Structure of the δ-Chain of Chimpanzee Haemoglobin A<Subscript>2</Subscript>

STUDIES of adult¹ and foetal² haemoglobin from the chimpanzee (Pan troglodytes) have shown that the amino-acid compositions of tryptic and chymotryptic peptides of the α, β and γ-chains are indistinguishable from those of man. The primary structures of chimpanzee α, β and γ-chains are therefore almost certainly identical to the homologous human chains. The two types of γ-chains found in man³, ^Gγ and ^Aγ, with glycine and alanine in position γ136, respectively, are likewise present in the chimpanzee².     

The effect of NBD-Cl in nucleotide-binding of the major subunit α and B of the motor proteins F<Subscript>1</Subscript>F<Subscript>O</Subscript> ATP synthase and A<Subscript>1</Subscript>A<Subscript>O</Subscript> ATP synthase

Subunit α of the Escherichia coli F₁F_O ATP synthase has been produced, and its low-resolution structure has been determined. The monodispersity of α allowed the studies of nucleotide-binding and inhibitory effect of 4-Chloro-7-nitrobenzofurazan (NBD-Cl) to ATP/ADP-binding. Binding constants (K _d ) of 1.6 μM of bound MgATP-ATTO-647N and 2.9 μM of MgADP-ATTO-647N have been determined from fluorescence correlation spectroscopy data. A concentration of 51 μM and 55 μM of NBD-Cl dropped the MgATP-ATTO-647N and MgADP-ATTO-647N binding capacity to 50% (IC₅₀), respectively. In contrast, no effect was observed in the presence of N,N′-dicyclohexylcarbodiimide. As subunit α is the homologue of subunit B of the A₁A_O ATP synthase, the interaction of NBD-Cl with B of the A-ATP synthase from Methanosarcina mazei Gö1 has also been shown. The data reveal a reduction of nucleotide-binding of B due to NBD-Cl, resulting in IC₅₀ values of 41 μM and 42 μM for MgATP-ATTO-647N and MgADP-ATTO-647N, respectively.     

Computational studies of the binding site of α<Subscript>1A</Subscript>-adrenoceptor antagonists

Aimed at achieving a good understanding of the 3-dimensional structures of human α_1A-adrenoceptor (α_1A-AR), we have successfully developed its homology model based on the crystal structure of β₂-AR. Subsequent structural refinements were performed to mimic the receptor’s natural membrane environment by using molecular mechanics (MM) and molecular dynamics (MD) simulations in the GBSW implicit membrane model. Through molecular docking and further simulations, possible binding modes of subtype-selective α_1A-AR antagonists, Silodosin, RWJ-69736 and (+)SNAP-7915, were examined. Results of the modeling and docking studies are qualitatively consistent with available experimental data from mutagenesis studies. The homology model built should be very useful for designing more potent subtype-selective α_1A-AR antagonists and for guiding further mutagenesis studies. Figure The superposition of β₂-AR crystal structure (gold ribbons) and α_1A-AR homology model (blue ribbons)     

NMR solution structure of the N-terminal domain of subunit E (E<Subscript>1–52</Subscript>) of A<Subscript>1</Subscript>A<Subscript>O</Subscript> ATP synthase from <Emphasis Type="Italic">Methanocaldococcus jannaschii</Emphasis>

The N-termini of E and H of A₁A_O ATP synthase have been shown to interact and an NMR structure of N-terminal H_1–47 has been solved recently. In order to understand the E-H assembly and the N-terminal structure of E, the truncated construct E_1–52 of Methanocaldococcus jannaschii A₁A_O ATP synthase was produced, purified and the solution structure of E_1–52 was determined by NMR spectroscopy. The protein is 60.5 Å in length and forms an α helix between the residues 8–48. The molecule is amphipathic with a strip of hydrophobic residues, discussed as a possible helix-helix interaction with neighboring subunit H.     

