Functional identification of toxin-antitoxin molecules from Helicobacter pylori 26695 and structural elucidation of the molecular interactions

Bacterial toxin-antitoxin (TA) systems are associated with many important cellular processes including antibiotic resistance and microorganism virulence. Here, we identify and structurally characterize TA molecules from the gastric pathogen, Helicobacter pylori. The HP0894 protein had been previously suggested, through our structural genomics approach, to be a putative toxin molecule. In this study, the intrinsic RNase activity and the bacterial cell growth-arresting activity of HP0894 were established. The RNA-binding surface was identified at three residue clusters: (Lys(8) and Ser(9)), (Lys(50)-Lys(54) and Glu(58)), and (Arg(80) and His(84)-Phe(88)). In particular, the -UA- and -CA- sequences in RNA were preferentially cleaved by HP0894, and residues Lys(52), Trp(53), and Ser(85)-Lys(87) were observed to be the main contributors to sequence recognition. The action of HP0894 could be inhibited by the HP0895 protein, and the HP0894-HP0895 complex formed an oligomer with a binding stoichiometry of 1:1. The N and C termini of HP0894 constituted the binding sites to HP0895. In contrast, the unstructured C-terminal region of HP0895 was responsible for binding to HP0894 and underwent a conformational change in the process. Finally, DNA binding activity was observed for both HP0895 and the HP0894-0895 complex but not for HP0894 alone. Taken together, it is concluded that the HP0894-HP0895 protein couple is a TA system in H. pylori, where HP0894 is a toxin with an RNase function, whereas HP0895 is an antitoxin functioning by binding to both the toxin and DNA.     

Identification of Chromosomal HP0892-HP0893 Toxin-Antitoxin Proteins in Helicobacter pylori and Structural Elucidation of Their Protein-Protein Interaction

Bacterial chromosomal toxin-antitoxin (TA) systems have been proposed not only to play an important role in the stress response, but also to be associated with antibiotic resistance. Here, we identified the chromosomal HP0892-HP0893 TA proteins in the gastric pathogen, Helicobacter pylori, and structurally characterized their protein-protein interaction. Previously, HP0892 protein was suggested to be a putative TA toxin based on its structural similarity to other RelE family TA toxins. In this study, we demonstrated that HP0892 binds to HP0893 strongly with a stoichiometry of 1:1, and HP0892-HP0893 interaction occurs mainly between the N-terminal secondary structure elements of HP0892 and the C-terminal region of HP0893. HP0892 cleaved mRNA in vitro, preferentially at the 5′ end of A or G, and the RNase activity of HP0892 was inhibited by HP0893. In addition, heterologous expression of HP0892 in Escherichia coli cells led to cell growth arrest, and the cell toxicity of HP0892 was neutralized by co-expression with HP0893. From these results and a structural comparison with other TA toxins, it is concluded that HP0892 is a toxin with intrinsic RNase activity and HP0893 is an antitoxin against HP0892 from a TA system of H. pylori. It has been known that hp0893 gene and another TA antitoxin gene, hp0895, of H. pylori, are both genomic open reading frames that correspond to genes that are potentially expressed in response to interactions with the human gastric mucosa. Therefore, it is highly probable that TA systems of H. pylori are involved in virulence of H. pylori.     

Synthesis of indole analogs as potent β-glucuronidase inhibitors

Natural products are the main source of motivation to design and synthesize new molecules for drug development. Designing new molecules against β-glucuronidase inhibitory is utmost essential. In this study indole analogs (1–35) were synthesized, characterized using various spectroscopic techniques including ¹H NMR and EI-MS and evaluated for their β-glucuronidase inhibitory activity. Most compounds were identified as potent inhibitors for the enzyme with IC₅₀ values ranging between 0.50 and 53.40 μM, with reference to standard d-saccharic acid 1,4-lactone (IC₅₀ = 48.4 ± 1.25 μM). Structure-activity relationship had been also established. The results obtained from docking studies for the most active compound 10 showed that hydrogen bond donor features as well as hydrogen bonding with (Oε1) of nucleophilic residue Glu540 is believed to be the most importance interaction in the inhibition activity. It was also observed that hydroxyl at fourth position of benzylidene ring acts as a hydrogen bond donor and interacts with hydroxyl (OH) on the side chain of catalysis residue Tyr508. The enzyme-ligand complexed were being stabilized through electrostatic π-anion interaction with acid-base catalyst Glu451 (3.96 Å) and thus preventing Glu451 from functioning as proton donor residue.     

