Determination of base and backbone contributions to the thermodynamics of premelting and melting transitions in B DNA

In previous papers of this series the temperature-dependent Raman spectra of poly(dA)·poly(dT) and poly(dA–dT)·poly(dA–dT) were used to characterize structurally the melting and premelting transitions in DNAs containing consecutive A·T and alternating A·T/T·A base pairs. Here, we describe procedures for obtaining thermodynamic parameters from the Raman data. The method exploits base-specific and backbone-specific Raman markers to determine separate thermodynamic contributions of A, T and deoxyribosyl-phosphate moieties to premelting and melting transitions. Key findings include the following: (i) Both poly(dA)·poly(dT) and poly(dA–dT)· poly(dA–dT) exhibit robust premelting transitions, due predominantly to backbone conformational changes. (ii) The significant van’t Hoff premelting enthalpies of poly(dA)·poly(dT) [ΔH_vH^pm = 18.0 ± 1.6 kcal·mol^–1 (kilocalories per mole cooperative unit)] and poly(dA–dT)·poly(dA–dT) (ΔH_vH^pm = 13.4 ± 2.5 kcal·mol^–1) differ by an amount (~4.6 kcal·mol^–1) estimated as the contribution from three-centered inter-base hydrogen bonding in (dA)_n·(dT)_n tracts. (iii) The overall stacking free energy of poly(dA)· poly(dT) [–6.88 kcal·mol_bp^–1 (kilocalories per mole base pair)] is greater than that of poly(dA–dT)· poly(dA–dT) (–6.31 kcal·mol_bp^–1). (iv) The difference between stacking free energies of A and T is significant in poly(dA)·poly(dT) (ΔΔG_st = 0.8 ± 0.3 kcal· mol_bp^–1), but marginal in poly(dA–dT)·poly(dA–dT) (ΔΔG_st = 0.3 ± 0.3 kcal·mol_bp^–1). (v) In poly(dA)· poly(dT), the van’t Hoff parameters for melting of A (ΔH_vH^A = 407 ± 23 kcal·mol^–1, ΔS_vH^A = 1166 ± 67 cal·°K^–1·mol^–1, ΔG_vH(25°C)^A = 60.0 ± 3.2 kcal·mol^–1) are clearly distinguished from those of T (ΔH_vH^T = 185 ± 38 kcal·mol^–1, ΔS_vH^T = 516 ± 109 cal·°K^–1·mol^–1, ΔG_vH(25°C)^T = 27.1 ± 5.5 kcal·mol^–1). (vi) Similar relative differences are observed in poly(dA–dT)· poly(dA–dT) (ΔH_vH^A = 333 ± 54 kcal·mol^–1, ΔS_vH^A = 961 ± 157 cal·°K^–1·mol^–1, ΔG_vH(25°C)^A = 45.0 ± 7.6 kcal· mol^–1; ΔH_vH^T = 213 ± 30 kcal·mol^–1, ΔS_vH^T = 617 ± 86 cal·°K^–1·mol^–1, ΔG_vH(25°C)^T = 29.3 ± 4.9 kcal·mol^–1). The methodology employed here distinguishes thermodynamic contributions of base stacking, base pairing and backbone conformational ordering in the molecular mechanism of double-helical B DNA formation.     

Protein Mobility in the Cytoplasm of Escherichia coli

The rate of protein diffusion in bacterial cytoplasm may constrain a variety of cellular functions and limit the rates of many biochemical reactions in vivo. In this paper, we report noninvasive measurements of the apparent diffusion coefficient of green fluorescent protein (GFP) in the cytoplasm of Escherichia coli. These measurements were made in two ways: by photobleaching of GFP fluorescence and by photoactivation of a red-emitting fluorescent state of GFP (M. B. Elowitz, M. G. Surette, P. E. Wolf, J. Stock, and S. Leibler, Curr. Biol. 7:809–812, 1997). The apparent diffusion coefficient, D_a, of GFP in E. coli DH5α was found to be 7.7 ± 2.5 μm²/s. A 72-kDa fusion protein composed of GFP and a cytoplasmically localized maltose binding protein domain moves more slowly, with D_a of 2.5 ± 0.6 μm²/s. In addition, GFP mobility can depend strongly on at least two factors: first, D_a is reduced to 3.6 ± 0.7 μm²/s at high levels of GFP expression; second, the addition to GFP of a small tag consisting of six histidine residues reduces D_a to 4.0 ± 2.0 μm²/s. Thus, a single effective cytoplasmic viscosity cannot explain all values of D_a reported here. These measurements have implications for the understanding of intracellular biochemical networks.     

