Synthetic genes for human muscle-type adenylate kinase in Escherichia coli

An artificial gene coding for the human muscle-type cytosolicadenylate kinase (hAK1) was chemically synthesized and directlyexpressed in Escherichia coli under the control of trp promoter.The DNA duplex of 596 bp was designed and constructed from 40oligonucleotide fragments of typically 30 nucleotides in length.Twelve unique restriction sites were fairly evenly spaced inthe synthetic gene to facilitate site-specific mutagenesis atany part of this recombinant protein. The genes for mutant hAK1(Tyr 95 – Phe 95, Y95F hAK1; Arg 97 – Ala 97, R97AhAK1) were constructed by cassette mutagenesis and utilizedrestriction sites incorporated in the hAK1 gene. The recombinanthAK1 was purified to homogeneity by a two-step chromatographicprocedure with a good yield, and showed the same adenylate kinaseactivity as that of authentic hAK1. preliminary kinetic studiesshow that the enzymatic activity (V_{max app,cor})     

The Role of Structure and Biophysical Properties in the Pleiotropic Effects of Statins

Statins are a class of drugs used to lower low-density lipoprotein cholesterol and are amongst the most prescribed medications worldwide. Most statins work as a competitive inhibitor of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGR), but statin intolerance from pleiotropic effects have been proposed to arise from non-specific binding due to poor enzyme-ligand sensitivity. Yet, research into the physicochemical properties of statins, and their interactions with off-target sites, has not progressed much over the past few decades. Here, we present a concise perspective on the role of statins in lowering serum cholesterol levels, and how their reported interactions with phospholipid membranes offer a crucial insight into the mechanism of some of the more commonly observed pleiotropic effects of statin administration. Lipophilicity, which governs hepatoselectivity, is directly related to the molecular structure of statins, which dictates interaction with and transport through membranes. The structure of statins is therefore a clinically important consideration in the treatment of hypercholesterolaemia. This review integrates the recent biophysical studies of statins with the literature on the physiological effects and provides new insights into the mechanistic cause of statin pleiotropy, and prospective means of understanding the cholesterol-independent effects of statins.     

Adenylate Kinase Isozyme 3 Regulates Mitochondrial Energy Metabolism and Knockout Alters HeLa Cell Metabolism

The balance between oxidative phosphorylation and glycolysis is important for cancer cell growth and survival, and changes in energy metabolism are an emerging therapeutic target. Adenylate kinase (AK) regulates adenine nucleotide metabolism, maintaining intracellular nucleotide metabolic homeostasis. In this study, we focused on AK3, the isozyme localized in the mitochondrial matrix that reversibly mediates the following reaction: Mg²⁺ GTP + AMP ⇌ Mg²⁺ GDP + ADP. Additionally, we analyzed AK3-knockout (KO) HeLa cells, which showed reduced proliferation and were detected at an increased number in the G1 phase. A metabolomic analysis showed decreased ATP; increased glycolytic metabolites such as glucose 6 phosphate (G6P), fructose 6 phosphate (F6P), and phosphoenolpyruvate (PEP); and decreased levels of tricarboxylic acid (TCA) cycle metabolites in AK3KO cells. An intracellular ATP evaluation of AK3KO HeLa cells transfected with ATeam plasmid, an ATP sensor, showed decreased whole cell levels. Levels of mitochondrial DNA (mtDNA), a complementary response to mitochondrial failure, were increased in AK3KO HeLa cells. Oxidative stress levels increased with changes in gene expression, evidenced as an increase in related enzymes such as superoxide dismutase 2 (SOD2) and SOD3. Phosphoenolpyruvate carboxykinase 2 (PCK2) expression and PEP levels increased, whereas PCK2 inhibition affected AK3KO HeLa cells more than wild-type (WT) cells. Therefore, we concluded that increased PCK2 expression may be complementary to increased GDP, which was found to be deficient through AK3KO. This study demonstrated the importance of AK3 in mitochondrial matrix energy metabolism.     