In vitro and in vivo ACE inhibitory of pistachio hydrolysates and in silico mechanism of identified peptide binding with ACE

The ACE inhibitory activity of pistachio (Pistacia vera L.) kernel's hydrolysates by gastrointestinal enzymes was studied. Results indicated that hydrolysate successively hydrolyzed by pepsin and trypsin, Pe–Tr–H, presented in vitro ACE inhibitory activity as IC₅₀ 0.87 ± 0.04 mg/ml. The Pe–Tr–H can in vivo decrease around 22 mmHg in systolic blood pressure (SBP) and 16 mmHg in the diastolic blood pressure (DBP) at 4 h after the oral administration, however the pistachio kernel powder can slightly lower SBP and DBP. The Pe–Tr–H with the highest activity was then separated by ultrafiltration membrane of 3 kDa, size exclusion chromatography on Sephadex G-15 and G-10 columns and reversed phase high-performance liquid chromatography (RP-HPLC) consecutively. A novel ACE inhibitory peptide, ACKEP, with the IC₅₀ value of 126 μM, was identified by MALDI–TOF/TOF system. ACKEP has the same C-terminal residue as Lisinopril and Enalapril, which plays a key role in binding with ACE. The binding mechanism was explored at a molecular basis by docking experiments, which revealed that seven residues from ACE active site (His383, His387, Glu384, Arg522, Asp358, Ala356 and Asn70) and two atoms of ACKEP (O5, H60) greatly contributed to the combinative stabilization.     

Crystal Structure of the Antitoxin-Toxin Protein Complex RelB-RelE from Methanococcus jannaschii

Here we present the crystal structure of the Methanococcus jannaschii RelE-RelB (RelBE) toxin-antitoxin (TA) protein complex determined by the MIRAS (multiple isomorphous replacement with anomalous signal) method. The genes encoding this TA system are located in the chromosome of this archaeon and involved in stress response. RelE acts as an endoribonuclease that cleaves mRNA on the ribosome, and we compare the RelBE complex to the known structures of other TA systems belonging to this group and to endoribonucleases. M. jannaschii RelBE forms a heterotetramer with the antitoxin in the centre of the complex, a configuration that differs vastly from the heterotetramer structure of the previously published RelBE from another archaeon, Pyrococcus horikoshii. The long N-terminal α-helix of the tightly bound M. jannaschii antitoxin RelB covers the presumed active site of the toxin RelE that is formed by a central β-sheet, a loop on one side and a C-terminal α-helix on the other side. The active site of the M. jannaschii toxin RelE harbours positive charges that are thought to neutralize the negative charges of the substrate mRNA, including Arg62 that was changed to Ser62 by the Escherichia coli expression system, thereby leading to inactive toxin RelE. Comparative studies suggest that Asp43 and His79 are also involved in the activity of the toxin.     

STK15 rs2273535 polymorphism and cancer risk: A meta-analysis of 74,896 subjects

Background: It has been suggested that the serine/threonine kinase 15 (STK15) T91A rs2273535 polymorphism is associated with susceptibility to cancer. However, the results are conflicting. We performed this meta-analysis to derive a more precise estimation of the relationship. Methods: PubMed was searched to select studies. Case–control studies containing available genotype frequencies of the STK15 rs2273535 polymorphism were chosen, and the odds ratio (OR) with its 95% confidence interval (CI) was utilized to assess the strength of association. Results: 52 studies – including 34,057 cases and 40,839 controls – were identified. A significant effect of the STK15 rs2273535 polymorphism on cancer risk was found (AA vs. TT: OR = 1.13, 95%CI = 1.01–1.26, P_{heterogeneity} < 0.001; AA vs. TA/TT: OR = 1.12, 95%CI = 1.02–1.22, P_{heterogeneity} < 0.001; TA/AA vs. TT: OR = 1.06, 95%CI = 1.01–1.12, P_{heterogeneity} < 0.001). Stratified analysis by cancer type revealed that the STK rs2273535 polymorphism may contribute to the risk of breast cancer (AA vs. TT: OR = 1.21, 95%CI = 1.01–1.44, P_{heterogeneity} = 0.002), colorectal cancer (AA vs. TA/TT: OR = 1.24, 95%CI = 1.05–1.47, P_{heterogeneity} = 0.124), and esophageal cancer (AA vs. TA/TT: OR = 1.19, 95%CI = 1.02–1.39, P_{heterogeneity} = 0.148). Further subgroup analysis by ethnicity indicated that there was a statistically increased cancer risk in Asians (AA vs. TA/TT: OR = 1.20, 95%CI = 1.05–1.37, P_{heterogeneity} = 0.004). Conclusion: This meta-analysis suggests that the STK15 rs2273535 polymorphism is a candidate gene polymorphism for cancer susceptibility, especially in Asian populations.     