Crystal structure of d(GCGAAAGCT) containing a parallel-stranded duplex with homo base pairs and an anti-parallel duplex with Watson-Crick base pairs

A DNA fragment d(GCGAAAGCT), known to adopt a stable mini-hairpin structure in solution, has been crystallized in the space group I4₁22 with the unit-cell dimensions a = b = 53.4 Å and c = 54.0 Å, and the crystal structure has been determined at 2.5 Å resolution. The four nucleotide residues CGAA of the first half of the oligomer form a parallel duplex with another half through the homo base pairs, C₂:C₂⁺ (singly-protonated between the Watson– Crick sites), G₃:G₃ (between the minor groove sites), A₄:A₄ (between the major groove sites) and A₅:A₅ (between the Watson–Crick sites). The two strands remaining in the half of the parallel duplex are split away in different directions, and they pair in an anti-parallel B-form duplex with the second half extending from a neighboring parallel duplex, so that an infinite column is formed in a head-to-tail fashion along the c-axis. It seems that a hexa-ammine cobalt cation supports such a branched and bent conformation of the oligomer. One end of the parallel duplex is stacked on the corresponding end of the adjacent parallel duplex; between them, the guanine base of the first residue is stacked on the fourth ribose of another duplex.     

UVB Exposure of Farm Animals: Study on a Food-Based Strategy to Bridge the Gap between Current Vitamin D Intakes and Dietary Targets

Vitamin D deficiency is a global health problem. This study aimed to investigate the efficacy of ultraviolet (UV) B radiation for improving vitamin D₃ content of eggs and meat. In a two-factorial design hens that received diets with 0 (-D₃) or 3,000 IU (+D₃) vitamin D₃/kg were non-exposed (-UVB) or exposed to UVB radiation (+UVB) for 3 h daily over 4 weeks. Data show that UVB radiation was very effective in raising the vitamin D₃ content of egg yolk and meat. Egg yolk from +UVB/−D₃ hens had a higher vitamin D₃ content (17.5±7.2 µg/100 g dry matter (DM)) than those from the –UVB/+D₃ group (5.2±2.4 µg/100 g DM, p<0.01). Vitamin D₃ content in egg yolk of vitamin D₃-supplemented hens could be further increased by UVB radiation (32.4±10.9 µg/100 g DM). The content of 25-hydroxyvitamin D₃ (25(OH)D₃) in the egg yolk also increased in response to UVB, although less pronounced than vitamin D₃. Meat revealed about 4-fold higher vitamin D₃ contents in response to UVB than to dietary vitamin D₃ (p<0.001). In conclusion, exposure of hens to UVB is an efficient approach to provide consumers with vitamin D₃-enriched foods from animal sources.     

The crystal structure of an alternating RNA heptamer r(GUAUACA) forming a six base-paired duplex with 3'-end adenine overhangs

The crystal structure of an alternating RNA heptamer r(GUAUACA) has been determined to 2.0 Å resolution and refined to an R_work of 17.1% and R_free of 18.5% using 2797 reflections. The heptamer crystallized in the space group C222 with a unit cell of a = 25.74, b = 106.58, c = 30.26 Å and two independent strands in the asymmetric unit. Each heptamer forms a duplex with its symmetry-related strand and each duplex contains six Watson–Crick base pairs and 3′-end adenosine overhangs. Therefore, two kinds of duplex (duplex 1 and duplex 2) are formed. Duplexes 1 stack on each other forming a pseudo-continuous column, which is typical of the RNA packing mode, while duplex 2 is typical of A-DNA packing with its termini in abutting interactions. Overhang adenine residues stack within the duplexes with C3′-endo sugar pucker and C2′-endo sugar pucker in duplexes 1 and 2, respectively. A Na⁺ ion in the crystal lattice is water bridged to two N1 atoms of symmetry-related A7 bases.     

