Discovery of a novel chemotype of potent human ENaC blockers using a bioisostere approach. Part 1: quaternary amines

We report the identification of a novel series of human epithelial sodium channel (ENaC) blockers that are structurally distinct from the pyrazinoyl guanidine chemotype found in prototypical ENaC blockers such as amiloride. Following a rational design hypothesis a series of quaternary amines were prepared and evaluated for their ability to block ion transport via ENaC in human bronchial epithelial cells (HBECs). Compound 11 has an IC(50) of 200nM and is efficacious in the Guinea-pig tracheal potential difference (TPD) model of ENaC blockade with an ED(50) of 44μgkg(-1) at 1h. As such, pyrazinoyl quaternary amines represent the first examples of a promising new class of human ENaC blockers.     

The degree of coupling of nuclear rotation in binucleate 3T3 cells

In order to test the existence of mechanical coupling between the rotational movements of two adjacent nuclei, we prepared binucleate 3T3 cells and observed their nuclear movements by near infrared microscopy and recorded them with time-lapse video techniques. We found that 49 out of 110 (44%) of the selected binucleate cells expressed nuclear rotation. Rotation could occur in just one of the nuclei while the second nucleus remained stationary (31/110) or in both nuclei simultaneously (18/110). In almost all cases where both nuclei rotated simultaneously (15/110) they did so at different speeds and in opposite directions. The nuclei were observed to rotate in the same direction in only three of the examples. The results are consistent with a weak mechanical interaction between a rotating nucleus and its neighbor. Consistent with our previous observations in mononucleate cells, we did not find a characteristic position of the centrosphere or a special distribution of the microtubules or the intermediate filaments in binucleate cells with rotating nuclei. There was an absence of long, well-formed microfilament bundles beneath the nuclei during rotation, even in the local region beneath the rotating nucleus in those cells with one rotating and one stationary nucleus. Also consistent with observations of mononucleate cells, nuclear rotation was inhibited by treatment with colcemid, although the ability of the nuclei to rotate was eventually restored when the colcemid-containing medium was replaced with normal medium.     

RING-POSITION AND FREQUENCY OF ADJACENT DISTRIBUTION OF MEIOTIC CHROMOSOMES IN RHOEO SPATHACEA

The morphological sequence of the twelve chromosomes around the ring as worked out by Sax is reaffirmed with slight corrections of the centromere position on three chromosomes: Aa, fA, and Dd. Adjacent distribution was found in 53/120 MI PMC (44.2%). Ring-position analysis was achieved in 34 of the 53. There were 127 chromosomes and 66 arm-pairs involved in adjacent distribution in these 34 MI PMC. Adjacent distributions occurred at random among the twelve chromosome positions and among the twelve arm-pair positions. There were eleven instances among the 66 arm-pairs (16.7%) of adjacent distribution despite free ends due to chiasma failure. Up to four consecutive chromosomes may pass to the same pole. Not all cells with 6–6 distribution are genetically balanced. Distribution of 7–5 occurred in 24/120 AI PMC (20.0%). Another nine (7.5%) in the same sample had one or more lagging chromosomes. At MI, three PMC had 8–4 distribution, but none such were seen at AI.     

Reaction centers from three herbicide-resistant mutants of Rhodobacter sphaeroides 2.4.1: sequence analysis and preliminary characterization

