angiogenesis in mice. Moreover, melatonin diminished the expression of the HIF-1α protein within the tumor mass during tumorigenesis. Our findings suggest that melatonin is a promising anti-angiogenic therapeutic agent targeting HIF-1α in cancer. Considering that HIF-1α is overexpressed in a majority of human cancers, melatonin could offer a potent therapeutic agent for cancer.

The 5-HT 6 receptor (5-HT 6 R) is one of the most recently cloned serotonin receptors, and it plays important roles in Alzheimer disease, depression, and learning and memory disorders. However, unlike the other serotonin receptors, the cellular mechanisms of 5-HT 6 R are poorly elucidated relative to its significance in human brain diseases. Here, using a yeast two-hybrid assay, we found that the human 5-HT 6 R interacts with Jun activation domain-binding protein-1 (Jab1). We also confirmed a physical interaction between 5-HT 6 R and Jab1 using glutathione S-transferase pulldown, fluorescence resonance energy transfer, co-immunoprecipitation, and immunocyto(histo)-chemistry assays. The manipulation of Jab1 expression using Jab1 small interference RNA decreased 5-HT 6 R-mediated activity and cell membrane expression of 5-HT 6 R, whereas overexpression of Jab1 produced no significant effect. In addition, we demonstrated that the activation of 5-HT 6 R induced the translocation of Jab1 into the nucleus and increased c-Jun phosphorylation and the interaction between Jab1 and c-Jun. Furthermore, we found that 5-HT 6 R and Jab1 were up-regulated in middle cerebral artery occlusion-induced focal cerebral ischemic rats and in cultured cells exposed to hypoxic insults, suggesting possible protective roles for 5-HT 6 R and Jab1. These findings suggest that Jab1 provides a novel signal transduction pathway for 5-HT 6 R and may play an important role in 5-HT 6 Rmediated behavior changes in the brain.Neurotransmitters and neurohormones regulate diverse and myriad brain functions ranging from rapid modulation of ligand-gated ion channels to long term modulation such as gene expression, behavior, and mood changes. Serotonin (5-HT) 2 is known to be one of the key neurotransmitters related to mood changes. The serotonergic system has also been implicated in the neurobiological control of learning and memory. Therefore, it emerges as a key player in affective, cognitive, complex sensory, and motor functions (1). 5-HT mediates its diverse physiological responses through seven distinct receptor families: the 5-HT 1 , 5-HT 2 , 5-HT 3 , 5-HT 4 , 5-HT 5 , 5-HT 6 , and 5-HT 7 receptors. With the exception of the 5-HT 3 receptor, all of these receptors are members of the G-protein-coupled receptor (GPCR) superfamily (2). Among the 5-HT receptors, the 5-HT 6 receptor (5-HT 6 R) is coupled to a stimulatory G␣ (G␣ S ) protein. This receptor increases cAMP formation and then activates cAMP-dependent protein kinase (3). 5-HT 6 R is abundantly distributed in the brain, especially in the limbic region (4), and it has a high affinity for antipsychotic compounds and tricyclic antidepressants (5). These preliminary reports imply that 5-HT 6 R has a significant role in the control of mood and emotion in the central nervous system. To date, most findings suggest that 5-HT 6 R plays crucial roles in neurological disorders, including Alzheimer disease, depression, and learning and memory disorders (6 -9). Selective antagonists of 5-HT 6 R improved me...

We herein report cytotoxicity of advanced glycation end-products (AGEs) on pancreatic beta cells. AGEs stimulated reactive oxygen species (ROS) generation but did not arrest proliferation of the INS-1 cell line. Pancreatic beta cell lines or primary cultured islets possess a receptor for AGE (RAGE), and its expression increased after AGE treatment. TUNEL staining and FACS analysis using annexin V/PI antibodies showed that apoptosis increased in INS-1 cells or primary cultured islets when incubated with BSA conjugated with glyceraldehyde (AGE2) or glucoaldehyde (AGE3), compared with those conjugated with glucose (AGE1). Reaction of INS-1 cells to Ki67, which is a cellular marker for proliferation, was also increased after AGE treatment. The ability of primary cultured islets to secrete insulin was retained even after AGE treatment under either low or high glucose conditions. The antiserum against RAGE partially prevented AGE-induced cellular events. Treatment of beta cells with the antioxidant metallothionein results in a significant reduction in pathologic changes. AGEs might be able to induce apoptosis as well as proliferation of pancreatic beta cell lines or primary cultured islets. Moreover, antibody array showed that RAD51 and RAD52 were significantly decreased in AGE2-treated INS-1 cells. AGEs might inhibit homologous DNA recombination for repairing DNA of INS-1 cells damaged by ROS generation. It might be suggested that treatment of AGEs resulted in ROS production and apoptosis through their receptor on pancreatic beta cells. AGEs might deteriorate function of pancreatic beta cells in patients with long-term hyperglycemia.