Determination of DNA minor groove width in distamycin-DNA complexes by solid-state NMR

We have performed solid-state ³¹P-¹⁹F REDOR nuclear magnetic resonance (NMR) experiments to monitor changes in minor groove width of the oligonucleotide d(CGCAAA^2′FUTGGC)·d(GCCAAT(pS)TT GCG) (A₃T₂) upon binding of the drug distamycin A at different stoichiometries. In the hydrated solid-state sample, the minor groove width for the unbound DNA, measured as the ^2′FU7–pS19 inter-label distance, was 9.4 ± 0.7 Å, comparable to that found for similar A:T-rich DNAs. Binding of a single drug molecule is observed to cause a 2.4 Å decrease in groove width. Subsequent addition of a second drug molecule results in a larger conformational change, expanding this minor groove width to 13.6 Å, consistent with the results of a previous solution NMR study of the 2:1 complex. These ³¹P-¹⁹F REDOR results demonstrate the ability of solid-state NMR to measure distances of 7–14 Å in DNA–drug complexes and provide the first example of a direct spectroscopic measurement of minor groove width in nucleic acids.     

Monovalent Ion Selectivity Sequences of the Rat Connexin43 Gap Junction Channel

The relative permeability sequences of the rat connexin 43 (rCx43) gap junction channel to seven cations and chloride were examined by double whole cell patch clamp recording of single gap junction channel currents in rCx43 transfected neuroblastoma 2A (N2A) cell pairs. The measured maximal single channel slope conductances (γ_j, in pS) of the junctional current-voltage relationships in 115 mM XCl were RbCl (103) ≥ CsCl (102) > KCl (97) > NaCl (79) ≥ LiCl (78) > TMACl (65) > TEACl (53) and for 115 mM KY were KBr (105) > KCl (97) > Kacetate (77) > Kglutamate (61). The single channel conductance-aqueous mobility relationships for the test cations and anions were linear. However, the predicted minimum anionic and cationic conductances of these plots did not accurately predict the rCx43 channel conductance in 115 mM KCl. Instead, the conductance of the rCx43 channel in 115 mM KCl was accurately predicted from cationic and anionic conductance-mobility plots by applying a mobility scaling factor D_x/D_o, which depends upon the relative radii of the permeant ions to an estimated pore radius. Relative permeabilities were determined for all of the monovalent cations and anions tested from asymmetric salt reversal potential measurements and the Goldman-Hodgkin-Katz voltage equation. These experiments estimate the relative chloride to potassium permeability to be 0.13. The relationship between the relative cation permeability and hydrated radius was modeled using the hydrodynamic equation assuming a pore radius of 6.3 ± 0.4 Å. Our data quantitatively demonstrate that the rCx43 gap junction channel is permeable to monovalent atomic and organic cations and anions and the relative permeability sequences are consistent with an Eisenman sequence II or I, respectively. These predictions about the rCx43 channel pore provide a useful basis for future investigations into the structural determinants of the conductance and permeability properties of the connexin channel pore.     

Patch Clamp on the Luminal Membrane of Exocrine Gland Acini from Frog Skin (Rana esculenta) Reveals the Presence of Cystic Fibrosis Transmembrane Conductance Regulator–like Cl? Channels Activated by Cyclic AMP