Many herbicides that inhibit photosynthesis in plants also inhibit photosynthesis in bacteria. We have isolated three mutants of the photosynthetic bacterium Rhodobacter sphaeroides that were selected for increased resistance to the herbicide terbutryne. All three mutants also showed increased resistance to the known electron transfer inhibitor o-phenanthroline. The primary structures of the mutants were determined by recombinant DNA techniques. All mutations were located on the gene coding for the L-subunit resulting in these changes Ile²²⁹ Met, Ser²²³ Pro and Tyr²²² Gly. The mutations of Ser²²³ is analogous to the mutation of Ser²⁶⁴ in the D1 subunit of photosystem II in green plants, strengthening the functional analogy between D1 and the bacterial L-subunit. The changed amino acids of the mutant strains form part of the binding pocket for the secondary quinone, Q _b . This is consistent with the idea that the herbicides are competitive inhibitors for the Q _b binding site. The reaction centers of the mutants were characterized with respect to electron transfer rates, inhibition constants of terbutryne and o-phenanthroline, and binding constants of the quinone UQ₀ and the inhibitors. By correlating these results with the three-dimensional structure obtained from x-ray analysis by Allen et al. (1987a, 1987b), the likely positions of o-phenanthroline and terbutryne were deduced. These correspond to the positions deduced by Michel et al. (1986a) for Rhodopseudomonas viridis.Abbreviations ATP adenosine 5-triphosphate - Bchl bacteriochlorophyll - Bphe bacteriopheophytin - bp basepair - cyt c²⁺ reduced form of cytochrome c - DEAE diethylami-noethyl - EDTA ethylenediamine tetraacetic acid - Fe²⁺ non-heme iron atom - LDAO lauryl dimethylamine oxide - Pipes piperazine-N,N-bis-2-ethane-sulfonic acid - PSII photosystem II - RC reaction center - SDS sodium dodecylsulfate - Tris tris(hydroxy-methyl)aminomethane - UQ₀ 2,3-dimethoxy-5-methyl benzoquinone - UQ₁₀ ubiquinone 50     

Aged BALB/c mice as a model for increased severity of severe acute respiratory syndrome in elderly humans

Advanced age has repeatedly been identified as an independent correlate of adverse outcome and a predictor of mortality in cases of severe acute respiratory syndrome (SARS). SARS-associated mortality may exceed 50% for persons aged 60 years or older. Heightened susceptibility of the elderly to severe SARS and the ability of SARS coronavirus to replicate in mice led us to examine whether aged mice might be susceptible to disease. We report here that viral replication in aged mice was associated with clinical illness and pneumonia, demonstrating an age-related susceptibility to SARS disease in animals that parallels the human experience.     

X-Ray structure determination of three mutants of the bacterial photosynthetic reaction centers from Rb. sphaeroides; altered proton transfer pathways

In the photosynthetic reaction center (RC) from Rhodobacter sphaeroides, the reduction of a bound quinone molecule Q(B) is coupled with proton uptake. When Asp-L213 is replaced by Asn, proton transfer is inhibited. Proton transfer was restored by two second-site revertant mutations, Arg-M233-->Cys and Arg-H177-->His. Kinetic effects of Cd(2+) on proton transfer showed that the entry point in revertant RCs to be the same as in the native RC. The structures of the parental and two revertant RCs were determined at resolutions of 2.10, 1.80, and 2.75 A. From the structures, we were able to delineate alternate proton transfer pathways in the revertants. The main changes occur near Glu-H173, which allow it to substitute for the missing Asp-L213. The electrostatic changes near Glu-H173 cause it to be a good proton donor and acceptor, and the structural changes create a cavity which accommodates water molecules that connect Glu-H173 to other proton transfer components.     

Synaptic abnormalities in a Drosophila model of Alzheimer’s disease

Alzheimer’s disease (AD) is an age-related neurodegenerative disease characterized by memory loss and decreased synaptic function. Advances in transgenic animal models of AD have facilitated our understanding of this disorder, and have aided in the development, speed and efficiency of testing potential therapeutics. Recently, we have described the characterization of a novel model of AD in the fruit fly, Drosophila melanogaster, where we expressed the human AD-associated proteins APP and BACE in the central nervous system of the fly. Here we describe synaptic defects in the larval neuromuscular junction (NMJ) in this model. Our results indicate that expression of human APP and BACE at the larval NMJ leads to defective larval locomotion behavior, decreased presynaptic connections, altered mitochondrial localization in presynaptic motor neurons and decreased postsynaptic protein levels. Treating larvae expressing APP and BACE with the γ-secretase inhibitor L-685,458 suppresses the behavioral defects as well as the pre- and postsynaptic defects. We suggest that this model will be useful to assess and model the synaptic dysfunction normally associated with AD, and will also serve as a powerful in vivo tool for rapid testing of potential therapeutics for AD.KEY WORDS: APP, Alzheimer’s disease, Drosophila, BACE, Synapse, NMJ