Identification of a Novel HIV-1 Inhibitor Targeting Vif-dependent Degradation of Human APOBEC3G Protein

APOBEC3G (A3G) is a cellular cytidine deaminase that restricts HIV-1 replication by inducing G-to-A hypermutation in viral DNA and by deamination-independent mechanisms. HIV-1 Vif binds to A3G, resulting in its degradation via the 26 S proteasome. Therefore, this interaction represents a potential therapeutic target. To identify compounds that inhibit interaction between A3G and HIV-1 Vif in a high throughput format, we developed a homogeneous time-resolved fluorescence resonance energy transfer assay. A 307,520 compound library from the NIH Molecular Libraries Small Molecule Repository was screened. Secondary screens to evaluate dose-response performance and off-target effects, cell-based assays to identify compounds that attenuate Vif-dependent degradation of A3G, and assays testing antiviral activity in peripheral blood mononuclear cells and T cells were employed. One compound, N.41, showed potent antiviral activity in A3G(+) but not in A3G(−) T cells and had an IC₅₀ as low as 8.4 μm and a TC₅₀ of >100 μm when tested against HIV-1_Ba-L replication in peripheral blood mononuclear cells. N.41 inhibited the Vif-A3G interaction and increased cellular A3G levels and incorporation of A3G into virions, thereby attenuating virus infectivity in a Vif-dependent manner. N.41 activity was also species- and Vif-dependent. Preliminary structure-activity relationship studies suggest that a hydroxyl moiety located at a phenylamino group is critical for N.41 anti-HIV activity and identified N.41 analogs with better potency (IC₅₀ as low as 4.2 μm). These findings identify a new lead compound that attenuates HIV replication by liberating A3G from Vif regulation and increasing its innate antiviral activity.     

Coalescent theory for a completely random mating monoecious population

Consider a large random mating monoecious diploid population that has N individuals in each generation. Let us assume that at time 0 a random sample of ninfinity. It is then possible to obtain a generalization of coalescent theory for haploid populations if the distribution of G1 has a finite second moment and E[G(1)(3)]/N-->0 as N-->infinity.     

Transmembrane topology of human glucose 6-phosphate transporter.

Glycogen storage disease type 1b is caused by a deficiency in a glucose 6-phosphate transporter (G6PT) that translocates glucose 6-phosphate from the cytoplasm to the endoplasmic reticulum lumen where the active site of glucose 6-phosphatase is situated. Using amino- and carboxyl-terminal tagged G6PT, we demonstrate that proteolytic digestion of intact microsomes resulted in the cleavage of both tags, indicating that both termini of G6PT face the cytoplasm. This is consistent with ten and twelve transmembrane domain models for G6PT predicted by hydropathy analyses. A region of G6PT corresponding to amino acid residues 50-71, which constitute a transmembrane segment in the twelve-domain model, are situated in a 51-residue luminal loop in the ten-domain model. To determine which of these two models is correct, we generated two G6PT mutants, T53N and S55N, that created a potential Asn-linked glycosylation site at residues 53-55 (N53SS) or 55-57 (N55QS), respectively. N53SS or N55QS would be glycosylated only if it is situated in a luminal loop larger than 33 residues as predicted by the ten-domain model. Whereas wild-type G6PT is not a glycoprotein, both T53N and S55N mutants are glycosylated, strongly supporting the ten-helical model for G6PT.     

Photosynthesis and nitrogen utilization in exponentially growing nitrogen-limited cultures of Lemna gibba

The photosynthetic performance and nitrogen utilization of Lemna gibba L. G3 adapted to limited nitrogen supply was studied. The plants were adapted to two levels of nitrogen limitation where the nitrogen addition rates were calculated to sustain relative growth rates (RGR) of 0.15 day^?1 and 0.25 day^?1, respectively. The photosynthetic performance of these cultures was compared to nitrogen-sufficient cultures with an average RGR of 0.32 day^?1. Plants transferred from nitrogen-sufficient conditions attained RGR values corresponding to the nitrogen addition rates after 6 to 10 days. Light-saturated net photosynthesis declined during adaptation according to the drop in growth rate, and a concomitant decrease in the respiration rate was recorded. The efficiency of net photosynthesis on a dry weight basis increased with increased nitrogen supply, whereas it was the same in all cultures when expressed on a chlorophyll basis. The light compensation point was unaffected by the nitrogen regime. Limited nitrogen supply resulted in an increased proportion of dry matter in the roots, which led to decreased leaf area ratios. The net assimilation rates also decreased, but not to the same extent as the leaf area ratios. Growth-limiting amounts of nitrogen were added to the cultures once daily, and the net influx of N was higher than the requirement for N, also in adapted cultures with a steady growth rate. This resulted in transient, periodic fluctuations in the NO₃^?, NH₄⁺ and amino acid pools. Also the rates of NO₃^? reduction and NH₄⁺ assimilation fluctuated as did the amino acid assimilation which paralleled NH₄⁺ assimilation. The role of flux rates over the plasmalemma and tonoplast for control of nitrogen assimilation rates are discussed.     

