16 alpha-substituted analogs of the antiprogestin RU486 induce a unique conformation in the human progesterone receptor resulting in mixed agonist activity.

Previously, we have shown that agonists and antagonists interact with distinct, though overlapping regions within the human progesterone receptor (hPR) resulting in the formation of structurally different complexes. Thus, a link was established between the structure of a ligand-receptor complex and biological activity. In this study, we have utilized a series of in vitro assays with which to study hPR pharmacology and have identified a third class of hPR ligands that induce a receptor conformation which is distinct from that induced by agonists or antagonists. Importantly, when assayed on PR-responsive target genes these compounds were shown to exhibit partial agonist activity; an activity that was influenced by cell context. Thus, as has been shown previously for estrogen receptor, the overall structure of the ligand-receptor complex is influenced by the nature of the ligand. It appears, therefore, that the observed differences in the activity of some PR and estrogen receptor ligands reflect the ability of the cellular transcription machinery to discriminate between the structurally different complexes that result following ligand interaction. These data support the increasingly favored hypothesis that different ligands can interact with different regions within the hormone binding domains of steroid hormone receptors resulting in different biologies.

The glycine binding site of the N-methyl-D-aspartate receptor subunit NR1: identification of novel determinants of co-agonist potentiation in the extracellular M3-M4 loop region.

The N-methyl-D-aspartate (NMDA) subtype of ionotropic glutamate receptors is a heterooligomeric membrane protein composed of homologous subunits. Here, the contribution of the M3-M4 loop of the NR1 subunit to the binding of glutamate and the co-agonist glycine was investigated by site-directed mutagenesis. Substitution of the phenylalanine residues at positions 735 or 736 of the M3-M4 loop produced a 15- to 30-fold reduction in apparent glycine affinity without affecting the binding of glutamate and the competitive glycine antagonist 7-chlorokynurenic acid; mutation of both residues caused a >100-fold decrease in glycine affinity. These residues are found in a C-terminal region of the M3-M4 loop that shows significant sequence similarity to bacterial amino acid-binding proteins. Epitope tagging revealed both the N-terminus and the M3-M4 loop to be exposed extracellularly, whereas a C-terminal epitope was localized intracellularly. These results indicate that the M3-M4 loop is part of the ligand-binding pocket of the NR1 subunit and provide the basis for a refined model of the glycine-binding site of the NMDA receptor.

Mutational properties of the primary aflatoxin B1-DNA adduct.

The mutagenic activity of the major DNA adduct formed by the liver carcinogen aflatoxin B1 (AFB1) was investigated in vivo. An oligonucleotide containing a single 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 (AFB1-N7-Gua) adduct was inserted into the single-stranded genome of bacteriophage M13. Replication in SOS-induced Escherichia coli yielded a mutation frequency for AFB1-N7-Gua of 4%. The predominant mutation was G --> T, identical to the principal mutation in human liver tumors believed to be induced by aflatoxin. The G --> T mutations of AFB1-N7-Gua, unlike those (if the AFB1-N7-Gua-derived apurinic site, were much more strongly dependent on MucAB than UmuDC, a pattern matching that in intact cells treated with the toxin. It is concluded that the AFB1-N7-Gua adduct, and not the apurinic site, has genetic requirements for mutagenesis that best explain mutations in aflatoxin-treated cells. While most mutations were targeted to the site of the lesion, a significant fraction (13%) occurred at the base 5' to the modified guanine. In contrast, the apurinic site-containing genome gave rise only to targeted mutations. The mutational asymmetry observed for AFB1-N7-Gua is consistent with structural models indicating that the aflatoxin moiety of the aflatoxin guanine adduct is covalently intercalated on the 5' face of the guanine residue. These results suggest a molecular mechanism that could explain an important step in the carcinogenicity of aflatoxin B1.

In vivo and in vitro characterization of the B1 and B2 zinc-binding domains from the acute promyelocytic leukemia protooncoprotein PML.