Chloride channels in the luminal membrane of exocrine gland acini from frog skin (Rana esculenta) constituted a single homogeneous population. In cell-attached patches, channels activated upon exposure to isoproterenol, forskolin, or dibutyryl-cAMP and isobutyl-1-methyl-xanthine rectified in the outward direction with a conductance of 10.0 ± 0.4 pS for outgoing currents. Channels in stimulated cells reversed at 0 mV applied potential, whereas channels in unstimulated cells reversed at depolarized potentials (28.1 ± 6.7 mV), indicating that Cl⁻ was above electrochemical equilibrium in unstimulated, but not in stimulated, cells. In excised inside-out patches with 25 mM Cl⁻ on the inside, activity of small (8-pS) linear Cl⁻-selective channels was dependent upon bath ATP (1.5 mM) and increased upon exposure to cAMP-dependent protein kinase. The channels displayed a single substate, located just below 2/3 of the full channel amplitude. Halide selectivity was identified as P_Br > P_I > P_Cl from the Goldman equation; however, the conductance sequence when either halide was permeating the channel was G_Cl > G_Br >> G_I. In inside-out patches, the channels were blocked reversibly by 5-nitro-2-(3-phenylpropylamino)benzoic acid, glibenclamide, and diphenylamine-2-carboxylic acid, whereas 4,4-diisothiocyanatostilbene-2,2-disulfonic acid blocked channel activity completely and irreversibly. Single-channel kinetics revealed one open state (mean lifetime = 158 ± 72 ms) and two closed states (lifetimes: 12 ± 4 and 224 ± 31 ms, respectively). Power density spectra had a double-Lorentzian form with corner frequencies 0.85 ± 0.11 and 27.9 ± 2.9 Hz, respectively. These channels are considered homologous to the cystic fibrosis transmembrane conductance regulator Cl⁻ channel, which has been localized to the submucosal skin glands in Xenopus by immunohistochemistry (Engelhardt, J.F., S.S. Smith, E. Allen, J.R. Yankaskas, D.C. Dawson, and J.M. Wilson. 1994. Am. J. Physiol. 267: C491–C500) and, when stimulated by cAMP-dependent phosphorylation, are suggested to function in chloride secretion.     

Crystal Structure of the Vaccinia Virus Uracil-DNA Glycosylase in Complex with DNA

Vaccinia virus polymerase holoenzyme is composed of the DNA polymerase catalytic subunit E9 associated with its heterodimeric co-factor A20·D4 required for processive genome synthesis. Although A20 has no known enzymatic activity, D4 is an active uracil-DNA glycosylase (UNG). The presence of a repair enzyme as a component of the viral replication machinery suggests that, for poxviruses, DNA synthesis and base excision repair is coupled. We present the 2.7 Å crystal structure of the complex formed by D4 and the first 50 amino acids of A20 (D4·A20_1–50) bound to a 10-mer DNA duplex containing an abasic site resulting from the cleavage of a uracil base. Comparison of the viral complex with its human counterpart revealed major divergences in the contacts between protein and DNA and in the enzyme orientation on the DNA. However, the conformation of the dsDNA within both structures is very similar, suggesting a dominant role of the DNA conformation for UNG function. In contrast to human UNG, D4 appears rigid, and we do not observe a conformational change upon DNA binding. We also studied the interaction of D4·A20_1–50 with different DNA oligomers by surface plasmon resonance. D4 binds weakly to nonspecific DNA and to uracil-containing substrates but binds abasic sites with a K_d of <1.4 μm. This second DNA complex structure of a family I UNG gives new insight into the role of D4 as a co-factor of vaccinia virus DNA polymerase and allows a better understanding of the structural determinants required for UNG action.     

The diffusion-solubility of oxygen in lipid bilayers

The product, D_oα, of the oxygen diffusion coefficient, D_o, and the oxygen solubility, α, is determined in phosphatidylcholine bilayers at temperatures above the lipid phase transitions from ESR spin-exchange measurements. The resulting values of D_oα are in good agreement with those obtained from fluorescence-quenching experiments. The use of fatty acid spin labels makes it possible to measure D_oα as a function of the coordinate perpendicular to the bilayer surface. The results indicate that D_oα is a strong function of this coordinate; it is greatest in the bilayer center and least near the bilayer head groups.     