Characterization of Aspartate Kinase from Corynebacterium pekinense and the Critical Site of Arg169

Aspartate kinase (AK) is the key enzyme in the biosynthesis of aspartate-derived amino acids. Recombinant AK was efficiently purified and systematically characterized through analysis under optimal conditions combined with steady-state kinetics study. Homogeneous AK was predicted as a decamer with a molecular weight of ~48 kDa and a half-life of 4.5 h. The enzymatic activity was enhanced by ethanol and Ni²⁺. Moreover, steady-state kinetic study confirmed that AK is an allosteric enzyme, and its activity was inhibited by allosteric inhibitors, such as Lys, Met, and Thr. Theoretical results indicated the binding mode of AK and showed that Arg169 is an important residue in substrate binding, catalytic domain, and inhibitor binding. The values of the kinetic parameter V_max of R169 mutants, namely, R169Y, R169P, R169D, and R169H AK, with l-aspartate as the substrate, were 4.71-, 2.25-, 2.57-, and 2.13-fold higher, respectively, than that of the wild-type AK. Furthermore, experimental and theoretical data showed that Arg169 formed a hydrogen bond with Glu92, which functions as the entrance gate. This study provides a basis to develop new enzymes and elucidate the corresponding amino acid production.     

Cholesterol-Dependent Energy Transfer between Fluorescent Proteins—Insights into Protein Proximity of APP and BACE1 in Different Membranes in Niemann-Pick Type C Disease Cells

Förster resonance energy transfer (FRET) -based techniques have recently been applied to study the interactions between β-site APP-cleaving enzyme-GFP (BACE1-GFP) and amyloid precursor protein-mRFP (APP-mRFP) in U373 glioblastoma cells. In this context, the role of APP-BACE1 proximity in Alzheimer’s disease (AD) pathogenesis has been discussed. FRET was found to depend on intracellular cholesterol levels and associated alterations in membrane stiffness. Here, NPC1 null cells (CHO-NPC1^−/−), exhibiting increased cholesterol levels and disturbed cholesterol transport similar to that observed in Niemann-Pick type C disease (NPC), were used to analyze the influence of altered cholesterol levels on APP-BACE1 proximity. Fluorescence lifetime measurements of whole CHO-wild type (WT) and CHO-NPC1^−/− cells (EPI-illumination microscopy), as well as their plasma membranes (total internal reflection fluorescence microscopy, TIRFM), were performed. Additionally, generalized polarization (GP) measurements of CHO-WT and CHO-NPC1^−/− cells incubated with the fluorescence marker laurdan were performed to determine membrane stiffness of plasma- and intracellular-membranes. CHO-NPC1^−/− cells showed higher membrane stiffness at intracellular- but not plasma-membranes, equivalent to cholesterol accumulation in late endosomes/lysosomes. Along with higher membrane stiffness, the FRET efficiency between BACE1-GFP and APP-mRFP was reduced at intracellular membranes, but not within the plasma membrane of CHO-NPC1^−/−. Our data show that FRET combined with TIRF is a powerful technique to determine protein proximity and membrane fluidity in cellular models of neurodegenerative diseases.     

Inhibitory Effect of Statins on Inflammation-Related Pathways in Human Abdominal Aortic Aneurysm Tissue

HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase inhibitors (statins) have been suggested to attenuate abdominal aortic aneurysm (AAA) growth. However, the effects of statins in human AAA tissues are not fully elucidated. The aim of this study was to investigate the direct effects of statins on proinflammatory molecules in human AAA walls in ex vivo culture. Simvastatin strongly inhibited the activation of nuclear factor (NF)-κB induced by tumor necrosis factor (TNF)-α in human AAA walls, but showed little effect on c-jun N-terminal kinase (JNK) activation. Simvastatin, as well as pitavastatin significantly reduced the secretion of matrix metalloproteinase (MMP)-9, monocyte chemoattractant protein (MCP)-2 and epithelial neutrophil-activating peptide (CXCL5) under both basal and TNF-α-stimulated conditions. Similar to statins, the Rac1 inhibitor NSC23766 significantly inhibited the activation of NF-κB, accompanied by a decreased secretion of MMP-9, MCP-2 and CXCL5. Moreover, the effect of simvastatin and the JNK inhibitor SP600125 was additive in inhibiting the secretion of MMP-9, MCP-2 and CXCL5. These findings indicate that statins preferentially inhibit the Rac1/NF-κB pathway to suppress MMP-9 and chemokine secretion in human AAA, suggesting a mechanism for the potential effect of statins in attenuating AAA progression.     