Differential expression of GABA<Subscript>A</Subscript> receptor π subunit in cultured rat alveolar epithelial cells

Although type A -aminobutyric acid (GABA) receptors (ligand-gated Cl^– channels) have been extensively studied in the central nervous system, no information is available on this receptor in lung cells. We have examined the expression of GABA_A receptor -subunit (GABRP) during the trans-differentiation between rat alveolar epithelial type II cells and type I cells. Rat alveolar type II cells, when cultured on plastic plates, gradually trans-differentiated into type-I-like cells and lost their GABRP mRNA expression. However, the GABRP mRNA was partially retained in the type II cells cultured on Matrigel. Keratinocyte growth factor (a mitogen of type II cells) increased GABRP expression. A detached collagen gel maintained the GABRP mRNA to a level close to that of the freshly isolated type II cells. An air–liquid interface culture system, mimicking in vivo conditions in the lung, significantly up-regulated the expression of GABRP mRNA and protein. mRNAs of the GABA_A receptor 1-, 3-, 2-, 2-, and 3-subunits were also detected in rat type II cells. These results suggest that GABRP expression is differentially regulated by culture substrata, growth factor, detached gel, and an air-apical surface.This work was supported by NIH R01 HL-52146, R01 NIH-071628, and OCAST HR01-093, and AHA heartland affiliate 0255992Z (to L.L.). N.J. was supported by an AHA heartland affiliate pre-doctoral fellowship (0315256Z).     

α<Subscript>2A</Subscript>- and α<Subscript>2C</Subscript>-Adrenoceptors as Potential Targets for Dopamine and Dopamine Receptor Ligands

The poor norepinephrine innervation and high density of Gi/o-coupled α_2A- and α_2C-adrenoceptors in the striatum and the dense striatal dopamine innervation have prompted the possibility that dopamine could be an effective adrenoceptor ligand. Nevertheless, the reported adrenoceptor agonistic properties of dopamine are still inconclusive. In this study, we analyzed the binding of norepinephrine, dopamine, and several compounds reported as selective dopamine D₂-like receptor ligands, such as the D₃ receptor agonist 7-OH-PIPAT and the D₄ receptor agonist RO-105824, to α₂-adrenoceptors in cortical and striatal tissue, which express α_2A-adrenoceptors and both α_2A- and α_2C-adrenoceptors, respectively. The affinity of dopamine for α₂-adrenoceptors was found to be similar to that for D₁-like and D₂-like receptors. Moreover, the exogenous dopamine receptor ligands also showed high affinity for α_2A- and α_2C-adrenoceptors. Their ability to activate Gi/o proteins through α_2A- and α_2C-adrenoceptors was also analyzed in transfected cells with bioluminescent resonance energy transfer techniques. The relative ligand potencies and efficacies were dependent on the Gi/o protein subtype. Furthermore, dopamine binding to α₂-adrenoceptors was functional, inducing changes in dynamic mass redistribution, adenylyl cyclase activity, and ERK1/2 phosphorylation. Binding events were further studied with computer modeling of ligand docking. Docking of dopamine at α_2A- and α_2C-adrenoceptors was nearly identical to its binding to the crystallized D₃ receptor. Therefore, we provide conclusive evidence that α_2A- and α_2C-adrenoceptors are functional receptors for norepinephrine, dopamine, and other previously assumed selective D₂-like receptor ligands, which calls for revisiting previous studies with those ligands.     

Deciphering the binding mode of Zolpidem to GABA<Subscript>A</Subscript> α<Subscript>1</Subscript> receptor – insights from molecular dynamics simulation

To investigate the binding mode of Zolpidem to GABA(A) and to delineate the conformational changes induced upon agonist binding, we carried out atomistic molecular dynamics simulation using the ligand binding domain of GABA(A) α(1) receptor. Comparative molecular dynamics simulation of the apo and the holo form of GABA(A) receptor revealed that γ(2)/α(1) interface housing the benzodiazepine binding site undergoes distinct conformational changes upon Zolpidem binding. We notice that C loop of the α(1) subunit experiences an inward motion toward the vestibule and the F loop of γ(2) sways away from the vestibule, an observation that rationalizes Zolpidem as an alpha1 selective agonist. Energy decomposition analysis carried out was able to highlight the important residues implicated in Zolpidem binding, which were largely in congruence with the experimental data. The simulation study disclosed herein provides a meaningful insight into Zolpidem-GABA(A)R interactions and helps to arrive at a binding mode hypothesis with implications for drug design.     