Cytoplasmic Compartmentalization of the Protein and Ribonucleic Acid Species of Vesicular Stomatitis Virus

The cytoplasmic sites of synthesis in L cells of the protein and ribonucleic acid species of vesicular stomatitis virus were studied by polyacrylamide gel electrophoresis after fractionation of membrane and other cytoplasmic components by the Caliguiri-Tamm technique. The viral spike protein (glycoprotein G) was found primarily associated with a smooth membrane fraction which is rich in plasma membrane; the G protein was also present in fractions containing rough endoplasmic reticulum. The nonglycosylated envelope protein S (also called M) was found in the smooth membrane fractions but was more abundant in endoplasmic reticulum-enriched fractions. Longer labeling resulted in detection of nucleoprotein N, as well as other minor nucleocapsid proteins L and NS₁, in the cellular membrane fractions. The N protein appeared to be made in membrane-free cytoplasm along with progeny ribonucleic acid and later became associated with membrane containing G and S viral proteins.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C,and <Superscript>15</Superscript>N backbone chemical shift assignments of 4E-BP1<Subscript>44–87</Subscript> and 4E-BP1<Subscript>44–87</Subscript> bound to eIF4E

The eukaryotic translational initiation factor 4G (eIF4G) interacts with the cap-binding protein eIF4E through a consensus binding motif, Y(X)₄LΦ (where X is any amino acid and Φ is a hydrophobic residue). 4E binding proteins (4E-BPs), which also contain a Y(X)₄LΦ motif, regulate the eIF4E/eIF4G interaction. The non- or minimally-phosphorylated form of 4E-BP1 binds eIF4E, preventing eIF4E from interacting with eIF4G, thus inhibiting translation initiation. 4EGI-1, a small molecule inhibitor of the eIF4E/eIF4G interaction that is under investigation as a novel anti-cancer drug, has a dual activity; it disrupts the eIF4E/eIF4G interaction and stabilizes the binding of 4E-BP1 to eIF4E. Here, we report the complete backbone NMR resonance assignment of an unliganded 4E-BP1 fragment (4E-BP1_44–87). We also report the near complete backbone assignment of the same fragment in complex to eIF4E/m⁷GTP (excluding the assignment of the last C-terminus residue, D87). The chemical shift data constitute a prerequisite to understanding the mechanism of action of translation initiation inhibitors, including 4EGI-1, that modulate the eIF4E/4E-BP1 interaction.     

Stream acidification increases nitrogen uptake by leaf biofilms: implications at the ecosystem scale

1. While anthropogenic stream acidification is known to lower species diversity and impair decomposition, its effects on nutrient cycling remain unclear. The influence of acid‐stress on microbial physiology can have implications for carbon (C) and nitrogen (N) cycles, linking environmental conditions to ecosystem processes. 2. We collected leaf biofilms from streams spanning a gradient of pH (5.1–6.7), related to chronic acidification, to investigate the relationship between qCO₂ (biomass‐specific respiration; mg CO₂‐C g^?1 fungal C h^?1), a known indicator of stress, and biomass‐specific N uptake (μg NH₄‐N mg^?1 fungal biomass h^?1) at two levels of N availability (25 and 100 μg NH₄‐N L^?1) in experimental microcosms. 3. Strong patterns of increasing qCO₂ (i.e. increasing stress) and increasing microbial N uptake were observed with a decrease in ambient (i.e. chronic) stream pH at both levels of N availability. However, fungal biomass was lower on leaves from more acidic streams, resulting in lower overall respiration and N uptake when rates were standardized by leaf biomass. 4. Results suggest that chronic acidification decreases fungal metabolic efficiency because, under acid conditions, these organisms allocate more resources to maintenance and survival and increase their removal of N, possibly via increased exoenzyme production. At the same time, greater N availability enhanced N uptake without influencing CO₂ production, implying increased growth efficiency. 5. At the ecosystem level, reductions in growth because of chronic acidification reduce microbial biomass and may impair decomposition and N uptake; however, in systems where N is initially scarce, increased N availability may alleviate these effects. Ecosystem response to chronic stressors may be better understood by a greater focus on microbial physiology, coupled elemental cycling, and responses across several scales of investigation.     

