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Purpose: To investigate the intraocular properties and toxicity of IMS2186, a small molecule developed as an anti-choroidal neovascularization (anti-CNV) drug., Materials and Methods: Cellular toxicity and mechanism of action was tested on cell lines in vitro. Intraocular studies used rabbits for drug dissolution as well as toxicity and rats for the treatment study as well as the toxicity confirmation study. Rabbits' eyes were injected with 2.5 mg of IMS2186 and observed for 36 weeks. Laser-induced CNV in rats was treated with IMS2186, Kenalog, or phosphate-buffered saline (pBS). Fluorescein angiography (FA) and immunohistochemical processing of the globes was performed., Results: The anti-proliferative IC(50) of IMS2186 for human fibroblast cells was 1.0-3.0 microM and 0.3-3.0 microM for human cancer cells; the IC(50) of IMS2186 to inhibit endothelial tube formation was 0.1-0.3 microM. The IC(50) of IMS2186 for inhibiting the production of pro-inflammatory cytokines was 0.3-1 microM. The IC(50) of IMS2186 for inhibiting macrophage migration was 1 micrM. These biological properties were not species specific. IMS2186 can be formulated as a suspension for long-lasting release and when delivered intraocularly, no intraocular toxicity was observed by slit lamp exam, fundus exam, intraocular pressure measurements, or by electroretinography. FA showed a reduction in the leakage in eyes treated with IMS2186 and triamcinolone acetonide; DAPI staining also showed significantly less cellularity in IMS2186-treated lesions as compared to PBS (p = 0.0025)., Conclusion: IMS2186 may be a safe intraocular therapeutic agent for intraocular proliferation and angiogenesis.

A cataluminescence (CTL) sensor using Al2O3 nanowires as the sensing material was developed for the determination of trace pinacolyl alcohol in air samples based on the catalytic chemiluminescence (CL) of pinacolyl alcohol on Al2O3 nanowires. Eight catalysts were examined and the CL intensity on Al2O3 nanowires prepared by supercritical fluid drying was the strongest. This novel CL sensor showed high sensitivity and selectivity to gaseous pinacolyl alcohol at optimal temperature of 340 degrees C. Quantitative analysis was performed at a wavelength of 460 nm. The linear range of CTL intensity versus concentration of gaseous pinacolyl alcohol was 0.09 x 10(-6) to 2.56 x 10(-6) g mL(-1) (r=0.9983, n=6) with a detection limit (3 sigma) of 0.0053 x 10(-6) g mL(-1). None or only very low levels of interference were observed while the foreign substances such as water vapor, ethanol, ammonia, chloroform, benzene, nitrogen dioxide, methylbenzene, hydrochloric acid, methanol and butanol were passing through the sensor. The response time of the sensor is less than 100 s, and the sensor had a long lifetime more than 60 h. The sensor would be potentially applied to analysis of the nerve agents such as Soman.

Bilateral frontoparietal polymicrogyria (BFPP) is a rare genetic disease characterized by cortical malformation associated with GPR56 mutations of frameshift, splicing, and point mutations (Science 303:2033). All the missense point mutations are located in the regions predicted to be exposed at the cell surface, e.g. the N-terminal extracellular domain (ECD), the proteolytic site (GPS), and the extracellular loops of transmembrane domain (TM), implying functionally important interaction among these domains. Wild type GPR56 protein is cleaved at the GPCR protein cleavage site (GPS) and gives rise to two subunits (ECD and TM), which are transported to cell surface. We have shown that GPR56 GPS mutant protein is defective in cleavage and surface localization, while non-GPS mutant proteins are cleaved normally but still defective in surface localization. Furthermore, all the mutant proteins demonstrated different glycosylation pattern from that of wild-type protein. PNGase F and Endo H sensitivity assays suggests that the mutant proteins are trapped in endoplasmic reticulum (ER), preventing them from trafficking to Golgi where further glycosylation modification usually occurs before destination to cell surface. Therefore, the loss-of-function of all these missense mutations is primarily caused by their failure to localize to cell surface.

