The voltage gates of connexin channels are sensitive to CO(2)

Cx45 channel sensitivity to CO(2), transjunctional voltage (V(j)) and inhibition of calmodulin (CaM) expression was tested in oocytes by dual voltage-clamp. Cx45 channels are very sensitive to V(j) and close preferentially by the slow gate, likely the same as the chemical gate. With CO(2)-induced drop in junctional conductance (G(j)), the speed of V(j)-dependent inactivation of junctional current (I(j)) and V(j) sensitivity increased. With 40 mV V(j), the tau of single exponential I(j) decay reversibly decreased by approximately 40% with CO(2), and G(j steady state)/G(j peak) decreased multiphasically, indicating that kinetics and V(j) sensitivity of chemical/slow-V(j) gating are altered by changes in [H(+)](i) and/or [Ca(2+)](i). With 15 min exposure to CO(2), G(j) dropped to 0% in controls and by approximately 17% following CaM expression inhibition; similarly, V(j) sensitivity decreased significantly. This indicates that the speed and sensitivity of V(j)-dependent inactivation of Cx45 channels are increased by CO(2), and that CaM plays a role in gating. Cx32 channels behaved similarly, but the drop in both G(j steady state)/G(j peak) and tau with CO(2) matched more closely that of G(j peak). In contrast, sensitivity and speed of V(j) gating of Cx40 and Cx26 channels decreased, rather than increased, with CO(2) application.     

Crystal structures and functional studies of T4moD, the toluene 4-monooxygenase catalytic effector protein

Toluene 4-monooxygenase (T4MO) is a four-component complex that catalyzes the regiospecific, NADH-dependent hydroxylation of toluene to yield p-cresol. The catalytic effector (T4moD) of this complex is a 102-residue protein devoid of metals or organic cofactors. It forms a complex with the diiron hydroxylase component (T4moH) that influences both the kinetics and regiospecificity of catalysis. Here, we report crystal structures for native T4moD and two engineered variants with either four (DeltaN4-) or 10 (DeltaN10-) residues removed from the N-terminal at 2.1-, 1.7-, and 1.9-A resolution, respectively. The crystal structures have C-alpha root-mean-squared differences of less than 0.8 A for the central core consisting of residues 11-98, showing that alterations of the N-terminal have little influence on the folded core of the protein. The central core has the same fold topology as observed in the NMR structures of T4moD, the methane monooxygenase effector protein (MmoB) from two methanotrophs, and the phenol hydroxylase effector protein (DmpM). However, the root-mean-squared differences between comparable C-alpha positions in the X-ray structures and the NMR structures vary from approximately 1.8 A to greater than 6 A. The X-ray structures exhibit an estimated overall coordinate error from 0.095 (0.094) A based on the R-value (R free) for the highest resolution DeltaN4-T4moD structure to 0.211 (0.196) A for the native T4moD structure. Catalytic studies of the DeltaN4-, DeltaN7-, and DeltaN10- variants of T4moD show statistically insignificant changes in k(cat), K(M), k(cat)/K(M), and K(I) relative to the native protein. Moreover, there was no significant change in the regiospecificity of toluene oxidation with any of the T4moD variants. The relative insensitivity to changes in the N-terminal region distinguishes T4moD from the MmoB homologues, which each require the approximately 33 residue N-terminal region for catalytic activity.     

Antisense oligodeoxynucleotides directed against a novel angiotensinogen mRNA-stabilizing protein reduce blood pressure in spontaneously hypertensive rats