Two-H-Bonded and One-H-Bonded Structure Alternations in Collagen

Abstract

This paper concerns the conformational variability of collagen as related to the concrete tripeptides (GXY)_n constituting its primary structure. The previously elaborated model (V.G. Tumanyan, N.G. Esipova, Biophysics 28, 1021–1025, 1983) with two nets of hydrogen bonds is useful for tripeptides where X is an amino acid. If X is an imino acid, the common one-bonded Rich & Crick model is valid. In this work, compound sequences including tripeptides of different types are considered. Molecular mechanics is used to assess the conformations of the junction regions when a structure with two nets of hydrogen bonds precedes the structure with one net, and vice versa. Thus, all types of sequences typical for natural collagen are covered. It is shown that the combined model representing an alternation of the two-H-bonded model and the one-H-bonded Rich & Crick model is satisfactory stere-ochemically, and provides more favorable energy in comparison with the continuous one-H-bonded model. Besides, a more favorable hydration of the molecule occurs in this case. Some conclusions are made about interchain and intrachain ionic bonds. Thus, it is deduced for the concrete fibrillar protein how a one-dimensional structure determines three-dimensional structure. The macromolecular structure thus suggested is in accord with the experimental data on hydrogen exchange.     

Understanding crop physiology to select breeding targets and improve crop management under increasing atmospheric CO2 concentrations

The present overview paper reviews knowledge on plant metabolism under elevated atmospheric CO₂ concentrations (e[CO₂]) with regard to underpinning options for the management of crop production systems and the selection of crop traits beneficial for future conditions.Better understanding of intra-specific variability in responses to e[CO₂] is of great importance to breed or select best possible genotypes for future conditions. Yield increases per 100 μL L⁻¹ increase in [CO₂] varied between none and over 30% among varieties of important crops. Carbon source–sink relationships are believed to play a major role in determining the ability of a plant to utilise e[CO₂] and avoid downward acclimation of photosynthesis upon prolonged e[CO₂] exposure. Corresponding traits (e.g. tillering capacity, stem carbohydrate storage capacity, or seed size and numbers) are currently under investigation in Free Air Carbon dioxide Enrichment (FACE) facilities, such as AGFACE (Australian Grains FACE).The stimulatory effect of e[CO₂] on plant growth is dependent on adequate nutrient supply. For example, N concentrations in plant tissues generally decrease under e[CO₂], which in leaves is commonly related to a decrease in Rubisco concentration and activity, and therefore linked to photosynthetic downward acclimation. This effect is also of direct concern for food production where decreased N and protein content can have negative effects on product quality (e.g. grain protein). Plant nutrient metabolism appears to adjust to a new physiological equilibrium under e[CO₂] which limits the extent to which nutrient application can ameliorate the situation. What the control points are for an adjustment of plant N metabolism is unclear. Rubisco metabolism in leaves, N assimilation, N translocation or N uptake are all potential key steps that may be inhibited or downregulated under e[CO₂]. To achieve the best possible growth response whilst maintaining product quality, it is important to understand plant nutrient metabolism under e[CO₂].Comparatively little is known about mechanisms of potential changes in plant stress tolerance under e[CO₂]. Defence metabolites such as antioxidants are, in part, directly linked to primary carbohydrate mechanism and so potentially impacted by e[CO₂]. It is unknown whether photoprotective and antioxidative defence systems, key to plant stress tolerance, will be affected, and if so, whether the response will be strengthened or weakened by e[CO₂]. Better understanding of underlying principles is particularly important because it is virtually impossible to test all possible stress factor combinations with e[CO₂] in realistic field settings.     