We identified pyrvinium pamoate, an old anthelminthic medicine, which preferentially inhibits anchorage-independent growth of cancer cells over anchorage-dependent growth (approximately 10 fold). It was also reported by others to have anti-tumor activity in vivo and selective toxicity against cancer cells under glucose starvation in vitro, but with unknown mechanism. Here, we provide evidence that pyrvinium suppresses the transcriptional activation of GRP78 and GRP94 induced by glucose deprivation or 2-deoxyglucose (2DG, a glycolysis inhibitor), but not by tunicamycin or A23187. Other UPR pathways induced by glucose starvation, e.g. XBP-1, ATF4, were also found suppressed by pyrvinium. Constitutive expression of GRP78 via transgene partially protected cells from pyrvinium induced cell death under glucose starvation, suggesting that suppression of the UPR is involved in pyrvinium mediated cytotoxicity under glucose starvation. Xenograft experiments showed rather marginal overall anti-tumor activity for pyrvinium as a monotherapy. However, the combination of pyrvinium and Doxorubicin demonstrated significantly enhanced efficacy in vivo, supporting a mechanistic treatment concept based on tumor hypoglycemia and UPR.

Adiponectin is an anti-diabetic hormone secreted by adipocytes. Circulating adiponectin levels are lower in obese and type II diabetic patients than in healthy people. Weight loss or thiazolidinedione treatment increases plasma adiponectin levels. Animal models and human studies suggest that elevated adiponectin levels increase insulin sensitivity. We screened a library of drug-like compounds and natural products for novel agents enhancing adiponectin production. We identified isoginkgetin, a compound derived from the leaves of Ginkgo biloba, to up-regulate adiponectin secretion with potency comparable to that of rosiglitazone, a known modulator of adiponectin production. However, unlike rosiglitazone, peroxisome proliferators-activated receptor gamma activity seems not required for the action of isoginkgetin, and isoginkgetin has only a slight effect on adipogenesis, which makes it an attractive candidate for anti-diabetic treatment. Further investigation revealed that both isoginkgetin and rosiglitazone activate AMP-activated protein kinase (AMPK) in adipocytes. Our findings suggest a novel mechanism for the elevation of adiponectin by isoginkgetin, which is different from that of rosiglitazone. Furthermore, this novel mechanism for adiponectin regulation involving AMPK can potentially facilitate new understanding of metabolic diseases and identification of new targets, as well as agents that increase plasma adiponectin levels.

GPR56 is an orphan G protein - coupled receptor, mutations of which have recently been associated with bilateral frontoparietal polymicrogyria, a rare neurologic disease that has implications in brain development. However, no phenotype beyond central nervous system has yet been described for the GPR56-null mutations despite abundant GPR56 expression in many non - central nervous system adult tissues. In the present study, we show that higher GPR56 expression is correlated with the cellular transformation phenotypes of several cancer tissues compared with their normal counterparts, implying a potential oncogenic function. RNA interference-mediated GPR56 silencing results in apoptosis induction and reduced anchorage-independent growth of cancer cells via increased anoikis, whereas cDNA overexpression resulted in increased foci formation in mouse fibroblast NIH3T3 cell line. When GPR56 silencing was induced in vivo in several xenograft tumor models, significant tumor responses (including regression) were observed, suggesting the potential of targeting GPR56 in the development of tumor therapies. The expression profiling of GPR56-silenced A2058 melanoma cell line revealed several genes whose expression was affected by GPR56 silencing, particularly those in the integrin-mediated signaling and cell adhesion pathways. The potential role of GPR56 in cancer cell adhesion was further confirmed by the observation that GPR56 silencing also reduced cell adhesion to the extracellular matrix, which is consistent with the observed increase in anoikis and reduction in anchorage-independent growth phenotypes. The oncogenic potential and apparent absence of physiologic defects in adult human tissues lacking GPR56, as well as the targetable nature of G protein - coupled receptor by small molecule or antibody, make GPR56 an attractive drug target for the development of cancer therapies.

Human xenograft tumor models are widely used for efficacy evaluation of potential cancer targets. siRNA is usually stably introduced into tumor cells prior to transplantation. However, silencing of the cancer therapeutic target usually results in reduced cell growth/survival in vitro and/or failure to establish tumors in vivo, thus hindering tumor response-based efficacy evaluation. The present study explored a new tumor response model based on regulated RNAi, which is more relevant from a clinical standpoint. As a proof of principle, an inducible lentiviral RNAi vector was used to silence the known cancer therapeutic target mTOR upon induction with Doxycycline (DOX). The responses to DOX-induced mTOR silencing were tested both in vitro and in vivo for prostate cancer PC3 models. Significant reduction in cancer cell survival was observed due to cell cycle arrest and apoptosis when mTOR silencing was induced in vitro. mTOR silencing also caused tumor regression for the early-staged PC3 tumors (100% tumor regressed and 45% became tumor-free). The advanced-staged tumors also demonstrated significant responses (100% regressed). Therefore, our results demonstrate the powerful utility of this new inducible xenograft tumor model for efficacy evaluation of cancer targets, and it provides a direct in vivo efficacy validation of mTOR as a cancer therapeutic target.

