Head skeleton malformations in zebrafish (Danio rerio) to assess adverse effects of mixtures of compounds.

The EU-EuroMix project adopted the strategy of the European Food Safety Authority (EFSA) for cumulative risk assessment, which limits the number of chemicals to consider in a mixture to those that induce a specific toxicological phenotype. These so-called cumulative assessment groups (CAGs) are refined at several levels, including the target organ and specific phenotype. Here, we explore the zebrafish embryo as a test model for quantitative evaluation in one such CAG, skeletal malformations, through exposure to test compounds 0-120 hpf and alcian blue cartilage staining at 120 hpf, focusing on the head skeleton.

Discovery of Potent 17β-Hydroxywithanolides for Castration-Resistant Prostate Cancer by High-Throughput Screening of a Natural Products Library for Androgen-Induced Gene Expression Inhibitors.

Prostate cancer (PC) is the second most prevalent cancer among men in Western societies, and those who develop metastatic castration-resistant PC (CRPC) invariably succumb to the disease. The need for effective treatments for CRPC is a pressing concern, especially due to limited durable responses with currently employed therapies. Here, we demonstrate the successful application of a high-throughput gene-expression profiling assay directly targeting genes of the androgen receptor pathway to screen a natural products library leading to the identification of 17β-hydroxywithanolides 1-5, of which physachenolide D (5) exhibited potent and selective in vitro activity against two PC cell lines, LNCaP and PC-3.

High-throughput, high-sensitivity analysis of gene expression in Arabidopsis.

High-throughput gene expression analysis of genes expressed during salt stress was performed using a novel multiplexed quantitative nuclease protection assay that involves customized DNA microarrays printed within the individual wells of 96-well plates. The levels of expression of the transcripts from 16 different genes were quantified within crude homogenates prepared from Arabidopsis (Arabidopsis thaliana) plants also grown in a 96-well plate format. Examples are provided of the high degree of reproducibility of quantitative dose-response data and of the sensitivity of detection of changes in gene expression within limiting amounts of tissue.

Heterologous desensitization of the human endothelin A and neurokinin A receptors in Xenopus laevis oocytes.

Endothelin 1 (ET1) desensitizes endothelin A receptor for 90-110 min while neurokinin A (NKA) desensitizes neurokinin A receptor for 25-35 min in Xenopus laevis oocytes. In the present study, endothelin A receptor and neurokinin A receptor were coexpressed in Xenopus laevis oocytes in an effort to characterize heterologous desensitization of the receptors that activate phospholipase C-beta. ET1 desensitizes both the endothelin A receptor and the neurokinin A receptor for 90-110 min, whereas stimulation with NKA desensitizes the same two receptors for only 25-35 min.

Regions of the human neurokinin A receptor involved in the generation of second messengers and in receptor desensitization.

Deletion analysis was used to study sites of human Neurokinin A receptor (HNKAR) necessary for signal transduction in CHO cells. Deletion of 62 and 81 amino acids from the c-terminus of HNKAR forms mutant receptors HNKAR delta 62 and HNKAR delta 81, which bind neurokinin A with high affinity but are functionally different. Wild type HNKAR and HNKAR delta 62 are functionally active whereas HNKAR delta 81 is functionally inactive.

Prolonged desensitization of the human endothelin A receptor in Xenopus oocytes. Comparative studies with the human neurokinin A receptor.

Human endothelin (ET) A receptor (hETAR) is a G-protein-mediated receptor that binds ET1 with high affinity and ET2 and ET3 with lower affinities. ET1 is the most potent endogenous vasoconstrictor known at this time. When expressed in Xenopus laevis oocytes, hETAR is rapidly desensitized after stimulation with ET1.

Desensitization of human endothelin A receptor.

The response of G-protein-coupled receptors is modulated by homologous desensitization. Because endothelin A receptor (ETA) plays a part in vasoconstriction, the extent of desensitization and resensitization of endothelin responsiveness was studied. A cDNA clone encoding human ETA receptor was isolated based on similarities to bovine endothelin A receptor.