A novel angiotensin I-converting enzyme (ACE) inhibitory peptide from a marine Chlorella ellipsoidea and its antihypertensive effect in spontaneously hypertensive rats

Marine Chlorella ellipsoidea protein was hydrolyzed using Protamex, Kojizyme, Neutrase, Flavourzyme, Alcalase, trypsin, α-chymotrypsin, pepsin and papain. Alcalase-proteolytic hydrolysate exhibited the highest ACE inhibitory activity among them and was fractionated into three ranges of molecular weight (below 5 kDa, 5–10 kDa and above 10 kDa). The below 5 kDa fraction showed the highest ACE inhibitory activity and was used for subsequent purification steps. During consecutive purification, a potent ACE inhibitory peptide from marine C. ellipsoidea, which was composed of 4 amino acids, Val–Glu–Gly–Tyr (MW: 467.2 Da, IC₅₀ value: 128.4 μM), was isolated. Lineweaver–Burk plots suggest that the peptide purified acts as a competitive inhibitor against ACE and stable against gastrointestinal enzymes of pepsin, trypsin and α-chymotrypsin. Furthermore, antihypertensive effect in spontaneously hypertensive rats (SHRs) also revealed that oral administration of purified peptide can decrease systolic blood pressure significantly. The results suggest that marine C. ellipsoidea would be an attractive raw material for the manufacture of antihypertensive nutraceutical ingredients.     

Structural and Mechanistic Analysis of the Choline Sulfatase from Sinorhizobium melliloti: A Class I Sulfatase Specific for an Alkyl Sulfate Ester

Hydrolysis of organic sulfate esters proceeds by two distinct mechanisms, water attacking at either sulfur (S–O bond cleavage) or carbon (C–O bond cleavage). In primary and secondary alkyl sulfates, attack at carbon is favored, whereas in aromatic sulfates and sulfated sugars, attack at sulfur is preferred. This mechanistic distinction is mirrored in the classification of enzymes that catalyze sulfate ester hydrolysis: arylsulfatases (ASs) catalyze S–O cleavage in sulfate sugars and arylsulfates, and alkyl sulfatases break the C–O bond of alkyl sulfates. Sinorhizobium meliloti choline sulfatase (SmCS) efficiently catalyzes the hydrolysis of alkyl sulfate choline-O-sulfate (k_cat/K_M = 4.8 × 10³ s^? 1 M^? 1) as well as arylsulfate 4-nitrophenyl sulfate (k_cat/K_M = 12 s^? 1 M^? 1). Its 2.8-Å resolution X-ray structure shows a buried, largely hydrophobic active site in which a conserved glutamate (Glu386) plays a role in recognition of the quaternary ammonium group of the choline substrate. SmCS structurally resembles members of the alkaline phosphatase superfamily, being most closely related to dimeric ASs and tetrameric phosphonate monoester hydrolases. Although > 70% of the amino acids between protomers align structurally (RMSDs 1.79–1.99 Å), the oligomeric structures show distinctly different packing and protomer–protomer interfaces. The latter also play an important role in active site formation. Mutagenesis of the conserved active site residues typical for ASs, H₂¹⁸O-labeling studies and the observation of catalytically promiscuous behavior toward phosphoesters confirm the close relation to alkaline phosphatase superfamily members and suggest that SmCS is an AS that catalyzes S–O cleavage in alkyl sulfate esters with extreme catalytic proficiency.     

