Novel HCV NS5B polymerase inhibitors derived from 4-(1',1'-dioxo-1',4'-dihydro-1'lambda(6)-benzo[1',2',4']thiadiazin-3'-yl)-5-hydroxy-2H-pyridazin-3-ones. Part 5: Exploration of pyridazinones containing 6-amino-substituents

The synthesis of 4-(1',1'-dioxo-1',4'-dihydro-1'lambda(6)-benzo[1',2',4']thiadiazin-3'-yl)-5-hydroxy-2H-pyridazin-3-ones bearing 6-amino substituents as potent inhibitors of the HCV RNA-dependent RNA polymerase (NS5B) is described. Several of these agents also display potent antiviral activity in cell culture experiments (EC(50)<0.10 microM). In vitro DMPK data (microsome t(1/2), Caco-2 P(app)) for many of the compounds are also disclosed, and a crystal structure of a representative inhibitor complexed with the NS5B protein is discussed.     

Intraluminal pressure oscillation enhances subsequent airway contraction in isolated bronchial segments.

A period of deep inspiration in humans has been shown to attenuate subsequent bronchoconstriction, a phenomenon termed bronchoprotection. The bronchoprotective effect of deep inspiration may be caused though a depression in the force production of airway smooth muscle (ASM). We determined the response of whole airway segments and isolated ASM to a period of cyclic stretches. Isovolumetric contraction to electrical field stimulation (EFS) was assessed in porcine bronchial segments before and after intraluminal pressure oscillation from 5 to 25 cmH(2)O for 10 min at 0.5 Hz. Morphometry showed that this pressure oscillation stretched ASM length by 21%. After pressure oscillation, the response to EFS was not reduced but instead was modestly enhanced (P < 0.01). Airway responses to EFS returned to preoscillation levels 10 min after the end of oscillation. The increase in EFS response after pressure oscillation was not altered by the addition of indomethacin. In a separate experiment, we assessed isometric force in isolated ASM strips before and after length oscillation. The amplitude, frequency, and duration of length oscillation were similar to those induced in bronchial segments. In contrast to bronchial segments, length oscillation of ASM produced a significant depression in isometric force induced by EFS (P < 0.01). These results suggest that the response of ASM to length oscillation is modified by the airway wall. They also suggest that the phenomenon of bronchoprotection reported in some in vivo studies may not be an intrinsic property of the airway.     

Modelling of Ca2+-Activated Chloride Current in Tracheal Smooth Muscle Cells

Stimulation of airway myocytes by contractile agents such as acetylcholine (ACh) activates a Ca²⁺-activated Cl^– current (I_ClCa) which may play a key role in calcium homeostasis of airway myocytes and hence in airway reactivity. The aim of the present study was to model I_ClCa in airway smooth muscle cells using a computerised model previously designed for simulation of cardiac myocyte functioning. Modelling was based on a simple resistor-battery permeation model combined with multiple binding site activation by calcium. In order to validate the model, a combination of equations, used to mimic [Ca²⁺]_i response to ACh stimulation, were incorporated into the model. The results indicate that the model developed in this article accounts for experimental recordings and electrophysiological characteristics of this current in airway smooth muscle cells, with parameter values consistent with those calculated from experimental data. Such a model may thus be used to predict I_ClCa functioning, though additional experimental data from airway myocytes would be useful to more accurately determine some parameter values of the model.     

Direct Profiling of Environmental Microbial Populations by Thermal Dissociation Analysis of Native rRNAs Hybridized to Oligonucleotide Microarrays

Oligonucleotide microarrays were used to profile directly extracted rRNA from environmental microbial populations without PCR amplification. In our initial inspection of two distinct estuarine study sites, the hybridization patterns were reproducible and varied between estuarine sediments of differing salinities. The determination of a thermal dissociation curve (i.e., melting profile) for each probe-target duplex provided information on hybridization specificity, which is essential for confirming adequate discrimination between target and nontarget sequences.     

CD8 T cells inhibit IgE via dendritic cell IL-12 induction that promotes Th1 T cell counter-regulation.