Epidermal growth factor receptor monoclonal antibodies inhibit the growth of lung cancer cell lines.

The ability of monoclonal antibody (MAb 108), an immunoglobulin G (IgG)2a against the epidermal growth factor receptor (EGF-R), to interact with lung cancer cell lines was investigated. 125I-EGF bound with high affinity to non-small-cell lung cancer (NSCLC) cells, and MAb 108 inhibited specific binding of nine NSCLC cell lines in a dose-dependent manner (IC50 = 0.3-3 micrograms per ml).

Cloning and chromosomal localization of a human endothelin ETA receptor.

A cDNA clone encoding a human endothelin receptor was isolated from a placenta cDNA library. The deduced amino acid sequence of the clone is 94% identical to the bovine endothelin ETA receptor and represents the human homologue. The human endothelin ETA receptor gene was localized to chromosome 4 by analysis of its segregation pattern in rodent-human hybrids.

Cloning and expression of the human substance K receptor and analysis of its role in mitogenesis.

The primary structure of the human substance K receptor was established from the sequences of complementary DNA clones isolated from a human jejunal complementary DNA library. It consists of 398 amino acids, including seven putative transmembrane regions. The gene for the human substance K receptor was localized to chromosome region 10p13-10q23, a region with frequent chromosomal abnormalities.

Characterization of the detergent solubilized receptor for gastrin-releasing peptide.

Properties of detergent solubilized gastrin-releasing peptide receptor were investigated. Swiss 3T3 membranes were covalently labeled with [125I]GRP and homobifunctional cross-linkers. A major labeled protein of 75 kDa was resolved using SDS-polyacrylamide gel electrophoresis.

Lung carcinoid cell lines have bombesin-like peptides and EGF receptors.

The biochemical properties of lung cancer cell lines were investigated. Bombesin-like peptides were present in three small cell lung cancer (SCLC) cell lines examined and three of four lung carcinoids but not in five non-small cell lung cancer (NSCLC) cell lines. Therefore SCLC and some lung carcinoids, but not NSCLC, are enriched in neuroendocrine properties.

Casein kinase II enhances the DNA binding activity of serum response factor.

Serum response factor (SRF) is a mammalian transcription factor that binds to the serum response element in the enhancer of the c-fos proto-oncogene and thus may mediate serum-induction of c-fos transcription. We report here that the DNA binding activity of recombinant SRF made in Escherichia coli can be greatly enhanced by incubation of the protein with HeLa cell nuclear extract. The enhancing activity is ATP or GTP dependent and cofractionates with a protein kinase that phosphorylates SRF on a specific tryptic peptide.

Solubilization of the receptor for the neuropeptide gastrin-releasing peptide (bombesin) with functional ligand binding properties.

The receptor for the neuropeptide gastrin-releasing peptide, the mammalian homologue of bombesin, was solubilized from rat brain and Swiss 3T3 cells by using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS) and the cholesteryl hemisuccinate ester (CHS). Only the combination of the detergent CHAPS and the cholesteryl ester CHS in a glycerol-containing buffer satisfactorily preserved the binding activity upon solubilization. Specific binding activity was only solubilized from cell lines and tissue preparations known to express the GRP receptor.

High-affinity epidermal growth factor binding is specifically reduced by a monoclonal antibody, and appears necessary for early responses.

We have tested the effects of an mAb directed against the protein core of the extracellular domain of the human EGF receptor (mAb108), on the binding of EGF, and on the early responses of cells to EGF presentation. We used NIH 3T3 cells devoid of murine EGF receptor, transfected with a cDNA encoding the full-length human EGF receptor gene, and fully responsive to EGF. The binding to saturation of mAb108 to the surface of these cells at 4 degrees C and at other temperatures specifically reduced high-affinity binding of EGF, but did not change the dissociation constant or the estimated number of binding sites for low-affinity binding of EGF.

Anti-epidermal growth factor receptor antibodies inhibit the autocrine-stimulated growth of MDA-468 human breast cancer cells.