We have previously reported that hypertension in the young spontaneously hypertensive rat (SHR) is associated with an elevation in tissue angiotensinogen and a novel polysomal protein known to stabilize angiotensinogen mRNA. In our current study we determined the role of the mRNA-stabilizing protein in the regulation of tissue angiotensinogen expression and mean arterial pressure (MAP) in the SHR utilizing antisense oligodeoxynucleotide (AON) inhibition. Three AONs (RNASTAAS1, position 31-50; RNASTAAS2, position 21-40; RNASTAAS3, position 143-162 of the cDNA coding for the polysomal protein) were administered intravenously (dose 450, 900, and 1,800 microg/kg; 1 dosage/day over 3 days) in conscious, chronically instrumented male SHRs at the age of 7 wk. Control SHRs received corresponding scrambled oligodeoxynucleotide sequences (SCR1, SCR2, SCR3). Each animal received the increasing dose schedule. RNASTAAS2 resulted in a reduced expression of the polysomal protein to 21% (liver), 12% (brain), 27% (heart), 18% (renal cortex), and 22% (renal medulla) of control. Angiotensinogen expression was inhibited to 54% (liver), 41% (brain), 68% (heart), 52% (renal cortex), and 74% (renal medulla) compared with control SHRs. Decreases in plasma concentrations of angiotensinogen and plasma renin activities were associated with a significant decrease in MAP from 147 +/- 6 mmHg (after SCR2) to 106 +/- 4 mmHg after RNASTAAS2. The effects of the two other AONs on MAP were less (RNASTAAS1, -31 mmHg; RNASTAAS3, -16 mmHg) with corresponding decreases in mRNAs coding for angiotensinogen and the polysomal protein. A significant decrease in intracellular concentrations of the polysomal protein accompanied AON inhibition. The magnitude of effects (-15 to -41 mmHg) was comparable to the effects of captopril (100 mg x kg(-1) x day(-1) for 3 days: -32 mmHg) and an AT(1) receptor antagonist (L-158809, 1.5 mg x kg(-1) x day(-1) for 3 days: -36 mmHg). These data suggest an important role of the mRNA-stabilizing protein for hepatic and extrahepatic angiotensinogen expression and MAP in the SHR.     

The Voltage Gates of Connexin Channels are Sensitive to CO2

Cx45 channel sensitivity to CO₂, transjunctional voltage (V_j) and inhibition of calmodulin (CaM) expression was tested in oocytes by dual voltage-clamp. Cx45 channels are very sensitive to V_j and close preferentially by the slow gate, likely the same as the chemical gate. With CO₂-induced drop in junctional conductance (G_j), the speed of V_j-dependent inactivation of junctional current (I_j) and V_j sensitivity increased. With 40 mV V_j, the τ of single exponential I_j decay reversibly decreased by ∼40% with CO₂, and G_{j steady state}/G_{j peak} decreased multiphasically, indicating that kinetics and V_j sensitivity of chemical/slow-V_j gating are altered by changes in [H⁺]_i and/or [Ca²⁺]_i. With 15 min exposure to CO₂, G_j dropped to 0% in controls and by ∼17% following CaM expression inhibition; similarly, V_j sensitivity decreased significantly. This indicates that the speed and sensitivity of V_j-dependent inactivation of Cx45 channels are increased by CO₂, and that CaM plays a role in gating. Cx32 channels behaved similarly, but the drop in both G_{j steady state}/G_{j peak} and τ with CO₂ matched more closely that of G_{j peak}. In contrast, sensitivity and speed of V_j gating of Cx40 and Cx26 channels decreased, rather than increased, with CO₂ application.     

Markers in time and space: A review of the last decade of plant phylogeographic approaches

Plant studies comprise a relatively small proportion of the phylogeographic literature, likely as a consequence of the fundamental challenges posed by the complex genomic structures and life history strategies of these organisms. Comparative plastomics (i.e., comparisons of mutation rates within and among regions of the chloroplast genome) across plant lineages has led to an increased understanding of which markers are likely to provide the most information at low taxonomic levels. However, the extent to which the results of such work have influenced the literature has not been fully assessed, nor has the extent to which plant phylogeographers explicitly analyse markers in time and space, both of which are integral components of the field. Here, we reviewed more than 400 publications from the last decade of plant phylogeography to specifically address the following questions: (i) What is the phylogenetic breadth of studies to date? (ii) What molecular markers have been used, and why were they chosen? (iii) What kinds of markers are most frequently used and in what combinations? (iv) How frequently are divergence time estimation and ecological niche modelling used in plant phylogeography? Our results indicate that chloroplast DNA sequence data remain the primary tool of choice, followed distantly by nuclear DNA sequences and microsatellites. Less than half (42%) of all studies use divergence time estimation, while even fewer use ecological niche modelling (14%). We discuss the implications of our findings, as well as the need for community standards on data reporting.     