Acute promyelocytic leukemia (APL) has been ascribed to a chromosomal translocation event which results in a fusion protein comprising the PML protein and retinoic acid receptor alpha. PML is normally a component of a nuclear multiprotein complex which is disrupted in the APL disease state. Here, two newly defined cysteine/histidine-rich protein motifs called the B-box (B1 and B2) from PML have been characterized in terms of their effect on PML nuclear body formation, their dimerization, and their biophysical properties. We have shown that both peptides bind Zn2+, which induces changes in the peptides' structures. We demonstrate that mutants in both B1 and B2 do not form PML nuclear bodies in vivo and have a phenotype that is different from that observed in the APL disease state. Interestingly, these mutations do not affect the ability of wild-type PML to dimerize with mutant proteins in vitro, suggesting that the B1 and B2 domains are involved in an additional interaction central to PML nuclear body formation. This report in conjunction with our previous work demonstrates that the PML RING-Bl/B2 motif plays a fundamental role in formation of a large multiprotein complex, a function that may be common to those unrelated proteins which contain the motif.

Agonist-induced desensitization of dopamine D1 receptor-stimulated adenylyl cyclase activity is temporally and biochemically separated from D1 receptor internalization.

The regulation of the dopamine D1 receptor was investigated by using c-myc epitope-tagged D1 receptors expressed in Sf9 (fall armyworm ovary) cells. Treatment of D1 receptors with 10 microM dopamine for 15 min led to a loss of the dopamine-detected high-affinity state of the receptor accompanying a 40% reduction in the ability of the receptor to mediate maximal dopamine stimulation of adenylyl cyclase activity. After 60 min of agonist exposure, 45 min after the occurrence of desensitization, 28% of the cell surface receptors were internalized into an intracellular light vesicular membrane fraction as determined by radioligand binding and supported by photoaffinity labeling, immunocytochemical staining, and immunoblot analysis. Pretreatment of cells with concanavalin A or sucrose completely blocked agonist-induced D1 receptor internalization without preventing agonist-induced desensitization, indicating a biochemical separation of these processes. Collectively, these findings indicate that the desensitization of D1 receptor-coupled adenylyl cyclase activity and D1 receptor internalization are temporarily and biochemically distinct mechanisms regulating D1 receptor function following agonist activation.

A nonpeptide agonist of the invertebrate receptor for SchistoFLRFamide (PDVDHVFLRFamide), a member of a subfamily of insect FMRFamide-related peptides.

We describe a nonpeptide mimetic analog of an invertebrate peptide receptor. Benzethonium chloride (Bztc) is an agonist of the SchistoFLRFamide (PDVDHVFLRFamide) receptors found on locust oviducts. Bztc competitively displaces [125I-labeled Y1]SchistoFLRFamide binding to both high- and low-affinity receptors of membrane preparations. Bztc mimics the physiological effects of SchistoFLRFamide on locust oviduct, by inhibiting myogenic and induced contractions in a dose-dependent manner. Bztc is therefore recognized by the binding and activation regions of the SchistoFLRFamide receptors. This discovery provides a unique opportunity within insects to finally target a peptide receptor for the development of future pest management strategies.

In vitro autoradiography of receptor-activated G proteins in rat brain by agonist-stimulated guanylyl 5'-[gamma-[35S]thio]-triphosphate binding.