The response of malignant and nonmalignant human breast cell lines to the growth inhibitory effects of monoclonal antibodies against the epidermal growth factor (EGF) receptor was studied. A series of human breast cell lines, which express EGF receptor, were used: MDA-468, MDA-231, and Hs578T human breast cancer cells and the transformed human mammary epithelial cell lines 184A1N4 and 184A1N4-T that have been benzo[a]pyrene immortalized and further transformed with SV40T, respectively. Four antibodies of two different classes were tested: 225 immunoglobulin G (IgG), 108.

All autophosphorylation sites of epidermal growth factor (EGF) receptor and HER2/neu are located in their carboxyl-terminal tails. Identification of a novel site in EGF receptor.

Activation of the epidermal growth factor (EGF) receptor kinase leads to autophosphorylation and to the phosphorylation of various cellular substrates. The three known autophosphorylation sites of EGF receptor are located at the carboxyl-terminal tail where they probably act to compete with and thus modulate substrate phosphorylation. Mutational analysis and microsequencing techniques have been used to localize and identify new autophosphorylation site(s) of the EGF receptor.

Intranasal immunization with proteoliposomes protects against influenza.

Worldwide, influenza virus remains a serious disease which has successfully eluded numerous attempts to design a consistently effective vaccine. In part, these attempts have been thwarted because of a lack of basic understanding of the mechanisms which mediate protection and recovery from influenza infection. A better understanding of the roles of secretory antibody, serum antibody and cell mediated immunity vis-à-vis protection and recovery from influenza infection has allowed us more rationally to approach the design and administration of a vaccine for influenza.

Evidence that autophosphorylation of solubilized receptors for epidermal growth factor is mediated by intermolecular cross-phosphorylation.

Structurally distinguishable mutants of human epidermal growth factor receptor (EGFR) were used to investigate the mechanism of EGFR autophosphorylation. Mutant receptors generated by site-directed mutagenesis were expressed in transfected NIH 3T3 cells lacking endogenous receptors. After coincubation of cell lysates in the presence or absence of EGF, receptor immunoprecipitates were incubated with [gamma-32P]ATP.

Human glioblastoma cell lines have neuropeptide receptors for bombesin/gastrin-releasing peptide.

Bombesin/gastrin-releasing peptide receptors were characterized in human glioblastoma cell lines. [125I]Gastrin-releasing peptide or ([125I]Tyr4)bombesin bound with high affinity to these cell lines. Binding to cell line U-118 was time dependent, reversible, and specific.

Kinetic parameters of the protein tyrosine kinase activity of EGF-receptor mutants with individually altered autophosphorylation sites.

Epidermal growth factor (EGF)-receptor mutants in which individual autophosphorylation sites (Tyr1068, Tyr1148 or Tyr1173) have been replaced by phenylalanine residues were expressed in NIH-3T3 cells lacking endogenous EGF-receptors. Kinetic parameters of the kinase of wild-type and mutant receptors were compared. Both wild-type and mutant EGF-receptors had a Km(ATP) 1-3 microM for the autophosphorylation reaction, and a Km(ATP) of 3-7 microM for the phosphorylation of a peptide substrate.

Sporadic amplification of the HER2/neu protooncogene in adenocarcinomas of various tissues.

The HER2/neu protooncogene was found to be amplified in 6 of 109 primary adenocarcinoma tumors. No HER2/neu amplification was found in 29 other primary nonadenocarcinomatous tumors. In two colon tumors, in addition to the amplification, DNA rearrangement of HER2/neu gene was also observed.

Passive serum antibody causes temporary recovery from influenza virus infection of the nose, trachea and lung of nude mice.

BALB/c normal and nude mice were infected with a non-lethal mouse-passaged A/PC/1/73 (H3N2) influenza virus in order to assess the role of T cells on the course of disease of the nose, trachea and lung. The tracheal epithelium of both mouse strains was desquamated by 3 days after infection. Although normal regeneration began, nude mice never completed that regeneration whereas normal mice had fully regenerated tracheas by Day 14.