α<Subscript>1A</Subscript>- adrenergic receptor mediated pressor response to phenylephrine in anesthetized rat

To determine which subtype of α_1-adrenergic receptors plays a role in the regulation of blood pressure, with α_1--adrenergic receptor-mediated vasoconstriction in perfused hindlimb as a control, we compared the inhibitory effects of various aradrenergic receptor selective antagonists on the vasopressure responses to phenylephrine between the mean arterial pressure and hindlimb perfusion pressure in anesthetized rats. In Normotensive Wistar rats, the results showed that the inhibitory effects (dose ratios of ED₅₀, Dr) of α_-1adrenoceptor selective antagonist (prazosin, Dr 13.5 ± 3.6 vs. 15.1 ± 4.3, n = 11), /ga_1A-adrenoceptor selective antagonist (5-methyl-urapidil, Dr 2.4 ± 0.9 vs. 3.7 ± 2.3, n = 12; RS-17053, Dr 3.2 ± 1.6 vs. 4.4 ± 3.3, n =12) and α_1D- adrenoceptor selective antagonist (BMY7378, Dr 1.9 ±0.9 vs. 2.2 ± 0.8, n = 8) on phenylephrineinduced increases of perfusion pressure in the autoperfused femoral beds were the same as that in the mean arterial blood pressure in normotensive Wistar rats. The inhibitory effects of antagonists (RS-17053, Dr 3.4 ± 0.6 vs. 4.3 ± 0.9, n = 5; BMY7378, Dr 1.7 ± 0.5 vs. 1.7 ± 0.5, n = 8) in spontaneous hypertensive rats were similar with the Wistar rats. These results suggest that the mean arterial pressure induced by phenylephrine was mainly mediated by α_1A-adrenergic receptor in both the anesthetized Wistar rats and spontaneous hypertensive rats.     

The PsbQ′ protein affects the redox potential of the Q<Subscript>A</Subscript> in photosystem II

Red alga contains four extrinsic proteins in photosystem II (PSII), which are PsbO, PsbV, PsbU, and PsbQ′. Except for the PsbQ′, the composition is the same in cyanobacterial PSII. Reconstitution analysis of cyanobacterial PSII has shown that oxygen-evolving activity does not depend on the presence of PsbQ′. Recently, the structure of red algal PSII was elucidated. However, the role of PsbQ′ remains unknown. In this study, the function of the acceptor side of PSII was analyzed in PsbQ′-reconstituted PSII by redox titration of Q_A and thermoluminescence. The redox potential of Q_A was positively shifted when PsbQ′ was attached to the PSII. The positive shift of Q_A is thought to cause a decrease in the amount of triplet chlorophyll in PSII. On the basis of these results, we propose that PsbQ′ has a photoprotective function when irradiated with strong light.     

Neuronal adenosine A<Subscript>2A</Subscript> receptor overexpression is neuroprotective towards 3-nitropropionic acid-induced striatal toxicity: a rat model of Huntington’s disease

The A_2A adenosine receptor (A_2AR) is widely distributed on different cellular types in the brain, where it exerts a broad spectrum of pathophysiological functions, and for which a role in different neurodegenerative diseases has been hypothesized or demonstrated. To investigate the role of neuronal A_2ARs in neurodegeneration, we evaluated in vitro and in vivo the effect of the neurotoxin 3-nitropropionic acid (3-NP) in a transgenic rat strain overexpressing A_2ARs under the control of the neural-specific enolase promoter (NSEA_2A rats). We recorded extracellular field potentials (FP) in corticostriatal slice and found that the synaptotoxic effect of 3-NP was significantly reduced in NSEA_2A rats compared with wild-type animals (WT). In addition, after exposing corticostriatal slices to 3-NP 10 mM for 2 h, we found that striatal cell viability was significantly higher in NSEA_2A rats compared to control rats. These in vitro results were confirmed by in vivo experiments: daily treatment of female rats with 3-NP 10 mg/kg for 8 days induced a selective bilateral lesion in the striatum, which was significantly reduced in NSEA_2A compared to WT rats. These results demonstrate that the overexpression of the A_2AR selectively at the neuronal level reduced 3-NP-induced neurodegeneration, and suggest an important function of the neuronal A_2AR in the modulation of neurodegeneration.     