Agonists stimulate guanylyl 5'-[gamma-[35S]thio]-triphosphate (GTP[gamma-35S]) binding to receptor-coupled guanine nucleotide binding protein (G proteins) in cell membranes as revealed in the presence of excess GDP. We now report that this reaction can be used to neuroanatomically localize receptor-activated G proteins in brain sections by in vitro autoradiography of GTP[gamma-35S] binding. Using the mu opioid-selective peptide [D-Ala2,N-MePhe4,Gly5-ol]enkephalin (DAMGO) as an agonist in rat brain sections and isolated thalamic membranes, agonist stimulation of GTP[gamma-35S] binding required the presence of excess GDP (1-2 mM GDP in sections vs. 10-30 microM GDP in membranes) to decrease basal G-protein activity and reveal agonist-stimulated GTP[gamma-35S] binding. Similar concentrations of DAMGO were required to stimulate GTP[gamma-35S] binding in sections and membranes. To demonstrate the general applicability of the technique, agonist-stimulated GTP[gamma-35S] binding in tissue sections was assessed with agonists for the mu opioid (DAMGO), cannabinoid (WIN 55212-2), and gamma-aminobutyric acid type B (baclofen) receptors. For opioid and cannabinoid receptors, agonist stimulation of GTP[gamma-35S] binding was blocked by incubation with agonists in the presence of the appropriate antagonists (naloxone for mu opioid and SR-141716A for cannabinoid), thus demonstrating that the effect was specifically receptor mediated. The anatomical distribution of agonist-stimulated GTP[gamma-35S] binding qualitatively paralleled receptor distribution as determined by receptor binding autoradiography. However, quantitative differences suggest that variations in coupling efficiency may exist between different receptors in various brain regions. This technique provides a method of functional neuroanatomy that identifies changes in the activation of G proteins by specific receptors.

Potent interleukin 3 receptor agonist with selectively enhanced hematopoietic activity relative to recombinant human interleukin 3.

A systematic evaluation of structure-activity information led to the construction of genetically engineered interleukin 3 (IL-3) receptor agonists (synthokines) with enhanced hematopoietic potency. SC-55494, the most extensively characterized member of this series, exhibits 10- to 20-fold greater biological activity than recombinant human IL-3 (rhIL-3) in human hematopoietic cell proliferation and marrow colony-forming-unit assays. In contrast, SC-55494 is only twice as active as rhIL-3 in priming the synthesis of inflammatory mediators such as leukotriene C4 and triggering the release of histamine from peripheral blood leukocytes. The enhanced hematopoietic activity of SC-55494 correlates with a 60-fold increase in IL-3 alpha-subunit binding affinity and a 20-fold greater affinity for binding to alpha/beta receptor complexes on intact cells relative to rhIL-3. SC-55494 demonstrates a 5- to 10-fold enhanced hematopoietic response relative to its ability to activate the priming and release of inflammatory mediators. Therefore, SC-55494 may ameliorate the myeloablation of cancer therapeutic regimens while minimizing dose-limiting inflammatory side effects.

Determinação de biomarcadores de aflatoxina B1 e aplicabilidade na avaliação da eficiência de adsorventes em frangos de corte

