Donor-acceptor tetrahydrochrysenes, inherently fluorescent, high-affinity ligands for the estrogen receptor: binding and fluorescence characteristics and fluorometric assay of receptor.

We have examined the binding behavior and fluorescence characteristics of a series of novel ligands for the estrogen receptor (ER). These ligands are derivatives of 5,6,11,12-tetrahydrochrysene (THC), a structure that embodies a stilbene chromophore, found in many nonsteroidal estrogens, within a rigid tetracyclic system where it cannot easily be distorted from planarity, thus providing the conjugation and rigidity required for efficient fluorescence. Additional steric bulk, as trans-disposed ethyl substituents at the internal C-5 and C-11 positions, is required for the highest relative binding affinity (RBA), and the trans-5,11-diethyl-2,8-dihydroxy-THC derivative binds to ER with an affinity greater than that of estradiol. The replacement of one of the phenolic hydroxyl groups of this THC derivative with an electron-withdrawing group (COMe, COOMe, CONH2, CN, or NO2) yields unsymmetrical THCs with binding affinities 15-40% that of estradiol (E2). The fluorescence emission shifts from about 380 nm for the dihydroxy THC to 475-688 nm for the donor-acceptor THCs. The emission of these donor-acceptor THCs is highly solvatochromic and shifts to longer wavelengths as the solvent polarity increases. In ethanol, the fluorescence quantum yield of the first four of these compounds is high (phi f = 0.43-0.69), but the fifth compound, the nitro-THC, is almost nonemissive in protic solvents. When they are incubated with protein solutions containing ER (approximately 10(-9) M), the emission from the donor-acceptor THCs bound specifically to ER is in the 500-570-nm range, whereas fluorescence from non-receptor-bound fluorophores is in the 425-460-nm range. Thus, fluorescence from these probes bound specifically to ER could be measured under equilibrium conditions as well as after the removal of free and non-receptor-bound material by treatment with charcoal-dextran. This is one of the first demonstrations of ligands whose fluorescence is distinctly different when free, when bound to ER, or when bound to non-receptor proteins. It is also the first demonstration of ER assay by fluorescence under equilibrium conditions.     

An Ustilago maydis Mutant Partially Blocked in P45014DM Activity Is Hypersensitive to Azole Fungicides

An Ustilago maydis ergosterol biosynthesis mutant (A14) which is partially blocked in sterol 14alpha-demethylase (P45014DM) activity is described. This mutant accumulated the abnormal 14alpha-methyl sterols, eburicol, 14alpha-methylfecosterol, and obtusifoliol, along with significant amounts of ergosterol. Although the A14 mutant grew nearly as well as the wild type, it was impaired in cell extension growth, which indicated a dysfunction in apical cell wall synthesis. The mutant was also found to be hypersensitive to the azole fungicides penconazole and tebuconazole.     

Hexazonium pararosaniline as a fixative for animal tissues

Hexazonium pararosaniline is a valuable reagent that has been used in enzyme activity histochemistry for 50 years. It is an aqueous solution containing the tris-diazonium ion derived from pararosaniline, an aminotriarylmethane dye, and it contains an excess of nitrous acid that was not consumed in the diazotization reaction. Other investigators have found that immersion for 2 min in an acidic (pH 3.5) 0.0015 M hexazonium pararosaniline solution can protect cryostat sections of unfixed animal tissues from the deleterious effects of aqueous reagents such as buffered solutions used in immunohistochemistry, while preserving specific affinities for antibodies. In the present investigation hexazonium pararosaniline protected lymphoid tissue and striated muscle against the damaging effects of water or saline. The same protection was conferred on unfixed sections treated with dilute nitrous or hydrochloric acid in concentrations similar to those in hexazonium pararosaniline solutions. Model tissues (solutions, gels or films containing gelatin and/or bovine albumin) responded predictably to well known cross-linking (formaldehyde) or coagulant (mercuric chloride) fixatives. Hexazonium pararosaniline solutions prevented the dissolution of protein gels in water only after 9 or more days of contact, during which time considerable swelling occurred. It is concluded that there is no evidence for a “fixative” action of hexazonium pararosaniline. The protective effect on frozen sections of unfixed tissue is attributable probably to the low pH of the solution.     