Inhibition of photosynthetic oxygen evolution and electron transfer from the quinone acceptor Q<Subscript>A</Subscript><Superscript>−</Superscript> to Q<Subscript>B</Subscript> by iron deficiency

The effect of iron deficiency on photosynthetic electron transport in Photosystem II (PS II) was studied in leaves and thylakoid membranes of lettuce (Lactuca sativa, Romaine variety) plants. PS II electron transport was characterized by oxygen evolution and chlorophyll fluorescence parameters. Iron deficiency in the culture medium was shown to affect water oxidation and the advancement of the S-states. A decrease of maximal quantum yield of PS II and an increase of fluorescence intensity at step J and I of OJIP kinetics were also observed. Thermoluminescence measurements revealed that charge recombination between the quinone acceptor of PS II, Q_B, and the S₂ state of the Mn-cluster was strongly perturbed. Also the dark decay of Chl fluorescence after a single turnover white flash was greatly retarded indicating a slower rate of Q_A ⁻ reoxidation.     

Effect of the L- or D-aspartate on ecto-5′nucleotidase activity and on cellular viability in cultured neurons: participation of the adenosine A<Subscript>2A</Subscript> receptors

Summary. Glutamate increases the extracellular adenosine levels, an important endogenous neuromodulator. The neurotoxicity induced by glutamate increases the ecto-5′-nucleotidase activity in neurons, which produces adenosine from AMP. L- and D-aspartate (Asp) mimic most of the actions of glutamate in the N-methyl-D-aspartate (NMDA) receptors. In the present study, both amino acids stimulated the ecto-5′-nucleotidase activity in cerebellar granule cells. MK-801 and AP-5 prevented the L- and D-Asp-evoked activation of ecto-5′-nucleotidase. Both NMDA receptor antagonists prevented completely the damage induced by L-Asp, but partially the D-Asp-induced damage. The antagonist of adenosine A_2A receptors (ZM 241385) prevented totally the L- Asp-induced cellular death, but partially the neurotoxicity induced by D-Asp and the antagonist of adenosine A₁ receptors (CPT) had no effect. The results indicated a different involvement of NMDA receptors on the L- or D-Asp-evoked activation of ecto-5′-nucleotidase and on cellular damage. The adenosine formed from ecto-5′-nucleotidase stimulation preferentially acted on adenosine A_2A receptor which is probably co-operating with the neurotoxicity induced by amino acids.     

Protein kinase Cζ regulates phospholipase D activity in rat-1 fibroblasts expressing the α<Subscript>1A</Subscript> adrenergic receptor

Background  

Phenylephrine (PHE), an α₁ adrenergic receptor agonist, increases phospholipase D (PLD) activity, independent of classical and novel protein kinase C (PKC) isoforms, in rat-1 fibroblasts expressing α_1A adrenergic receptors. The aim of this study was to determine the contribution of atypical PKCζ to PLD activation in response to PHE in these cells.     

Insight into π-hole interactions containing the inorganic heterocyclic compounds S<Subscript>2</Subscript>N<Subscript>2</Subscript>/SN<Subscript>2</Subscript>P<Subscript>2</Subscript>

Similar to σ-hole interactions, the π-hole interaction has attracted much attention in recent years. According to the positive electrostatic potentials above and below the surface of inorganic heterocyclic compounds S₂N₂ and three SN₂P₂ isomers (heterocyclic compounds 1–4), and the negative electrostatic potential outside the X atom of XH₃ (X = N, P, As), S₂N₂/SN₂P₂?XH₃ (X = N, P, As) complexes were constructed and optimized at the MP2/aug-cc-pVTZ level. The X atom of XH₃ (X = N, P, As) is almost perpendicular to the ring of the heterocyclic compounds. The π-hole interaction energy becomes greater as the trend goes from 1?XH₃ to 4?XH₃. These π-hole interactions are weak and belong to “closed-shell” noncovalent interactions. According to the energy decomposition analysis, of the three attractive terms, the dispersion energy contributes more than the electrostatic energy. The polarization effect also plays an important role in the formation of π-hole complexes, with the contrasting phenomena of decreasing electronic density in the π-hole region and increasing electric density outside the X atom of XH₃ (X = N, P, As).