Adenylate Kinase 4—A Key Regulator of Proliferation and Metabolic Shift in Human Pulmonary Arterial Smooth Muscle Cells via Akt and HIF-1α Signaling Pathways

Increased proliferation of pulmonary arterial smooth muscle cells (PASMCs) in response to chronic hypoxia contributes to pulmonary vascular remodeling in pulmonary hypertension (PH). PH shares numerous similarities with cancer, including a metabolic shift towards glycolysis. In lung cancer, adenylate kinase 4 (AK4) promotes metabolic reprogramming and metastasis. Against this background, we show that AK4 regulates cell proliferation and energy metabolism of primary human PASMCs. We demonstrate that chronic hypoxia upregulates AK4 in PASMCs in a hypoxia-inducible factor-1α (HIF-1α)-dependent manner. RNA interference of AK4 decreases the viability and proliferation of PASMCs under both normoxia and chronic hypoxia. AK4 silencing in PASMCs augments mitochondrial respiration and reduces glycolytic metabolism. The observed effects are associated with reduced levels of phosphorylated protein kinase B (Akt) as well as HIF-1α, indicating the existence of an AK4-HIF-1α feedforward loop in hypoxic PASMCs. Finally, we show that AK4 levels are elevated in pulmonary vessels from patients with idiopathic pulmonary arterial hypertension (IPAH), and AK4 silencing decreases glycolytic metabolism of IPAH-PASMCs. We conclude that AK4 is a new metabolic regulator in PASMCs interacting with HIF-1α and Akt signaling pathways to drive the pro-proliferative and glycolytic phenotype of PH.     

Retinol inhibition of some proteolytic enzymes

Automated analyses were used to determine the effect of retinol on the activity of the following proteolytic enzymes: ficin (EC 3.4.4.12), bromelain (EC 3.4.4. 24), trypsin (EC 3.4.4.4.), chymotrypsin A (EC 3.4.4.5), papain (EC 3.4.4.10), clostridiopeptidase A (EC 3.4.4.19), pepsin (EC 3.4.4.1), cathepsin D (EC 3.4.4. 23) from rat-liver and rat-kidney lysosomes and the nonspecific proteolytic enzyme, pronase. Of these proteolytic enzymes only ficin, bromelain, and rat-kidney lysosomal cathepsin D were inhibited significantly by 1×10⁻⁴ M retinol. Some nonproteolytic enzymes not inhibited by retinol were acid phosphatase (EC 3.1.3.2), β-acetylglucosaminidase (EC 3.2.1.30), arylsulfatase (EC 3.1.6.1), and pyruvate kinase (EC 2.7.1.40). The inhibition of cathepsin D varied with the substrate used, being greater with hemoglobin than with ovalbumin or bovine serum albumin. Carotene and retinol inhibited ficin and cathepsin D to similar extents. Retinol inhibition of ficin was partially reversible. These studies of proteolytic enzyme inhibition by retinol serve as a simple model for studying retinol-protein interactions in vitro.     