Economic evaluation of water loss saving due to the biological control of water hyacinth at New Year’s Dam,Eastern Cape province,South Africa

Water hyacinth Eichhornia crassipes is considered the most damaging aquatic weed in the world. However, few studies have quantified the impact of this weed economically and ecologically, and even fewer studies have quantified the benefits of its control. This paper focuses on water loss saving as the benefit derived from biological control of this plant between 1990 and 2013 at New Year’s Dam, Alicedale, Eastern Cape, South Africa. Estimates of water loss due to evapotranspiration from water hyacinth vary significantly; therefore, the study used three different rates, high, medium and low. A conservative raw agriculture value of R 0.26 per m³ was used to calculate the benefits derived by the water saved. The present benefit and cost values were determined using 10% and 5% discount rates. The benefit/cost ratio at the low evapotranspiration rate was less than one, implying that biological control was not economically viable but, at the higher evapotranspiration rates, the return justified the costs of biological control. However, at the marginal value product of water, the inclusion of the costs of damage to infrastructure, or the adverse effects of water hyacinth on biodiversity, would justify the use of biological control, even at the low transpiration rate.     

The Tau/A152T mutation,a risk factor for frontotemporal‐spectrum disorders,leads to NR2B receptor‐mediated excitotoxicity

We report on a novel transgenic mouse model expressing human full‐length Tau with the Tau mutation A152T (hTau^AT), a risk factor for FTD‐spectrum disorders including PSP and CBD. Brain neurons reveal pathological Tau conformation, hyperphosphorylation, mis‐sorting, aggregation, neuronal degeneration, and progressive loss, most prominently in area CA3 of the hippocampus. The mossy fiber pathway shows enhanced basal synaptic transmission without changes in short‐ or long‐term plasticity. In organotypic hippocampal slices, extracellular glutamate increases early above control levels, followed by a rise in neurotoxicity. These changes are normalized by inhibiting neurotransmitter release or by blocking voltage‐gated sodium channels. CA3 neurons show elevated intracellular calcium during rest and after activity induction which is sensitive to NR2B antagonizing drugs, demonstrating a pivotal role of extrasynaptic NMDA receptors. Slices show pronounced epileptiform activity and axonal sprouting of mossy fibers. Excitotoxic neuronal death is ameliorated by ceftriaxone, which stimulates astrocytic glutamate uptake via the transporter EAAT2/GLT1. In summary, hTau^AT causes excitotoxicity mediated by NR2B‐containing NMDA receptors due to enhanced extracellular glutamate.     

In vitro evaluation of native entomopathogenic fungi and neem (Azadiractha indica) extracts on Spodoptera frugiperda

The control of Spodoptera frugiperda is based on synthetic insecticides, so some alternatives are the use of entomopathogenic fungi (EF) and neem extract. The objective of the study was to evaluate in vitro effectiveness of native EF and neem extracts on S. frugiperda larvae. Six EF were identified by DNA sequencing of ITS regions from three EF (Fusarium solani, Metarrhizium robertsii, Nigrospora spherica and Penicillium citrinum). They were evaluated in concentrations of 1 × 10⁸ spores/ mL. In addition, a second bioassay was carried out evaluating only F. solani, M. robertsii and N. sphaerica and the addition of vegetable oil. On the other hand, extraction of secondary metabolites from neem seed (Azadirachta indica) was carried out by performing, mass (g) and solvent volume (mL ethanol and water) combinations, which were subjected to microwaves and ultrasound. Subsequently, these extracts were evaluated in concentrations of 3%, 4% and 5%. A survival analysis was performed for each of the bioassays. With respect to the results of the first bioassay, F. solani obtained a probability of survival of 0.476 on the seventh day, while in the second bioassay, M. robertsii obtained 0.488 survival probability. This suggests that the expected percentage of larvae that stay alive on the sixth day is 48.8%. However, in the evaluation of the neem extract the combination 1:12/70% to 4% caused 84% mortality of larvae. The use of native HE and neem extracts has potential for the control of S. frugiperda.     

Mitochondrial DNA sequence divergence among Schizaphis graminum (Hemiptera: Aphididae) clones from cultivated and non-cultivated hosts: haplotype and host associations

A 1.0 kb region of the mitochondrial cytochrome oxidase subunit I gene from the greenbug aphid, Schizaphis graminum (Rondani), was sequenced for 24 field collected clones from non-cultivated and cultivated hosts. Maximum likelihood, maximum parsimony and neighbour-joining phylogenies were estimated for these clones, plus 12 previously sequenced clones. All three tests produced trees with identical topologies and confirmed the presence of three clades within S. graminum. Clones showed no relationship between biotype and mtDNA haplotype. At least one biotype was found in all three clades, suggesting exchange among clades of genetic material conditioning for crop virulence, or the sharing of a common ancestor. However, there was a relationship between host and haplotype. Clade 1 was the most homogeneous and contained 12 of 16 clones collected from cultivated hosts and five of the six collected from johnsongrass, Sorghum halepense, a congener of cultivated sorghum, S. bicolor. Four of the six clones collected from Agropyron spp. were found in clade 2. Clade 3 contained two clones from wheat, Triticum aestivum, and four from non-cultivated hosts other than Agropyron spp. A partitioning of populations by mtDNA haplotype and host suggests the occurrence of host adapted races in Schizaphis graminum.