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Graphical abstract Computed density difference plots for the complexes 3?NH ₃ (a ₁), 3?PH ₃ (b ₁), 3?AsH ₃ (c ₁) and electron density shifts for the complexes 3?NH ₃ (a ₂), 3?PH ₃ (b ₂),3?AsH ₃ (c ₂) on the 0.001 a.u. contour

     

Electronic and optical properties of C<Subscript>24</Subscript>, C<Subscript>12</Subscript>X<Subscript>6</Subscript>Y<Subscript>6</Subscript>, and X<Subscript>12</Subscript>Y<Subscript>12</Subscript> (X = B,Al and Y = N,P)

Utilizing first-principles calculations, we studied the electronic and optical properties of C₂₄, C₁₂X₆Y₆, and X₁₂Y₁₂ fullerenes (X?=?B, Al; Y?=?N, P). These fullerenes are energetically stable, as demonstrated by their negative cohesive energies. The energy gap of C₂₄ may be tuned by doping, and the B₁₂N₁₂ fullerene was found to have the largest energy gap. All of the fullerenes had finite optical gaps, suggesting that they are optical semiconductors, and they strongly absorb UV radiation, so they could be used in UV light protection devices. They could also be used in solar cells and LEDs due to their low reflectivities.

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Graphical abstract Possible applications of doped C₂₄ fullerene

     

Genome-wide linkage analysis of longitudinal phenotypes using σ<Superscript>2</Superscript><Subscript>A</Subscript> random effects (SSARs) fitted by Gibbs sampling

The study of change in intermediate phenotypes over time is important in genetics. In this paper we explore a new approach to phenotype definition in the genetic analysis of longitudinal phenotypes. We utilized data from the longitudinal Framingham Heart Study Family Cohort to investigate the familial aggregation and evidence for linkage to change in systolic blood pressure (SBP) over time. We used Gibbs sampling to derive sigma-squared-A-random-effects (SSARs) for the longitudinal phenotype, and then used these as a new phenotype in subsequent genome-wide linkage analyses. Additive genetic effects (sigma2A.time) were estimated to account for approximately 9.2% of the variance in the rate of change of SBP with age, while additive genetic effects (sigma2A) were estimated to account for approximately 43.9% of the variance in SBP at the mean age. The linkage results suggested that one or more major loci regulating change in SBP over time may localize to chromosomes 2, 3, 4, 6, 10, 11, 17, and 19. The results also suggested that one or more major loci regulating level of SBP may localize to chromosomes 3, 8, and 14. Our results support a genetic component to both SBP and change in SBP with age, and are consistent with a complex, multifactorial susceptibility to the development of hypertension. The use of SSARs derived from quantitative traits as input to a conventional linkage analysis appears to be valuable in the linkage analysis of genetically complex traits. We have now demonstrated in this paper the use of SSARs in the context of longitudinal family data.     

How Common Are Intragene Windows with <Emphasis Type="Italic">K</Emphasis><Subscript>A</Subscript> > <Emphasis Type="Italic">K</Emphasis><Subscript>S</Subscript> Owing to Purifying Selection on Synonymous Mutations?

One method for diagnosing the mode of sequence evolution considers the ratio of nonsynonymous substitutions per nonsynonymous site (K _A) to the corresponding figure for synonymous substitutions (K _S). A ratio (K _A/K _S) greater than unity is taken as evidence for positive selection. This, however, need not necessarily be the case. Notably, there is one instance of a high intragenic K _A/K _S peak, revealed by sliding window analysis and observed in two pairwise comparisons, better accounted for by localised purifying selection on synonymous mutations that affect splicing. Is this example exceptional? To address this we isolate intragenic domains with K _A/K _S > 1 from more than 1000 long mouse-rat orthologues. Approximately one K _A/K _S > 1 peak is found per 12–15 kb of coding sequence. Surprisingly, low synonymous substitution rates underpin more incidences than do high nonsynonymous rates. Several reasons, however, prevent us from supposing that the low synonymous rates reflect purifying selection on synonymous mutations. First, for many peaks, the null that the peak is no higher than expected given the underlying rates of evolution, cannot be rejected. Second, of 18 statistically significant incidences with unusually low K _S values, only 3 are repeatable across independent comparisons. At least two of these are within alternatively spliced exons. We conclude that repeatable statistically significant intragenic domains of low intragenic K _S are rare. As so few K _A/K _S peaks reflect increased rates of protein evolution and so few hold statistical support, we additionally conclude that sliding window analysis to infer domains of positive selection is highly error-prone.