The 4b-4c loop of excitatory amino acid transporter 1 containing four critical residues essential for substrate transport

Abstract

In the mammalians, the 4b-4c loop of excitatory amino acid transporters (EAATs) spans more than 50 amino-acid residues that are absent in glutamate transporter homologue of Pyrococcus horikoshii (Glt_Ph). This part of insertion is unique for metazoans and indispensable to the localization of EAATs. The excitatory amino acid transporter (EAAT) 1 is one of the two glial glutamate transporters, which are responsible for efficiently clearing glutamate from the synaptic cleft to prevent neurotoxicity and cell death. Although the crystal structure of EAAT1_cryst (a human thermostable EAAT1) was resolved in 2017, the structure-function relationship of the 4b-4c loop has not been elucidated in EAAT1_cryst. To investigate the role of the 4b-4c loop, we performed alanine-scanning mutagenesis in the mutants and observed dramatically decreased transport activities in T192A, Y194A, N242A, and G245A mutants. The surface expression of T192A and Y194A mutants even decreased by more than 80%, and most of them were detained in the cytoplasm. However, when T192 and Y194 were substituted with conservative residues, the transport activities and the surface expressions of T192S and Y194F were largely recovered, and their kinetic parameters (K_m values) were comparable to the wild-type EAAT1 as well. In contrast, N242 and G245 substituted with conservative residues could not rescue the uptake function, suggesting that N242 and G245 may play irreplaceable roles in the glutamate uptake process. These results indicate that the 4b-4c loop of EAAT1 may not only affect the glutamate uptake activity, but also influence the surface localization of EAAT1 by T192 and Y194.

Communicated by Ramaswamy H. Sarma     

Gel-based oligonucleotide microarray approach to analyze protein–ssDNA binding specificity

Gel-based oligonucleotide microarray approach was developed for quantitative profiling of binding affinity of a protein to single-stranded DNA (ssDNA). To demonstrate additional capabilities of this method, we analyzed the binding specificity of ribonuclease (RNase) binase from Bacillus intermedius (EC 3.1.27.3) to ssDNA using generic hexamer oligodeoxyribonucleotide microchip. Single-stranded octamer oligonucleotides were immobilized within 3D hemispherical gel pads. The octanucleotides in individual pads 5′-{N}N₁N₂N₃N₄N₅N₆{N}-3′ consisted of a fixed hexamer motif N₁N₂N₃N₄N₅N₆ in the middle and variable parts {N} at the ends, where {N} represent A, C, G and T in equal proportions. The chip has 4096 pads with a complete set of hexamer sequences. The affinity was determined by measuring dissociation of the RNase–ssDNA complexes with the temperature increasing from 0°C to 50°C in quasi-equilibrium conditions. RNase binase showed the highest sequence-specificity of binding to motifs 5′-NNG(A/T/C)GNN-3′ with the order of preference: GAG > GTG > GCG. High specificity towards G(A/T/C)G triplets was also confirmed by measuring fluorescent anisotropy of complexes of binase with selected oligodeoxyribonucleotides in solution. The affinity of RNase binase to other 3-nt sequences was also ranked. These results demonstrate the applicability of the method and provide the ground for further investigations of nonenzymatic functions of RNases.     

Analysis of penicillin V biosynthesis during fed-batch cultivations with a high-yielding strain of Penicillium chrysogenum

Metabolites (both intra- and extracellular) involved in penicillin biosynthesis were measured during fed-batch cultivations with a high-yielding strain of Penicillium chrysogenum. The fed-batch cultivations were carried out on a complex medium containing corn steep liqour. Three distinct phases were observed: (a) a rapid growth phase where free amino acids present in the medium are metabolized, (b) a linear growth phase, and (c) a stationary phase. The specific penicillin production (r _p) is initially high and, during the rapid growth phase, it increases slightly. During the linear growth phase r _p is approximately constant [4–6 mg penicillin V (g dry weight)^–1 h^–1 depending on the operating conditions], whereas it decreases during the stationary phase. During the cultivations the tripeptide Aad-Cys-Val (the first metabolite in penicillin biosynthesis) and 8-hydroxypenillic acid (formed by carboxylation of 6-aminopenicillanic acid, 6-APA) were found to accumulate in the medium, whereas the concentrations of isopenicillin N and 6-APA were found to be approximately constant and low. About 3% of the Aad-Cys-Val formed in the first step of the penicillin biosynthetic pathway is lost to the medium and 4% of the isopenicillin N formed in the second step of the pathway is lost as extracellular isopenicillin N, 6-APA or 8-hydroxypenillic acid. Also the cyclic form of -aminoadipic acid, 6-oxopiperidine-2-carboxylic acid, was found to accumulate in the medium and it was found to be formed in an approximately constant ratio to penicillin V of 6 mol/100 mol.     