Novel polyacetylenes containing pendant 1-pyrenyl groups: synthesis, characterization, and thermal and optical properties

We report the synthesis as well as the thermal, optical and photophysical properties of four different polyacetylenes with pendant 1-pyrenyl groups: poly(1-ethynylpyrene) (PEP), poly(1-(trimethylsilanylethynyl)pyrene) (PTMSEP), poly(1-(4-(trimethylsilanyl-buta-1,3-diynyl)pyrene) (PTMSBDP) and poly(1-buta-1,3-diynylpyrene) (PBDP). Polymerizations were carried out with W and Ta catalysts, respectively, for mono- and disubstituted monomers. Soluble poly(1-ethynylpyrene) with high molecular weights (up to 4×10⁵) and extended conjugation of the main chain was obtained with good yields. Lower molecular weights (up to 6×10³) were obtained with the other polymers. Oligomers and polymers displayed high thermal stability. From the absorption spectra of the various polymers, it is found that PEP possesses a higher degree of conjugation than the other polyacetylenes. Molecular interactions occur between pyrene units present in each polymer giving rise to an emission due to associated pyrenes. These interactions are affected by the steric hindrance present in the polymer backbone. Excitation spectra combined with fluorescence decay profiles show that these interactions occur in the ground state (excited complex).     

Statins and Bempedoic Acid: Different Actions of Cholesterol Inhibitors on Macrophage Activation

Statins represent the most prescribed class of drugs for the treatment of hypercholesterolemia. Effects that go beyond lipid-lowering actions have been suggested to contribute to their beneficial pharmacological properties. Whether and how statins act on macrophages has been a matter of debate. In the present study, we aimed at characterizing the impact of statins on macrophage polarization and comparing these to the effects of bempedoic acid, a recently registered drug for the treatment of hypercholesterolemia, which has been suggested to have a similar beneficial profile but fewer side effects. Treatment of primary murine macrophages with two different statins, i.e., simvastatin and cerivastatin, impaired phagocytotic activity and, concurrently, enhanced pro-inflammatory responses upon short-term lipopolysaccharide challenge, as characterized by an induction of tumor necrosis factor (TNF), interleukin (IL) 1β, and IL6. In contrast, no differences were observed under long-term inflammatory (M1) or anti-inflammatory (M2) conditions, and neither inducible NO synthase (iNOS) expression nor nitric oxide production was altered. Statin treatment led to extracellular-signal regulated kinase (ERK) activation, and the pro-inflammatory statin effects were abolished by ERK inhibition. Bempedoic acid only had a negligible impact on macrophage responses when compared with statins. Taken together, our data point toward an immunomodulatory effect of statins on macrophage polarization, which is absent upon bempedoic acid treatment.     

Functionalization of reduced graphene oxide nanosheets via stacking interactions with the fluorescent and water-soluble perylene bisimide-containing polymers

Reduced graphene oxide (RGO)-polymer composites were prepared via π-π stacking interactions of RGO with the perylene bisimide-containing poly(glyceryl acrylate) (PBIPGA). PBIPGA was synthesized via atom transfer radical polymerization (ATRP) of solketal acrylate (SA), using bifunctional N,N′-bis{2-[2-[(2-bromo-2-methylpropanoyl)oxy]ethoxy]ethyl}perylene-3,4,9,10-tetracarboxylic acid bisimide (PBI-Br) as the initiator, and subsequent hydrolysis of the acetal-protecting group. PBIPGA was characterized by ¹H NMR spectroscopy, gel permeation chromatography (GPC), UV-visible absorption spectroscopy and fluorescence spectroscopy. The presence of π-π stacking interactions between PBI and the RGO surface was suggested by fluorescence and UV-visible absorption spectroscopy results. The chemical states and π-π stacking interaction, morphology, and composition of RGO-PBIPGA composites were characterized, respectively, by X-ray photoelectron spectroscopy (XPS), transmission electron microscopy (TEM) and thermogravimetric analysis (TGA). The low cytotoxicity level of the RGO-PBIPGA composites was revealed by incubation with 3T3 fibroblasts in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays in vitro.     