Correlations between trial-to-trial fluctuations in the responses of individual sensory neurons and perceptual reports, commonly quantified with choice probability (CP), have been widely used as an important tool for assessing the contributions of neurons to behavior. These correlations are usually weak and often require a large number of trials for a reliable estimate. Therefore, working with measures such as CP warrants care in data analysis as well as rigorous controls during data collection. Here we identify potential confounds that can arise in data analysis and lead to biased estimates of CP, and suggest methods to avoid the bias. In particular, we show that the common practice of combining neuronal responses across different stimulus conditions with z-score normalization can result in an underestimation of CP when the ratio of the numbers of trials for the two behavioral response categories differs across the stimulus conditions. We also discuss the effects of using variable time intervals for quantifying neuronal response on CP measurements. Finally, we demonstrate that serious artifacts can arise in reaction time tasks that use varying measurement intervals if the mean neuronal response and mean behavioral performance vary over time within trials. To emphasize the importance of addressing these concerns in neurophysiological data, we present a set of data collected from V1 cells in macaque monkeys while the animals performed a detection task.

Dietary K+ intake may increase renal K+ excretion via increasing plasma [K+] and/or activating a mechanism independent of plasma [K+]. We evaluated these mechanisms during normal dietary K+ intake. After an overnight fast, [K+] and renal K+ excretion were measured in rats fed either 0% K+ or the normal 1% K+ diet. In a third group, rats were fed with the 0% K+ diet, and KCl was infused to match plasma [K+] profile to that of the 1% K+ diet group. The 1% K+ feeding significantly increased renal K+ excretion, associated with slight increases in plasma [K+], whereas the 0% K+ diet decreased K+ excretion, associated with decreases in plasma [K+]. In the KCl-infused 0% K+ diet group, renal K+ excretion was significantly less than that of the 1% K+ group, despite matched plasma [K+] profiles. We also examined whether dietary K+ alters plasma profiles of gut peptides, such as guanylin, uroguanylin, glucagon-like peptide 1, and glucose-dependent insulinotropic polypeptide, pituitary peptides, such as AVP, α-MSH, and γ-MSH, or aldosterone. Our data do not support a role for these hormones in the stimulation of renal K+ excretion during normal K+ intake. In conclusion, postprandial increases in renal K+ excretion cannot be fully accounted for by changes in plasma [K+] and that gut sensing of dietary K+ is an important component of the regulation of renal K+ excretion. Our studies on gut and pituitary peptide hormones suggest that there may be previously unknown humoral factors that stimulate renal K+ excretion during dietary K+ intake.

SummaryParagonimus westermani is a trematode parasite, which causes pulmonary and/or extrapulmonary granulomatous disease in humans. Successful invasion of the host tissue is critical for the survival of this tissue-invasive parasite. The enzymatic hydrolysis of host proteins is clearly a prerequisite of this process. In this study, we have investigated the functional roles of the excretory-secretory cysteine proteases of P. westermani newly excysted metacercariae (PwNEM) in tissue invasion. The 27 and 28 kDa enzymes (PwMc27 and PwMc28) purified from PwNEM excretory-secretory products (ESP), preferentially degraded fibrillar proteins, but not globular proteins. PwMc28 significantly facilitated the invasion of PwNEM into mouse peritoneum, whereas a diffusible cysteine protease inhibitor, trans -epoxysuccinyl-L -leuciloamido-(4-guanidino) butane (E-64) inhibited this process dose-dependently. Two distinct isoforms of PwMc28 (PwMc28a and PwMc28b), which exhibited two amino acid differences in their mature domains, were identified by tandem mass spectrometry and sequence analysis. Both enzymes were localized at the tegument on the anterior border and on the oral sucker, which suggests excretion-secretion via exocytosis or via the excretory canal network. The mRNA transcripts of PwMc28a and b were expressed abundantly during the active invasion/migration through the host's tissues, suggesting their relevant function to tissue invasion/migration in the definitive host.

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