Advances in assessing Sabellaria spinulosa reefs for ongoing monitoring

Standardized and repeatable data acquisition and analyses are required to enable the mapping and condition monitoring of reefs within Marine Protected Areas (MPAs). Changes in habitat condition must be reliably identified and reported to best support evidence‐based management. Biogenic reefs in temperate waters, that is, hard matter created by living organisms and raised above the seabed, provide food and shelter for many plant and animal species. This article explores the feasibility of habitat mapping, using remote sensing datasets, as well as metrics for repeatable and suitable assessment of areas of Sabellaria spinulosa for their status as biogenic reef. Data were gathered within the North Norfolk Sandbanks and Saturn Reef candidate Special Area of Conservation/Site of Community Importance in the southern North Sea. Six study areas were identified as potential locations of biogenic reef using previously acquired data, and these were targeted for further investigation using a combination of high resolution multibeam echosounder and sidescan sonar. Where potential S. spinulosa was identified from the acoustic data, a drop‐down camera system was employed for visual verification. Areas of known and potential S. spinulosa reef were mapped successfully at two of the six study areas, although future approaches should take careful consideration of the seabed morphology and predominant habitat backdrop to successfully interpret such data. Camera tows from S. spinulosa reef areas were broken up into 5‐s segments, with each segment scored for (a) average tube elevation; (b) average percentage cover; and (c) for the presence or absence of S. spinulosa. These metrics were utilized to create summary statistics, including a value of patchiness derived from presence/absence data, that is recommended for application as part of future monitoring programs. The application of this methodology could benefit wider assessments of similar threated or declining habitats such as intertidal Mytilus edulis beds on mixed and sandy sediments, Maerl beds, Modioulus modiolus beds, Ostrea edulis beds, and Zostera beds where patchiness may also be considered of environmental importance.     

Assessing Body Composition among 3‐ to 8‐Year‐Old Children: Anthropometry,BIA, and DXA

Objective: To examine the inter‐relationships of body composition variables derived from simple anthropometry [BMI and skinfolds (SFs)], bioelectrical impedance analysis (BIA), and dual energy x‐ray (DXA) in young children. Research Methods and Procedures: Seventy‐five children (41 girls, 34 boys) 3 to 8 years of age were assessed for body composition by the following methods: BMI, SF thickness, BIA, and DXA. DXA served as the criterion measure. Predicted percentage body fat (%BF), fat‐free mass (FFM; kilograms), and fat mass (FM; kilograms) were derived from SF equations [Slaughter (SL)1 and SL2, Deurenberg (D) and Dezenberg] and BIA. Indices of truncal fatness were also determined from anthropometry. Results: Repeated measures ANOVA showed significant differences among the methods for %BF, FFM, and FM. All methods, except the D equation (p = 0.08), significantly underestimated measured %BF (p < 0.05). In general, correlations between the BMI and estimated %BF were moderate (r = 0.61 to 0.75). Estimated %BF from the SL2 also showed a high correlation with DXA %BF (r = 0.82). In contrast, estimated %BF derived from SFs showed a low correlation with estimated %BF derived from BIA (r = 0.38); likewise, the correlation between DXA %BF and BIA %BF was low (r = 0.30). Correlations among indicators of truncal fatness ranged from 0.43 to 0.98. Discussion: The results suggest that BIA has limited utility in estimating body composition, whereas BMI and SFs seem to be more useful in estimating body composition during the adiposity rebound. However, all methods significantly underestimated body fatness as determined by DXA, and, overall, the various methods and prediction equations are not interchangeable.     

Design of selective PI3Kδ inhibitors using an iterative scaffold-hopping workflow

PI3Kδ mediates key immune cell signaling pathways and is a target of interest for multiple indications in immunology and oncology. Here we report a structure-based scaffold-hopping strategy for the design of chemically diverse PI3Kδ inhibitors. Using this strategy, we identified several scaffolds that can be combined to generate new PI3Kδ inhibitors with high potency and isoform selectivity. In particular, an oxindole-based scaffold was found to impart exquisite selectivity when combined with several hinge binding motifs.