Effect of supercritical CO2 flux,temperature and processing time on physicochemical and morphological properties of commercial reverse osmosis membranes

Coupling supercritical CO₂ (SC-CO₂) extraction with membrane separation leads to energy savings by recycling CO₂ at supercritical state while separating extract components. However, high pressure operating conditions may cause physicochemical and morphological changes in polymer membranes, which in turn can adversely affect membrane performance. In this study, the effect of different flux (50 and 200 kg/m² h), temperature (40 and 80 °C) and time (0–8 h) levels were investigated at 120 bar on two commercial reverse osmosis membranes, AK and SG using contact angle, ATR-FTIR and FE-SEM measurements. Contact angle of AK increased substantially with high flux and high temperature processing unlike SG. The peaks assigned to N–H and carbonyl groups at 1541, 1609 and 1663 cm⁻¹ showed the highest decrease in absorbance with high flux processing while high temperature was more effective on O–H groups between 2700 and 3700 cm⁻¹. AK membrane exhibited the formation of bead-like structures at different processing times and conditions. The effect of SC-CO₂ processing on the membranes varied depending on their chemical structure, which is important to understand for further process development.     

Rutaecarpine Increases Nitric Oxide Synthesis via eNOS Phosphorylation by TRPV1-Dependent CaMKII and CaMKKβ/AMPK Signaling Pathway in Human Endothelial Cells

Rutaecarpine (RUT) is a bioactive alkaloid isolated from the fruit of Evodia rutaecarpa that exerts a cellular protective effect. However, its protective effects on endothelial cells and its mechanism of action are still unclear. In this study, we demonstrated the effects of RUT on nitric oxide (NO) synthesis via endothelial nitric oxide synthase (eNOS) phosphorylation in endothelial cells and the underlying molecular mechanisms. RUT treatment promoted NO generation by increasing eNOS phosphorylation. Additionally, RUT induced an increase in intracellular Ca²⁺ concentration and phosphorylation of Ca²⁺/calmodulin-dependent protein kinase kinase β (CaMKKβ), AMP-activated protein kinase (AMPK), and Ca²⁺/calmodulin-dependent kinase II (CaMKII). Inhibition of transient receptor potential vanilloid type 1 (TRPV1) attenuated RUT-induced intracellular Ca²⁺ concentration and phosphorylation of CaMKII, CaMKKβ, AMPK, and eNOS. Treatment with KN-62 (a CaMKII inhibitor), Compound C (an AMPK inhibitor), and STO-609 (a CaMKKβ inhibitor) suppressed RUT-induced eNOS phosphorylation and NO generation. Interestingly, RUT attenuated the expression of ICAM-1 and VCAM-1 induced by TNF-α and inhibited the inflammation-related NF-κB signaling pathway. Taken together, these results suggest that RUT promotes NO synthesis and eNOS phosphorylation via the Ca²⁺/CaMKII and CaM/CaMKKβ/AMPK signaling pathways through TRPV1. These findings provide evidence that RUT prevents endothelial dysfunction and benefit cardiovascular health.     

Spectroscopic Investigation on the Interaction of Ferrocene Containing Hyperbranched Poly(amine) Ester with Model Plasma Protein

Mechanism of interaction between ferrocenyl-functionalized hyperbranched poly(amine) ester (HPAE-Fc) and model plasma protein-bovine serum albumin (BSA) was investigated using cyclicvoltammetry, differential pulsed voltammetry, fluorescence, UV–Vis absorption spectrometry and circular dichroism (CD). Some complicated interactions occurred between BSA and HPAE-Fc and the new redox centers appeared in the BSA/HPAE-Fc complex that changed and hindered the electron transfer of Fe/Fe²⁺. Stern–Volmer analysis of fluorescence quenching data showed that BSA fluorescence was statically quenched by HPAE-Fc, which implied that ground state complex formed between BSA and HPAE-Fc. The hydrogen bonds and vander Waals force play major roles in the reaction as indicated by the ¹H NMR and variable temperature ¹H NMR spectra and thermodynamic parameters. The distance, r between the donor (BSA) and acceptor (HPAE-Fc) was calculated based on the Forster’s theory of non-radiation energy transfer and was found to be 3.24 nm, which in turn indicated that HPAE-Fc can bind to BSA with high probability. Synchronous, three-dimensional fluorescence and CD studies indicated that the interaction of BSA with HPAE-Fc induced conformational changes in BSA with overall decrease in the α-helical structure and increase in β-pleated sheet structure. The molecular model of the interaction between HPAE-Fc and BSA was also presented according to the results in this study.     