Combinatorial gene inactivation using an RNAi library is a powerful approach to discovering novel functional genes. However, generation of a comprehensive RNAi library remains technically challenging. In this report, we describe a simple and novel approach to designing gene-family-specific RNAi libraries by targeting conserved motifs using degenerate oligonucleotides. We created an siRNA library in the pHUMU vector using partially randomized sequences targeting the consensus region in the ZnF_C4 signature motif of the nuclear hormone receptors and thus against the entire receptor superfamily. For proof of principle, we adapted a reporter assay to screen this library for receptors that might be involved in reducing amyloid beta peptide accumulation. We modified a previously described luciferase reporter assay to measure the amyloid beta precursor cleavages occurring only between beta- and gamma-secretase cleavage sites, thus excluding the major gamma-secretase activities that could generate neurotoxic Abeta peptides. Our screen using this assay identified siRNA vectors that specifically increase the Abeta40/42 cleavage and pointed to a potential receptor target, ROR-gamma. SiRNAs targeting other regions of ROR-gamma not only confirmed the observed reporter activity but also reduced the level of the toxic Abeta peptides. The results demonstrated a general principle for the creation and application of this RNAi library approach for functional gene discovery within a predefined protein family. The discovered negative effect of ROR-gamma on the degradation of the toxic Abeta peptides may also provide a potential drug target or targetable pathway for intervention of Alzheimer disease.

This article reviews the recent development in two areas of RNAi technology that could potentially shorten the otherwise long process of drug development. First, the combinatorial gene silencing built on the RNAi library, particularly the coming of age of 'RNAi gene' libraries, by overcoming numerous technical hurdles and also increasing understanding of miRNA biogenesis, is gradually establishing more effective approaches to identifying drug targets. Second, regulated RNAi gene expression provides tools for in vivo target validation for both on-target efficacy and potential on-target toxicity.:, (© 2006 Elsevier Ltd . All rights reserved.)

Using insulin-secreting cell line (INS)-1 cells stably expressing dihydropyridine-insensitive mutants of either Cav1.2 or Cav1.3, we previously demonstrated that Cav1.3 is preferentially coupled to insulin secretion and [Ca2+]i oscillations stimulated by 11.2 mM glucose. Using the same system, we found that insulin secretion in 7.5 mM glucose plus 1 mM 8-bromo-cAMP (8-Br-cAMP) is mediated by both Cav1.2 and Cav1.3. Treatment of INS-1 cells or INS-1 cells stably expressing Cav1.2/dihydropyridine-insensitive (DHPi) channels in the presence of 10 microM nifedipine, with effector-specific cAMP analogs 8-(4-chlorophenylthio)-2'-O-methyladenosine-cAMP [8-pCPT-2'-O-Me-cAMP; 100 microM; Exchange Protein directly Activated by cAMP 2 (Epac2)-selective] or N6-benzoyl-cAMP [50 microM; Protein Kinase A (PKA)-selective] partially increased insulin secretion. Secretion stimulated by a combination of the two cAMP analogs was additive and comparable with that stimulated by 1 mM 8-Br-cAMP. In INS-1 cells stably expressing Cav1.3/DHPi in the presence of 10 microM nifedipine, N6-benzoyl-cAMP, but not 8-pCPT-2'-O-Me-cAMP, significantly increased glucose-stimulated insulin secretion. However, the combination of N6-benzoyl-cAMP and 8-pCPT-2'-O-Me-cAMP significantly increased glucose-stimulated secretion compared with N6-benzoyl-cAMP alone. In INS-1 cells, 8-Br-cAMP potentiation of insulin secretion in 7.5 mM glucose is blocked by thapsigargin (1 microM) and ryanodine (0.5 microM). In contrast, ryanodine has no effect on insulin secretion or [Ca2+]i oscillations stimulated by 11.2 mM glucose in INS-1 cells. Our data suggest that both Cav1.2 and Cav1.3 mediate insulin secretion stimulated by 7.5 mM glucose and cAMP via a mechanism that requires internal stores of Ca2+. Furthermore, cAMP modulation of secretion mediated by Cav1.2 seems to involve both Epac2 and PKA independently. In contrast, cAMP modulation of Cav1.3-mediated secretion depends upon PKA activation, whereas the contribution of Epac2 is dependent upon PKA activation.