Primer-dependent synthesis by poliovirus RNA-dependent RNA polymerase (3Dpol)

Properties of poliovirus RNA-dependent RNA polymerase (3D^pol) including optimal conditions for primer extension, processivity and the rate of dissociation from primer-template (k_off) were examined in the presence and absence of viral protein 3AB. Primer-dependent polymerization was examined on templates of 407 or 1499 nt primed such that fully extended products would be 296 or 1388 nt, respectively. Maximal primer extension was achieved with low rNTP concentrations (50–100 µM) using pH 7 and low (<1 mM) MgCl₂ and KCl (<20 mM) concentrations. However, high activity (about half maximal) was also observed with 500 µM rNTPs providing that higher MgCl₂ levels (3–5 mM) were used. The enhancement observed with the former conditions appeared to result from a large increase in the initial level or active enzyme that associated with the primer. 3AB increased the number of extended primers at all conditions with no apparent change in processivity. The k_off values for the polymerase bound to primer-template were 0.011 ± 0.005 and 0.037 ± 0.006 min^–1 (average of four or more experiments ± SD) in the presence or absence of 3AB, respectively. The decrease in the presence of 3AB suggested an enhancement of polymerase binding or stability. However, binding was tight even without 3AB, consistent with the highly processive (at least several hundred nucleotides) nature of 3D^pol. The results support a mechanism whereby 3AB enhances the ability of 3D^pol to form a productive complex with the primer-template. Once formed, this complex is very stable resulting in highly processive synthesis.     

Comparison of Texture Features Derived from Static and Respiratory-Gated PET Images in Non-Small Cell Lung Cancer

Background

PET-based texture features have been used to quantify tumor heterogeneity due to their predictive power in treatment outcome. We investigated the sensitivity of texture features to tumor motion by comparing static (3D) and respiratory-gated (4D) PET imaging.

Methods

Twenty-six patients (34 lesions) received 3D and 4D [¹⁸F]FDG-PET scans before the chemo-radiotherapy. The acquired 4D data were retrospectively binned into five breathing phases to create the 4D image sequence. Texture features, including Maximal correlation coefficient (MCC), Long run low gray (LRLG), Coarseness, Contrast, and Busyness, were computed within the physician-defined tumor volume. The relative difference (δ_3D-4D) in each texture between the 3D- and 4D-PET imaging was calculated. Coefficient of variation (CV) was used to determine the variability in the textures between all 4D-PET phases. Correlations between tumor volume, motion amplitude, and δ_3D-4D were also assessed.

Results

4D-PET increased LRLG ( = 1%–2%, p<0.02), Busyness ( = 7%–19%, p<0.01), and decreased MCC ( = 1%–2%, p<7.5×10⁻³), Coarseness ( = 5%–10%, p<0.05) and Contrast ( = 4%–6%, p>0.08) compared to 3D-PET. Nearly negligible variability was found between the 4D phase bins with CV<5% for MCC, LRLG, and Coarseness. For Contrast and Busyness, moderate variability was found with CV = 9% and 10%, respectively. No strong correlation was found between the tumor volume and δ_3D-4D for the texture features. Motion amplitude had moderate impact on δ for MCC and Busyness and no impact for LRLG, Coarseness, and Contrast.

Conclusions

Significant differences were found in MCC, LRLG, Coarseness, and Busyness between 3D and 4D PET imaging. The variability between phase bins for MCC, LRLG, and Coarseness was negligible, suggesting that similar quantification can be obtained from all phases. Texture features, blurred out by respiratory motion during 3D-PET acquisition, can be better resolved by 4D-PET imaging. 4D-PET textures may have better prognostic value as they are less susceptible to tumor motion.     

Influence of spatial distribution and size of clones on the realized outcrossing rate of the marsh cinquefoil (Comarum palustre)

Background and Aims

Clonal growth is a common feature in flowering plants. As clone size increases, the selfing rate in self-compatible species is likely to increase due to more frequent geitono-pollination events (i.e. pollination among flowers within the same genet). This study investigated the breeding system of the marsh cinquefoil (Comarum palustre) and assessed spatial distribution of clones, clone size and architecture, and their effects on realized outcrossing rates. In addition, pollen dispersal was investigated in two patchy populations.