Assessing post-anthesis nitrogen uptake, distribution and utilisation in grain protein synthesis in barley (Hordeum vulgare L.) using 15N fertiliser and 15N proteinogenic and non-proteinogenic amino acids

We investigated the effects of nitrogen (N) availability during the vegetative phase on (a) post‐anthesis N uptake and (b) its translocation into ears in barley plants grown in a greenhouse at two levels of N: low (50 mg N kg^?1 sand) and optimal N supply (150 mg N kg^?1 sand). Plants in the two N treatments were fertilised with the same amount of labelled ¹⁵N [50 mg ¹⁵N kg^?1 sand at 10% ¹⁵N_exc (N_excess, i.e. N_exc, is defined as the abundance of enriched stable isotope minus the natural abundance of the isotope) applied as ¹⁵NH₄¹⁵NO₃] 10 days after anthesis (daa). In a separate experiment, the uptake and transport into ears of proteinogenic and non‐proteinogenic amino acids were studied to determine whether a relationship exists between amino acid transport into ears and their proteinogenic nature. Plants were fed with either ¹⁵N‐α‐alanine, a proteinogenic amino acid, or ¹⁵N‐α‐aminoisobutyric acid, a non‐proteinogenic amino acid. Both these amino acids were labelled at 95.6% ¹⁵N_exc. Results showed that N accumulations in stems, leaves and especially in ears were correlated with their dry matter (dm) weights. The application of 150 mg N kg^?1 sand significantly increased plant dm weight and total N accumulation in plants. During their filling period, ears absorbed N from both external (growth substrate) and internal (stored N in plants) sources. Nitrogen concentration in ears was higher in optimal N‐fed plants than in low N‐fed plants until 10 daa, but from 21 to 35 daa, differences were not detected. Conversely, ¹⁵N_exc in ears, leaves and stems was higher in low N‐fed plants than in optimal N‐fed plants. Ears acted as strong sink organ for the post‐anthesis N taken up from the soil independently of pre‐anthesis N nutrition: on average, 87% of the N taken up from the soil after anthesis was translocated and accumulated in ears. Low N‐fed plants continued to take up N from the post‐anthesis N fertiliser during the later grain‐filling period. The increase of pre‐anthesis N supply rate led to a decrease in the contribution of nitrogen derived from post‐anthesis ¹⁵N‐labelled fertiliser (N_dff) to total N in all aboveground organs, especially in ears where 44% and 22% of total N originated from post‐anthesis N uptake in low N‐fed and optimal N‐fed plants, respectively. The experiment with labelled amino acids showed that there was greater transport of proteinogenic amino acid into the ear (50% of total ¹⁵N) than non‐proteinogenic amino acid (39%). However, this transport of the non‐proteinogenic amino acids into ear suggested that the transport of N compounds from source (leaves) to sink organs (ear) might not be intrinsically regulated by their ability to be incorporated into storage protein of ears.     

Effective Size of Populations under Selection

Equations to approximate the effective size (N(e)) of populations under continued selection are obtained that include the possibility of partial full-sib mating and other systems such as assortative mating. The general equation for the case of equal number of sexes and constant number of breeding individuals (N) is N(e) = 4N/[2(1 - α(I)) + (S(k)(2) + 4Q(2)C(2)) (1 + α(I) + 2α(O))], where S(k)(2) is the variance of family size due to sampling without selection, C(2) is the variance of selective advantages among families (the squared coefficient of variation of the expected number of offspring per family), α(I) is the deviation from Hardy-Weinberg proportions, α(O) is the correlation between genes of male and female parents, and Q(2) is the term accounting for the cumulative effect of selection on an inherited trait. This is obtained as Q = 2/[2 - G(1 + r)], where G is the remaining proportion of genetic variance in selected individuals and r is the correlation of the expected selective values of male and female parents. The method is also extended to the general case of different numbers of male and female parents. The predictive value of the formulae is tested under a model of truncation selection with the infinitesimal model of gene effects, where C(2) and G are a function of the selection intensity, the heritability and the intraclass correlation of sibs. Under random mating r = α(I) = -1/(N - 1) and α(O) = 0. Under partial full-sib mating with an average proportion β of full-sib matings per generation, r & β and α(O) & α(I) & β/ (4 - 3β). The prediction equation is compared to other approximations based on the long-term contributions of ancestors to descendants. Finally, based on the approach followed, a system of mating (compensatory mating) is proposed to reduce rates of inbreeding without loss of response in selection programs in which selected individuals from the largest families are mated to those from the smallest families.     