Effects of hexadecylphosphocholine on protein kinase C and TPA-induced differentiation of HL60 cells

Several structural analogs of alkylphosphocholine (APC) were studied for their effects on protein kinase C (PKC) and 12-O-tetradecanoylphorbol-13-acetate (TPA) elicited biochemical and cellular events in HL60 cells. Hexadecylphosphocholine (He-PC₂), the APC prototype, inhibited PKC competitively with respect to phosphatidylserine and noncompetitively with respect to CaCl₂, both with an apparent K_i of about 15 μM. Inhibition of PKC by He-PC₂ was selective, since cyclic AMP dependent protein kinase and Ca²⁺/calmodulin dependent protein kinase II were relatively unaffected. He-PC₂ inhibited TPA-induced depletion of PKC and TPA-stimulated phosphorylation of cellular proteins in HL60 cells. TPA-induced differentiation of HL60 cells was also inhibited by He-PC₂, and this inhibition was synergistic or additive to the effects of 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine (H-7), a PKC inhibitor. The present findings are consistent with the hypothesis that inhibition of PKC might be related, in part, to the antineoplastic effect of He-PC₂ and ether lipid analogs such as ET-18-OCH₃ (1-octadecyl-2-methyl-glycero-3-phosphocholine).     

Spectrofluorometric and Molecular Docking Studies on the Binding of Curcumenol and Curcumenone to Human Serum Albumin

Curcumenol and curcumenone are two major constituents of the plants of medicinally important genus of Curcuma, and often govern the pharmacological effect of these plant extracts. These two compounds, isolated from C. zedoaria rhizomes were studied for their binding to human serum albumin (HSA) using the fluorescence quench titration method. Molecular docking was also performed to get a more detailed insight into their interaction with HSA at the binding site. Additions of these sesquiterpenes to HSA produced significant fluorescence quenching and blue shifts in the emission spectra of HSA. Analysis of the fluorescence data pointed toward moderate binding affinity between the ligands and HSA, with curcumenone showing a relatively higher binding constant (2.46 × 10⁵ M⁻¹) in comparison to curcumenol (1.97 × 10⁴ M⁻¹). Cluster analyses revealed that site I is the preferred binding site for both molecules with a minimum binding energy of −6.77 kcal·mol⁻¹. However, binding of these two molecules to site II cannot be ruled out as the binding energies were found to be −5.72 and −5.74 kcal·mol⁻¹ for curcumenol and curcumenone, respectively. The interactions of both ligands with HSA involved hydrophobic interactions as well as hydrogen bonding.     

Enhancement by ganglioside GT1b of annexin I phosphorylation in bovine mammary gland in the presence of phosphatidylserine and Ca2+

Ganglioside GT1b and, to a lesser extent, GD3, enhanced phosphorylation of a 36 kDa protein (the substrate of protein kinase C) in the particulate fraction from bovine mammary gland. Sialic acids, asialogangliosides, and GM3 were without effect, and GD1a conversely inhibited phosphorylation of the 36 kDa protein. The enhanced phosphorylation by GT1b required the simultaneous presence of phosphatidylserine (PS) and Ca²⁺. The 36 kDa protein reacted with anti-annexin I in Western blot analysis. Addition of purified annexin I to the reaction mixture containing the particulate fraction increased the extent of phosphorylated 36 kDa protein, and the phosphorylation was further enhanced by GT1b. The enhanced phosphorylation of annexin I by GT1b was also dependent on PS and Ca²⁺. When annexin I was phosphorylated by purified protein kinase C, GT1b inhibited the annexin I phosphorylation. Addition of epidermal growth factor or insulin to the particulate fraction had little effect on the enhancement. These results suggest that an enzyme or enzymes other than protein kinase C, epidermal growth factor receptor kinase, or insulin receptor kinase is responsible for the GT1b- and GD3-enhanced phosphorylation of annexin I in the presence of PS and Ca²⁺.     