Methods

The species'' breeding system was investigated under controlled conditions through hand pollinations (self- vs. cross-pollination). Using microsatellite markers, an assessment was made of the realized outcrossing rates and the genetic diversity in four natural populations, the clonal structure in two populations within five 15 × 15 m sampling plots following 0·5 × 0·5 m grids, and the pollen dispersal through paternity assignment tests in those two populations.

Key Results Comarum palustre

is a self-compatible species but only presents a low rate of spontaneous self-pollination. The occurrence of inbreeding depression was not detected at the seed set stage (δ_SS = 0·04). Clones were spatially clumped (A_C = 0·60–0·80), with intermediate to no intermingling of the ramets (D_C = 0·40–1·00). Genet size ranged from one to 171 ramets. Patchy populations had low outcrossing rates (t_m = 0·33–0·46). Large clones showed lower outcrossing rates than small clones. Pollen dispersal mainly occurred within patches as only 1–7 % of the pollination events occurred between patches of >25 m separation. Seedling recruitment events were detected.

Conclusions

Genet size together with distances between patches, through increasing geitono-pollination events, appeared to be important factors influencing realized outcrossing rates. The study also revealed seed flow allowing seedling recruitment, which may contribute to increasing the number of new patches, and potentially further enhance gene flow within populations.     

Single-trait and antagonistic index selection for litter size and body weight in mice

Individual selection based on female performance only was conducted in four lines of mice: L⁺ for increased litter size, W⁺ for increased 6-week body weight, L^-W⁺ for a selection index aimed at decreasing litter size and increasing 6-week body weight and L⁺W^- for a selection index aimed at increasing litter size and decreasing 6-week body weight. A fifth line (K) served as an unselected control. All litters were standardized to eight mice at one day of age. Expected heritability was based on twice the regression of offspring on dam (h²_d), which contains additive genetic variance due to direct (σ²_Ao) and maternal (σ²_Am) effects and their covariance (σ_AoAm). Responses and correlated responses were measured either deviated (method 1) or not deviated (method 2) from the control line. Realized heritabilities (h²_R) for litter size were 0.19 ± 0.04 (1) and 0.16 ± 0.03 (2), which were similar to h ²_d of 0.17 ± 0.04. The h² _R for 6-week body weight of 0.55 ± 0.07 (1) and 0.44 ± 0.07 (2) agreed with h²_d of 0.42 ± 0.02. Realized genetic correlations (r*_GR) between litter size and 6-week body weight calculated from the double-selection experiment were 0.52 ± 0.10 (1) and 0.52 ± 0.13 (2), which were not significantly different from the base population estimate of r* _Gd = 0.63 ± 0.14. Divergence (L^-W ⁺ minus L⁺W^-) in the antagonistic index selection lines was 0.21 ± 0.01 index units (I = 0.305 P_W - 0.436 P_L, where P _W and P_L are the phenotypic values for 6-week body weight and litter size, respectively.). The h² _R of index units of 0.14 ± 0.02 calculated from divergence agreed with h²_d of 0.14 ± 0.04. Divergences in litter size (-0.19 ± 0.07) and 6-week body weight (0.46 ± 0.10) were in the expected direction. Antagonistic index selection yielded about one-half the expected divergence in litter size, while divergence in 6-week body weight was only slightly less than expected. Realized genetic correlations indicated that litter size, 6-week body weight and index units each showed positive pleiotropy with 3-week body weight, postweaning gain and weight at vaginal introitus and negative pleiotropy with age at vaginal introitus. Sex ratio and several components of fitness (days from joining to parturition, percent fertile matings and percent perinatal survival) did not change significantly in the selected lines.     