Strong Patterns in Homooligomer Tracts Occurrences in Non-Coding and in Potential Regulatory Sites in Eukaryotic Genomes

Abstract

Previous studies of the dinucleotides flanking both the 5′ and 3′ ends of homooligomer tracts have shown that some flanks are consistently preferred over others (1,2). In the first preferred group, the homooligomer tracts are flanked by the same nucleotide and/or the complementary nucleotides, e.g., ATA_n, TTA_n, CCG_n, where n=2–5. Runs flanked by nucleotides with which they cannot base pair are distinctly disfavored. (In this group A/T_n are flanked by C and/or G; G_n/C_n are flanked by A/T, e.g., CGA_n, T_nGG, G., AT). The frequencies of runs flanked by AorT, and G or C (“mixed” group) are as expected. Here we seek the origin of this effect and its relevance to protein-DNA interactions. Surprisingly, within the first group, runs flanked by their complements with a pyrimidine-purine junction (e.g., TTA_n, C_nGG) are greatly preferred. The frequencies of their purine-pyrimidine junction mirror-images is just as expected. This effect, as well as additional ones enumerated below, is seen universally in eukaryotes and in prokaryotes, although it is stronger in the former. Detailed analysis of regulatory regions shows these strong trends, particularly in GC sequences. The potential relationship to DNA conformation and DNA-protein interaction is discussed.     

Elevated [CO2] effects on crops: Advances in understanding acclimation,nitrogen dynamics and interactions with drought and other organisms

Future rapid increases in atmospheric CO₂ concentration [CO₂] are expected, with values likely to reach ~550 ppm by mid‐century. This implies that every terrestrial plant will be exposed to nearly 40% more of one of the key resources determining plant growth. In this review we highlight selected areas of plant interactions with elevated [CO₂] (e[CO₂]), where recently published experiments challenge long‐held, simplified views. Focusing on crops, especially in more extreme and variable growing conditions, we highlight uncertainties associated with four specific areas. (1) While it is long known that photosynthesis can acclimate to e[CO₂], such acclimation is not consistently observed in field experiments. The influence of sink–source relations and nitrogen (N) limitation on acclimation is investigated and current knowledge about whether stomatal function or mesophyll conductance (g_m) acclimate independently is summarised. (2) We show how the response of N uptake to e[CO₂] is highly variable, even for one cultivar grown within the same field site, and how decreases in N concentrations ([N]) are observed consistently. Potential mechanisms contributing to [N] decreases under e[CO₂] are discussed and proposed solutions are addressed. (3) Based on recent results from crop field experiments in highly variable, non‐irrigated, water‐limited environments, we challenge the previous opinion that the relative CO₂ effect is larger under drier environmental conditions. (4) Finally, we summarise how changes in growth and nutrient concentrations due to e[CO₂] will influence relationships between crops and weeds, herbivores and pathogens in agricultural systems.     

The Active and the Inactive Form of the hAT1 Receptor Have an Identical Ligand-Binding Environment: An MPA Study on a Constitutively Active Angiotensin II Receptor Mutant

Several models of activation mechanisms were proposed for G protein-coupled receptors (GPCRs), yet no direct methods exist for their elucidation. The availability of constitutively active mutants has given an opportunity to study active receptor conformations within acceptable limits using models such as the angiotensin II type 1 (AT₁)¹ receptor mutant N111G-hAT₁ which displays an important constitutive activity. Recently, by using methionine proximity assay, we showed for the hAT₁ receptor that TMD III, VI, and VII form the ligand-binding pocket of the C-terminal amino acid of an antagonistic AngII analogue. In the present contribution, we investigated whether the same residues would also constitute the ligand-binding contacts in constitutively activated mutant (CAM) receptors. For this purpose, the same Met mutagenesis strategy was carried out on the N111G double mutants. Analysis of 43 receptors mutants in the N111G-hAT₁ series, photolabeled and CNBr digested, showed that there were only subtle structural changes between the wt-receptor and its constitutively active form.     