The mechanisms of reproductive malfunction of male mammals caused by 2,2-bis(4-chlorophenyl)-1,1-dichloroethylene (p,p'-DDE, hereafter DDE) remain unknown. To explore the effects of DDE on the expressions of transferrin (Tf) and androgen-binding protein (ABP), we isolated Sertoli cells from healthy immature rats (18-20 days SD rats), set up Sertoli cell cultures, evaluated the toxicity, and measured the expression levels of mRNA of Tf and ABP genes by the one-step reverse transcriptase polymerase chain reaction method after cultured Sertoli cells were in vitro exposed to DDE at different concentrations for 24 h. The results showed that the number and survival rate of Sertoli cells decreased sharply with increased doses of DDE. The expression level of Tf mRNA decreased, whereas ABP mRNA increased gradually with increased DDE doses. There existed an obvious dose-effect relationship between the concentration of DDE and the expression levels of Tf mRNA and ABP mRNA. These findings suggest that DDE may inhibit the expression of Tf and up-modulate expression of ABP in cultured rat Sertoli cells.

The mineralocorticoid receptor (MR) is expressed in kidney and plays a central role in the control of sodium, homeostatic fluid, and blood pressure. It has also been implicated in other functions in cardiovascular system, central nervous system, and adipose tissue. This study revealed a novel role of MR in the gene regulation related to hepatic glucose production. RNAi-mediated MR silencing led to a decrease in the expression of glucose-6-phosphatase (G6Pase), phosphoenolpyruvate carboxykinase, and fructose-1,6-bisphosphatase 1, the enzymes known to be involved in glucose production in liver. The MR-specific antagonists also down-regulated the expression of G6Pase, while the specific agonist enhanced G6Pase expression. These observations, for the first time, revealed a novel role for MR and its ligands in the regulation of de novo glucose synthesis in hepatocytes. It also suggests the potential of liver-specific MR modulation for the treatment of hyperglycemia.

HeLaHF is a non-transformed revertant of HeLa cells, likely resulting from the activation of a putative tumor suppressor(s). p53 protein was stabilized in this revertant and reactivated for certain transactivation functions. Although p53 stabilization has not conclusively been linked to the reversion, it is clear that the genes in p53 pathway are involved. The present study confirms the direct role of p53 in HeLaHF reversion by demonstrating that RNAi-mediated p53 silencing partially restores anchorage-independent growth potential of the revertant through the suppression of anoikis. In addition, we identified a novel gene, named PHTS, with putative tumor suppressor properties, and showed that this gene is also involved in HeLaHF reversion independently of the p53 pathway. Expression profiling revealed that PHTS is one of the genes that is up-regulated in HeLaHF but not in HeLa. It encodes a putative protein with CD59-like domains. RNAi-mediated PHTS silencing resulted in the partial restoration of transformation (anchorage-independent growth) in HeLaHF cells, similar to that of p53 gene silencing, implying its tumor suppressor effect. However, the observed increased transformation potential by PHTS silencing appears to be due to an increased anchorage-independent proliferation rate rather than suppression of anoikis, unlike the effect of p53 silencing. p53 silencing did not affect PHTS gene expression, and vice versa, suggesting PHTS may function in a new and p53-independent tumor suppressor pathway. Furthermore, over-expression of PHTS in different cancer cell lines, in addition to HeLa, reduces cell growth likely via induced apoptosis, confirming the broad PHTS tumor suppressor properties.

This paper describes a novel aerosol chemiluminescence-based detector, which can be coupled to liquid chromatography for the determination of the chemicals with weak optical absorbance in the UV-visible region. This aerosol chemiluminescence (CL)-based detector, in which HPLC effluent is converted to aerosol and then generated CL emission on the surface of porous alumina, is composed of three main processes: nebulization of HPLC effluent, CL emission on surface of porous alumina material, and optical detection. To demonstrate the utility of the aerosol chemiluminescence detector, some compounds such saccharides, poly(ethylene glycol)s, amino acids, and steroid pharmaceuticals are determined by the present aerosol chemiluminescence detection method. Compared with an evaporative light scattering detector, the proposed detector shows the following features: (a) extensive CL emissions on porous alumina by many compounds tested, which leads to the potential application for the determination of volatile and nonvolatile chemicals with or without UV-visible absorbance; (b) a CL mechanism based on the catalytic oxidation of analytes, not on the light scattering, which suggests the present detector be free from the interference of the inorganic and nonvolatile mobile-phase modifiers. The CL characteristics and effect of different parameters, such as temperature and nebulizer gas flow rate, were also discussed in this paper. Furthermore, this aerosol chemiluminescence-based detector was successfully applied to the determination of raffinose, glucose, sucrose, maltose, and alpha-lactose.