Towards Profiling of the G-Quadruplex Targeting Drugs in the Living Human Cells Using NMR Spectroscopy

Recently, the ¹H-detected in-cell NMR spectroscopy has emerged as a unique tool allowing the characterization of interactions between nucleic acid-based targets and drug-like molecules in living human cells. Here, we assess the application potential of ¹H and ¹⁹F-detected in-cell NMR spectroscopy to profile drugs/ligands targeting DNA G-quadruplexes, arguably the most studied class of anti-cancer drugs targeting nucleic acids. We show that the extension of the original in-cell NMR approach is not straightforward. The severe signal broadening and overlap of ¹H in-cell NMR spectra of polymorphic G-quadruplexes and their complexes complicate their quantitative interpretation. Nevertheless, the ¹H in-cell NMR can be used to identify drugs that, despite strong interaction in vitro, lose their ability to bind G-quadruplexes in the native environment. The in-cell NMR approach is adjusted to a recently developed 3,5-bis(trifluoromethyl)phenyl probe to monitor the intracellular interaction with ligands using ¹⁹F-detected in-cell NMR. The probe allows dissecting polymorphic mixture in terms of number and relative populations of individual G-quadruplex species, including ligand-bound and unbound forms in vitro and in cellulo. Despite the probe’s discussed limitations, the ¹⁹F-detected in-cell NMR appears to be a promising strategy to profile G-quadruplex–ligand interactions in the complex environment of living cells.     

Solubility parameters of two chlorinated polyethylene polymers

An equation that appears adequate for the calculation of the solubility parameter of random copolymers was tested with two nonrandom copolymers, chlorinated polyethylene (CPE) AK 227 and AK 243, containing 27.8% and 42.1% chlorine, respectively. Flory's equation relating the energy of interaction between polymer and diluent to the depression of the melting point of the pure polymer was used to estimate the solubility parameter of the polymers from solution temperature studies in chlorobenzene, toluene, o-xylene, and p-xylene. Using data obtained on PE as a criterion, agreement within 3% was obtained between the reported δ-value and that estimated from solution temperature studies when toluene, o-xylene, and p-xylene were used as diluents. In the case of the two CPE polymers, values for the gas constant and for the heat of fusion in units of cal/cm³ polymer °K and cal/cm³ polymer, respectively, were obtained by solving simultaneous equations. From the derived gas constant values, the weight of an average repeating unit of CPE polymer was obtained, 39.38 for AK 227 and 46.95 for AK 243, which compares favorably with values obtained using the expression n₁M₁ + n₂M₂ = M_x. For ΔH, the results showed that in going from PE to CPE, a reversal in the sign of ΔH occurs indicating that, in the diluents studied, the value of χ is positive in the case of PE and negative for the CPE polymers. Taking this into account, agreement between calculated δ-values and those estimated from solution temperature studies is within 2% for AK 227 (toluene, o-xylene, p-xylene) and within 1% for AK 243 when toluene and o-xylene were used as diluents. Anomalous results were obtained in the latter case when p-xylene and chlorobenzene were used as diluents and from solution temperature studies of PE in chlorobenzene. The results do indicate that the equation used to calculate the solubility parameter of random copolymers may also be used for nonrandom copolymers such as CPE.     

The Interfacial Interactions of Glycine and Short Glycine Peptides in Model Membrane Systems

The interactions of amino acids and peptides at model membrane interfaces have considerable implications for biological functions, with the ability to act as chemical messengers, hormones, neurotransmitters, and even as antibiotics and anticancer agents. In this study, glycine and the short glycine peptides diglycine, triglycine, and tetraglycine are studied with regards to their interactions at the model membrane interface of Aerosol-OT (AOT) reverse micelles via ¹H NMR spectroscopy, dynamic light scattering (DLS), and Langmuir trough measurements. It was found that with the exception of monomeric glycine, the peptides prefer to associate between the interface and bulk water pool of the reverse micelle. Monomeric glycine, however, resides with the N-terminus in the ordered interstitial water (stern layer) and the C-terminus located in the bulk water pool of the reverse micelle.