Th1 and Th2 cells are counterinhibitory; their balance determines allergic sensitization. We show here that CD8 T cell subsets break these rules as both T cytotoxic (Tc)1 and Tc2 cells promote Th1 over Th2 immunity. Using IL-12(-/-), IFN-gamma(-/-), and OVA(257-264)-specific Valpha2Vbeta5 TCR-transgenic mice, we have identified the key steps involved. OVA-specific IFN-gamma(-/-) CD8 T cells inhibited IgE responses equivalent to wild-type CD8 T cells (up to 98% suppression), indicating that CD8 T cell-derived IFN-gamma was not required. However, OVA-specific CD8 T cells could not inhibit IgE in IFN-gamma(-/-) recipients unless reconstituted with naive, wild-type CD4 T cells, suggesting that CD4 T cell-derived IFN-gamma did play a role. Transfer of either Tc1 or Tc2 Valpha2Vbeta5 TCR-transgenic CD8 T cells inhibited IgE and OVA-specific Th2 cells while promoting OVA-specific Th1 cell responses, suggesting a potential role for a type 1 inducing cytokine such as IL-12. CD8 T cells were shown to induce IL-12 in OVA(257-264)-pulsed dendritic cells (DC) in vitro. Furthermore, CD8 T cells were unable to inhibit IgE responses in IL-12(-/-) recipients without the addition of naive, wild-type DC, thus demonstrating a pivotal role for IL-12 in this mechanism. These data reveal a mechanism of IgE regulation in which CD8 T cells induce DC IL-12 by an IFN-gamma-independent process that subsequently induces Th1 and inhibits Th2 cells. Th1 cell IFN-gamma is the final step that inhibits B cell IgE class switching. This demonstrates a novel regulatory network through which CD8 T cells inhibit allergic sensitization.     

Single-base-pair discrimination of terminal mismatches by using oligonucleotide microarrays and neural network analyses.

The effects of single-base-pair near-terminal and terminal mismatches on the dissociation temperature (T(d)) and signal intensity of short DNA duplexes were determined by using oligonucleotide microarrays and neural network (NN) analyses. Two perfect-match probes and 29 probes having a single-base-pair mismatch at positions 1 to 5 from the 5' terminus of the probe were designed to target one of two short sequences representing 16S rRNA. Nonequilibrium dissociation rates (i.e., melting profiles) of all probe-target duplexes were determined simultaneously. Analysis of variance revealed that position of the mismatch, type of mismatch, and formamide concentration significantly affected the T(d) and signal intensity. Increasing the concentration of formamide in the washing buffer decreased the T(d) and signal intensity, and it decreased the variability of the signal. Although T(d)s of probe-target duplexes with mismatches in the first or second position were not significantly different from one another, duplexes with mismatches in the third to fifth positions had significantly lower T(d)s than those with mismatches in the first or second position. The trained NNs predicted the T(d) with high accuracies (R(2) = 0.93). However, the NNs predicted the signal intensity only moderately accurately (R(2) = 0.67), presumably due to increased noise in the signal intensity at low formamide concentrations. Sensitivity analysis revealed that the concentration of formamide explained most (75%) of the variability in T(d)s, followed by position of the mismatch (19%) and type of mismatch (6%). The results suggest that position of the mismatch at or near the 5' terminus plays a greater role in determining the T(d) and signal intensity of duplexes than the type of mismatch.     

A multiple watershed field test of hydrogeomorphic functional assessment of headwater streams—Variability in field measurements between independent teams

Ephemeral and intermittent headwater streams are under increasing pressure from disturbance and development. Rapid, repeatable assessment techniques are needed in order to gauge the condition of these stream systems. Several attributes of these headwater streams constrain the use of the most widely used macroinvertebrate or water quality stream assessment techniques. The hydrogeomorphic (HGM) functional assessment is a reference-based alternative technique. To evaluate this alternative, repeated assessments were conducted in eight high-gradient headwaters in West Virginia by four independent teams. Across-site and measurement variance among teams was assessed using a coefficient of variation (CV, expressed as percent). A variability of >50% CV, which suggests less repeatable results, occurred in only 13.8% of measurements, primarily associated with 2 of the 9 variables examined (snag density and substrate size). Between site measurement variance was the greatest at more highly disturbed sample locations, particularly with regard to the large woody debris, tree species richness, and channel bank erosion variables. Variables with the lowest CV were tree diameter, detrital cover, canopy cover, and channel embeddedness. Based on these results, measurements included when applying HGM approach to these streams should focus on direct measurements or directed estimates that yield a large response range across a spectrum of sites while maintaining consistent repeatability among different teams, with special attention paid to their use in highly disturbed sites.     

Measurement of 14CO2 evolution during tissue oxidation in vitro; an alternative to the Kontes well

1. An alternative method to the use of the disposable Kontes well for trapping 14CO2 produced in the course of biological oxidations is described. 2. A polyethylene miniature scintillation vial was used to contain the hyamine hydroxide-impregnated filter paper wick. 3. The two methods are compared in their abilities to trap 14CO2 produced directly by acidification of sodium [14C]bicarbonate and during beta-oxidation of 1[14C] palmitic acid. 4. The miniature vial and Kontes well methods showed similar efficiencies in the trapping of 14CO2 (97% and 95%, respectively, on average) the radioactivity of which was determined in the miniature vial using 5 ml only of scintillation fluid compared with a minimum of 10 ml required by the standard scintillation vial used to accommodate the Kontes well. 5. The technical advantages of the suggested miniature vial system, during both incubation and counting stages, are discussed.