As aflatoxinas são metabólitos secundários produzidos por fungos toxigênicos das espécies Aspergillus flavus, A. parasiticus e A. nomius. São amplamente encontradas em matérias-primas de rações animais, em especial o milho, e têm a capacidade de levar a quadros clínicos agudos ou crônicos de aflatoxicose, caracterizados por, desde a morte por hepatite aguda até a diminuição do desempenho zootécnico por diminuição de peso ou consumo de ração. A aflatoxina B1 tem sido considerada o metabólito mais perigoso, uma vez que possui alto poder hepatotóxico, além de ser mutagênica e carcinogênica. Atualmente a ciência trabalha rumo à descoberta de substâncias que sejam indicadoras confiáveis de contaminação por componentes tóxicos em homens e em animais, os chamados biomarcadores, que medem uma mudança celular, biológica ou molecular em um meio biológico (tecidos humanos, células ou fluídos) que fornecem informação a respeito de uma doença ou exposição a uma determinada substância. Sua detecção pode auxiliar na identificação, no diagnóstico e no tratamento de indivíduos afetados que podem estar sob risco, mas ainda não exibem os sintomas. Sendo assim, com o auxílio de análises que confirmem a patogenicidade da aflatoxina B1 (determinação da atividade de enzimas hepáticas, da avaliação da função renal, de hematologia, da dosagem de minerais séricos e da avaliação de desempenho zootécnico), o objetivo deste trabalho foi avaliar a aplicabilidade da determinação de resíduos hepáticos de aflatoxinas e do aduto sérico AFB1-lisina na avaliação da eficiência de adsorventes em frangos de corte. Utilizou-se 240 pintos de 1 dia, machos, de linhagem Cobb 500®, distribuídos aleatoriamente em 4 dietas experimentais: Controle Negativo: Ração Basal (RB); RB + 0,5% de adsorvente ((aluminosilicato de cálcio e sódio hidratado/HSCAS); RB + 0,5% de adsorvente + 500 µg de AFB1/kg de ração e; RB + 500 µg de AFB1/kg de ração.Os resultados experimentais mostram que o efeito deletério da AFB1, na concentração utilizada, é mais pronunciado que os efeitos protetores do HSCAS sobre os parâmetros de saúde dos animais. Não houve ação efetiva do adsorvente utilizado sobre quase nenhuma variável estudada, apenas para a redução das lesões histopatológicas em fígado, na redução da concentração de gama-glutamiltransferase (GGT), fósforo e aumento da contagem de hemáceas aos 21 dias de idade. Porém, influenciou positivamente a redução de resíduos hepáticos de aflatoxina G1 aos 21 dias e as concentrações de AFB1-lisina sérica aos 21 e aos 42 dias de idade. Estes dados são importantes porque permite concluir que, embora sintomatologicamente o HSCAS não tenha exercido função efetiva, molecularmente foi capaz de mostrar de eficácia sobre os alguns biomarcadores de aflatoxinas no organismo das aves

Neuroprotective effects of the cannabinoid agonist HU210 on retinal degeneration

Cannabinoids have been demonstrated to exert neuroprotective effects on different types of neuronal insults. Here we have addressed the therapeutic potential of the synthetic cannabinoid HU210 on photoreceptor degeneration, synaptic connectivity and functional activity of the retina in the transgenic P23H rat, an animal model for autosomal dominant retinitis pigmentosa (RP). In P23H rats administered with HU210 (100 μg/kg, i.p.) from P24 to P90, ERG recordings showed an amelioration of vision loss, as compared to vehicle-administered animals. Under scotopic conditions, the maximum a-wave amplitudes recorded at P60 and P90 were higher in HU210-treated animals, as compared to the values obtained in untreated animals. The scotopic b-waves were significantly higher in treated animals than in untreated rats at P30, P60 and P90. This attenuation of visual deterioration correlated with a delay in photoreceptor degeneration and the preservation of retinal cytoarchitecture. HU210-treated animals had 40% more photoreceptors than untreated animals. Presynaptic and postsynaptic elements, as well as the synaptic contacts between photoreceptors and bipolar or horizontal cells, were also preserved in HU210-treated P23H rats. These results indicate that HU210 preserves cone and rod structure and function, together with their contacts with postsynaptic neurons, in P23H rats. These data suggest that cannabinoids are potentially useful to delay retinal degeneration in RP patients.

Herbert Hoover national historic site, interpretive prospectus (1971) B1; Natural resources management plan (1980) B2; Cultural resources management plan (1981) B3; Annual statement for interpretation and visitor services B4; Snowmobile use, environmental assessment (EA) : environmental impact statement.

Mode of access: Internet.

Pipe Spring National Monument (N.M.), interim interpretive plan, second draft (1977) B1; Master plan (1978) B2; Development concept plan, environmental assessment (EA) B3; Statement for management (1981) B4; Record of decision, draft development concept plan and Finding of no significant impact (FONSI) (1982) : environmental impact statement.

Mode of access: Internet.

Lake Flower boat launch facility, Greater Adirondack RC&D area, Essex County, RC&D measure plan B1; Finding of no significant impact (FONSI) : environmental impact statement.

Mode of access: Internet.