A Model for the Action of Vinblastine in Vivo

A model for the action of vinblastine (VLB) on cells multiplying exponentially in vivo with a generation time, T_G, has been derived. It is based on the assumption that cells attempting to pass through mitosis in the presence of VLB lose their proliferative capacity and that this lethal effect occurs only when the cells are exposed to a concentration of VLB which is above a critical value, C_k. The model leads to two predictions. First, that the percentage of cells surviving at any time after exposure to a dose, D, of VLB is 100% if D < D_k and decreases to 0% after a time, T_G, following a dose D ≥ D_k·2^{T G/T1/2}, where D_k represents the dose of VLB required to produce the concentration C_k, and T_1/2 is the half-life of the VLB in vivo. Second, that the time, T_G, at which the percentage of cells surviving an exposure to VLB, at doses greater than D_k·2^{U G/T1/2}, decreases to zero should be equal to the generation time of the cells. Both of these predictions were confirmed experimentally which indicates that the model adequately explains the action of VLB in vivo.     

Role of Alveolar β2-Adrenergic Receptors on Lung Fluid Clearance and Exercise Ventilation in Healthy Humans

Background

In experimental conditions alveolar fluid clearance is controlled by alveolar β₂-adrenergic receptors. We hypothesized that if this occurs in humans, then non-selective β-blockers should reduce the membrane diffusing capacity (D_M), an index of lung interstitial fluid homeostasis. Moreover, we wondered whether this effect is potentiated by saline solution infusion, an intervention expected to cause interstitial lung edema. Since fluid retention within the lungs might trigger excessive ventilation during exercise, we also hypothesized that after the β₂-blockade ventilation increased in excess to CO₂ output and this was further enhanced by interstitial edema.

Methods and Results

22 healthy males took part in the study. On day 1, spirometry, lung diffusion for carbon monoxide (DLCO) including its subcomponents D_M and capillary volume (V_Cap), and cardiopulmonary exercise test were performed. On day 2, these tests were repeated after rapid 25 ml/kg saline infusion. Then, in random order 11 subjects were assigned to oral treatment with Carvedilol (CARV) and 11 to Bisoprolol (BISOPR). When heart rate fell at least by 10 beats·min⁻¹, the tests were repeated before (day 3) and after saline infusion (day 4). CARV but not BISOPR, decreased D_M (−13±7%, p = 0.001) and increased V_Cap (+20±22%, p = 0.016) and VE/VCO₂ slope (+12±8%, p<0.01). These changes further increased after saline: −18±13% for D_M (p<0.01), +44±28% for V_Cap (p<0.001), and +20±10% for VE/VCO₂ slope (p<0.001).

Conclusions

These findings support the hypothesis that in humans in vivo the β₂-alveolar receptors contribute to control alveolar fluid clearance and that interstitial lung fluid may trigger exercise hyperventilation.     

THE VIOLOGEN INDICATORS

The tabulation gives the normal potentials of the various indicators at 30°C.; referred to the normal hydrogen electrode, the accuracy is estimated to be ±0.002 volt. Normal potentials of the viologens at 30°C.: Methyl viologen –0.446 volts Ethyl viologen –0.449 volts Betaine viologen –0.444 volts Benzyl viologen –0.359 volts Supposing some solution brings about a coloration of one of these indicators to the extent of A per cent of the maximum color, the oxidation-reduction potential of this solution is E = E_o – 0.06 log See PDF for Equation where E_o is the normal potential according to the above tabulation. This normal potential is independent of pH.     

The external K+ concentration and mutations in the outer pore mouth affect the inhibition of kv1.5 current by Ni2+

By examining the consequences both of changes of [K⁺]_o and of point mutations in the outer pore mouth, our goal was to determine if the mechanism of the block of Kv1.5 ionic currents by external Ni²⁺ is similar to that for proton block. Ni²⁺ block is inhibited by increasing [K⁺]_o, by mutating a histidine residue in the pore turret (H463Q) or by mutating a residue near the pore mouth (R487V) that is the homolog of Shaker T449. Aside from a slight rightward shift of the Q-V curve, Ni²⁺ had no effect on gating currents. We propose that, as with H_o⁺, Ni²⁺ binding to H463 facilitates an outer pore inactivation process that is antagonized by K_o⁺ and that requires R487. However, whereas H_o⁺ substantially accelerates inactivation of residual currents, Ni²⁺ is much less potent, indicating incomplete overlap of the profiles of these two metal ions. Analyses with Co²⁺ and Mn²⁺, together with previous results, indicate that for the first-row transition metals the rank order for the inhibition of Kv1.5 in 0 mM K_o⁺ is Zn²⁺ (K_D ~ 0.07 mM) ≥ Ni²⁺ (K_D ~ 0.15 mM) > Co²⁺ (K_D ~ 1.4 mM) > Mn²⁺ (K_D > 10 mM).     