A novel copper(II) complex with a pendant Schiff base: An unprecedented monodentate bonding mode of the potentially tridentate ligand

The 1:1 condensation of 1-benzoylacetone and 1,2-diaminopropane yields 6-amino-3-methyl-1-phenyl-4-azahept-2-en-1-one (HL). When copper(II) perchlorate is added to the methanolic solution of HL, followed by triethylamine in 1:2:1 molar ratio, an unusual copper(II) complex, [Cu(L)(HL)]ClO₄, is separated out where the deprotonated ligand, L, is coordinated in the usual chelating tridentate manner but HL is coordinated to Cu(II) only through the amine N, i.e. it acts as a pendant ligand. The complex is characterized by X-ray crystal structure analysis.     

Anti-neuroinflammatory effects of grossamide from hemp seed via suppression of TLR-4-mediated NF-κB signaling pathways in lipopolysaccharide-stimulated BV2 microglia cells

Rac1 belongs to the Rho family of small GTPases and regulates actin cytoskeleton reorganization. T2R4 is a bitter taste receptor belonging to the G protein-coupled receptor family of proteins. In addition to mediating bitter taste perception from the tongue, T2R4s are found in extra-oral tissues, e.g., nasal epithelium, airways, brain, testis suggesting a much broader physiological function for these receptors. Anti-malarial drug and a bitter tasting compound, quinine, is a known agonist for T2R4, whereas BCML (Nα,Nα-Bis(carboxymethyl)-l-lysine) acts as an inverse agonist. Using western blot and Ca⁺⁺ mobilization assays, the effects of quinine on Rac1 activity in HEK293T cells stably expressing T2R4/Gα_16/44, T2R4, or Gα_16/44 and transiently transfected with HA-Rac1 were investigated. Quinine treatment caused a significant reduction in the amount of active Rac1, whereas in the presence of BCML, quinine failed to cause any significant change in active Rac1. No significant change in Rac1 activity was observed in BAPTA-AM plus quinine-treated Gα_16/44 cells, suggesting possibility of a pathway in addition to the canonical Ca⁺⁺-dependent pathway. A noticeable role for Gα_16/44 independent of T2R4 is observed in quinine-mediated Rac1 inactivation. Further, a significant difference in quinine-induced Ca⁺⁺ response in T2R4/Gα_16/44 or T2R4 cells was observed validating the partial role of calcium and importance of Gα_16/44. This study is the first to show an inhibitory downstream action of a T2R4 agonist on Rac1 function. Further investigation will help in better understanding the downstream signal transduction network of T2R4 and its extra-oral physiological roles.     

The role of cholesterol and sphingolipids in the dopamine D1 receptor and G protein distribution in the plasma membrane

G proteins are peripheral membrane proteins which interact with the inner side of the plasma membrane and form part of the signalling cascade activated by G protein-coupled receptors (GPCRs). Since many signalling proteins do not appear to be homogeneously distributed on the cell surface, they associate in particular membrane regions containing specific lipids. Therefore, protein–lipid interactions play a pivotal role in cell signalling. Our previous results showed that although Gαs and Gαi₃ prefer different types of membrane domains they are both co-localized with the D₁ receptor. In the present report we characterize the role of cholesterol and sphingolipids in the membrane localization of Gαs, Gαi₃ and their heterotrimers, as well as the D₁ receptor. We measured the lateral diffusion and membrane localization of investigated proteins using fluorescence recovery after photobleaching (FRAP) microscopy and fluorescence resonance energy transfer (FRET) detected by lifetime imaging microscopy (FLIM). The treatment with either methyl-β-cyclodextrin or Fumonisin B₁ led to the disruption of cholesterol–sphingolipids containing domains and changed the diffusion of Gαi₃ and the D₁ receptor but not of Gαs. Our results imply a sequestration of Gαs into cholesterol-independent solid-like membrane domains. Gαi₃ prefers cholesterol-dependent lipid rafts so it does not bind to those domains and its diffusion is reduced. In turn, the D₁ receptor exists in several different membrane localizations, depending on the receptor's conformation. We conclude that the inactive G protein heterotrimers are localized in the low-density membrane phase, from where they displace upon dissociation into the membrane-anchor- and subclass-specific lipid domain.