The link between Ca(2+) influx through the L-type calcium channels Ca(v)1.2 or Ca(v)1.3 and glucose- or KCl-induced [Ca(2+)](i) mobilization in INS-1 cells was assessed using the calcium indicator indo-1. Cells responded to 18 mM glucose or 50 mM KCl stimulation with different patterns in [Ca(2+)](i) increases, although both were inhibited by 10 microM nifedipine. Although KCl elicited a prolonged elevation in [Ca(2+)](i), glucose triggered oscillations in [Ca(2+)](i.) Ca(v)1.2/dihydropyridine-insensitive (DHPi) cells and Ca(v)1.3/DHPi cells, and stable INS-1 cell lines expressing either DHP-insensitive Ca(v)1.2 or Ca(v)1.3 channels showed normal responses to glucose. However, in 10 microM nifedipine, only Ca(v)1.3/DHPi cells maintained glucose-induced [Ca(2+)](i) oscillation. In contrast, both cell lines exhibited DHP-resistant [Ca(2+)](i) increases in response to KCl. The percentage of cells responding to glucose was not significantly decreased by nifedipine in Ca(v)1.3/DHPi cells but was greatly reduced in Ca(v)1.2/DHPi cells. In 10 microM nifedipine, KCl-elicited [Ca(2+)](i) elevation was retained in both Ca(v)1.2/DHPi and Ca(v)1.3/DHPi cells. In INS-1 cells expressing the intracellular II-III loop of Ca(v)1.3, glucose failed to elicit [Ca(2+)](i) changes, whereas INS-1 cells expressing the Ca(v)1.2 II-III loop responded to glucose with normal [Ca(2+)](i) oscillation. INS-1 cells expressing Ca(v)1.2/DHPi containing the II-III loop of Ca(v)1.3 demonstrated a nifedipine-resistant slow increase in [Ca(2+)](i) and nifedipine-resistant insulin secretion in response to glucose that was partially inhibited by diltiazem. Thus, whereas the II-III loop of Ca(v)1.3 may be involved in coupling Ca(2+) influx to insulin secretion, distinct structural domains are required to mediate the preferential coupling of Ca(v)1.3 to glucose-induced [Ca(2+)](i) oscillation.

L-Type Ca(2+) channel blockers inhibit glucose and KCl-stimulated insulin secretion by pancreatic beta cells. However, the role of the two distinct L-type channels expressed by beta cells, Ca(v)1.2 and Ca(v)1.3, in this process is not clear. Therefore, we stably transfected INS-1 cells with two mutant channel constructs, Ca(v)1.2DHPi or Ca(v)1.3 DHPi. Whole-cell patch-clamp recordings demonstrated that both mutant channels are insensitive to dihydropyridines (DHPs), but are blocked by diltiazem. INS-1 cells expressing Ca(v)1.3/DHPi maintained glucose- and KCl-stimulated insulin secretion in the presence of DHPs, whereas cells expressing Ca(v)1.2/DHPi demonstrated DHP resistance to only KCl-induced secretion. INS-1 cells were also stably transfected with cDNAs encoding the intracellular loop between domains II and III of either Ca(v)1.2 or Ca(v)1.3 (Ca(v)1.2/II-III or Ca(v)1.3/II-III). Glucose- and KCl-stimulated insulin secretion in Ca(v)1.2/II-III cells were not different from untransfected INS-1 cells. However, glucose-stimulated insulin secretion was completely inhibited and KCl-stimulated secretion was substantially resistant to inhibition by DHPs, but sensitive to omega-agatoxin IVA in Ca(v)1.3/II-III cells. Moreover, the L-type channel agonist FPL 64176 markedly enhanced KCl-stimulated secretion by Ca(v)1.3/II-III cells. Together, our results suggest that Ca(2+) influx via Ca(v)1.3 is preferentially coupled to glucose-stimulated insulin secretion in INS-1 cells.