Decreases in Molecular Diffusion,Perfusion Fraction and Perfusion-Related Diffusion in Fibrotic Livers: A Prospective Clinical Intravoxel Incoherent Motion MR Imaging Study

Purpose

This study was aimed to determine whether pure molecular-based diffusion coefficient (D) and perfusion-related diffusion parameters (perfusion fraction f, perfusion-related diffusion coefficient D*) differ in healthy livers and fibrotic livers through intra-voxel incoherent motion (IVIM) MR imaging.

Material and Methods

17 healthy volunteers and 34 patients with histopathologically confirmed liver fibrosis patients (stage 1 = 14, stage 2 = 8, stage 3& 4 = 12, METAVIR grading) were included. Liver MR imaging was performed at 1.5-T. IVIM diffusion weighted imaging sequence was based on standard single-shot DW spin echo-planar imaging, with ten b values of 10, 20, 40, 60, 80, 100, 150, 200, 400, 800 sec/mm² respectively. Pixel-wise realization and regions-of-interest based quantification of IVIM parameters were performed.

Results

D, f, and D* in healthy volunteer livers and patient livers were 1.096±0.155 vs 0.917±0.152 (10⁻³ mm²/s, p = 0.0015), 0.164±0.021 vs 0.123±0.029 (p<0.0001), and 13.085±2.943 vs 9.423±1.737 (10⁻³ mm²/s, p<0.0001) respectively, all significantly lower in fibrotic livers. As the fibrosis severity progressed, D, f, and D* values decreased, with a trend significant for f and D*.

Conclusion

Fibrotic liver is associated with lower pure molecular diffusion, lower perfusion volume fraction, and lower perfusion-related diffusion. The decrease of f and D* in the liver is significantly associated liver fibrosis severity.     

The Association of Maximum Body Weight on the Development of Type 2 Diabetes and Microvascular Complications: MAXWEL Study

Background

Obesity precedes the development of type 2 diabetes (T2D). However, the relationship between the magnitude and rate of weight gain to T2D development and complications, especially in non-White populations, has received less attention.

Methods and Findings

We determined the association of rate and magnitude of weight gain to age at T2D diagnosis (Age_T2D), HbA1c at T2D diagnosis (HbA1c_T2D), microalbuminuria, and diabetic retinopathy after adjusting for sex, BMI at age 20 years, lifestyles, family history of T2D and/or blood pressure and lipids in 2164 Korean subjects aged ≥30 years and newly diagnosed with diabetes. Body weight at age 20 years (Wt_20y) was obtained by recall or from participants’ medical, school, or military records. Participants recalled their maximum weight (Wt_max) prior to T2D diagnosis and age at maximum weight (Age_{max_wt}). The rate of weight gain (Rate_{max_wt}) was calculated from magnitude of weight gain (ΔWt = Wt_max–Wt_20y) divided by ΔTime (Age_{max_wt} –20 years). The mean Age_{max_wt} and Age_T2D were 41.5±10.9 years and 50.1±10.5 years, respectively. The Wt_20y and Wt_max were 59.9±10.5 kg and 72.9±11.4 kg, respectively. The Rate_{max_wt} was 0.56±0.50 kg/year. After adjusting for risk factors, greater ΔWt and higher Rate_{max_wt} were significantly associated with earlier Age_T2D, higher HbA1c_T2D after additional adjusting for Age_T2D, and microalbuminuria after further adjusting for HbA1c_T2D and lipid profiles. Greater ΔWt and higher Rate_{max_wt} were also significantly associated with diabetic retinopathy.

Conclusions

This finding supports public health recommendations to reduce the risk of T2D and its complications by preventing weight gain from early adulthood.