Transforming growth factor beta 1 (TGF-β1) modulates Epstein-Barr virus reactivation in absence of Helicobacter pylori infection in patients with gastric cancer

BackgroundTransforming growth factor-beta 1 (TGF-β1), a multifunctional cytokine, acts as a key factor for Epstein-Barr virus (EBV) reactivation. We investigated the role of TGF-β1 in latent and lytic stages of EBV in relation to Helicobacter pylori infection among patients with gastric cancer (GC) and peptic ulcer disease (PUD).MethodGastric mucosal TGF-β1 expression was determined in 95 EBV positive patients with gastroduodenal pathology [GC 40, PUD 19 and non-ulcer dyspepsia (NUD) 36] by quantitative real time PCR. Presence of H. pylori infection was diagnosed when either culture or any two of three tests (RUT, histopathology and specific ureA PCR) were positive. Serum level of TGF-β1 was detected among 60 patients using ELISA.ResultsMucosal TGF-β1 mRNA expression was detected in 85 of 95 EBV positive patients and it was significantly higher in patients with GC (p = 0.042). TGF-β1 expression tended to be higher among H. pylori non-infected than infected patients (3.80 ± 6.24 vs. 2.07 ± 2.50, p = 0.085). Both mRNA and serum level had significant association with lytic stage of EBV in absence of H. pylori infection when compared with its presence (5.21 ± 4.00 vs. 2.29 ± 2.89, p = 0.040 and 842.00 [669.55] vs. 662.63 [628.76], p = 0.049; respectively).ConclusionTGF-β1 expression was significantly associated with GC. TGF-β1 was higher both at expression and translational levels in lytic EBV infection without H. pylori suggests that H. pylori infection might play important role in preventing EBV reactivation through attenuated TGF-β1 expression. This might be a “wise host defense against EBV reactivation”.     

Hydrogen-bond network and pH sensitivity in transthyretin: Neutron crystal structure of human transthyretin

Transthyretin (TTR) is a tetrameric protein associated with human amyloidosis. In vitro, the formation of amyloid fibrils by TTR is known to be promoted by low pH. Here we show the neutron structure of TTR, focusing on the hydrogen bonds, protonation states and pH sensitivities. A large crystal was prepared at pD 7.4 for neutron protein crystallography. Neutron diffraction studies were conducted using the IBARAKI Biological Crystal Diffractometer with the time-of-flight method. The neutron structure solved at 2.0 Å resolution revealed the protonation states of His88 and the detailed hydrogen-bond network depending on the protonation states of His88. This hydrogen-bond network is composed of Thr75, Trp79, His88, Ser112, Pro113, Thr118-B and four water molecules, and is involved in both monomer–monomer and dimer–dimer interactions, suggesting that the double protonation of His88 by acidification breaks the hydrogen-bond network and causes the destabilization of the TTR tetramer. In addition, the comparison with X-ray structure at pH 4.0 indicated that the protonation occurred to Asp74, His88 and Glu89 at pH 4.0. Our neutron model provides insights into the molecular stability of TTR related to the hydrogen-bond network, the pH sensitivity and the CH···O weak hydrogen bond.     

Structures,magnetic properties,and XPS of cyanide-bridged NdIII/SmIII/GdIII–CrIII complexes

Synthesis, structural characterization, and spectroscopic and magnetic properties of three new cyano-bridged 3d–4f bimetallic complexes, Ln^III(DMF)₄(H₂O)₃Cr^III (CN)₆ · nH₂O (Ln = Nd, Sm, Gd), have been described. The Nd–Cr complex crystallizes in the monoclinic P2₁/n space group with the following unit cell parameters: a = 20.063(7) Å, b = 8.967(4) Å, c = 18.023(6) Å, b = 96.12(3)°, V = 3224(2) Å³, and Z = 4. The neodymium (III) ion, which adopts anti-prism eight-coordination environment, is linked to the [Cr^III(CN)₆]³⁻ moiety through a bridging cyanide ligand with Nd–N = 2.550(4) Å and Nd–N–C = 164.4(4)°. The variable-temperature (0.5 T at 2–300 K) and variable-field (0–5 T at 2 and 5 K) magnetic measurements reveal that the weak interaction of Gd–Cr complexes differs from that of Nd–Cr and Sm–Cr ones mainly because of the lack of orbital angular momentum. The XPS and diffuse reflectance electronic spectra were also measured to discuss charge transfer transitions concerning π-backdonation from the viewpoint of magneto-optical functions.     

Polymorphisms of endothelial nitric oxide synthase gene in systolic heart failure: an haplotype analysis

BackgroundEndothelial nitric oxide synthase (eNOS) gene polymorphisms have been associated with the pathogenesis of cardiovascular diseases, but few studies have evaluated the role of eNOS haplotypes on the risk and prognosis of heart failure (HF). This prospective study was designed to analyze the impact of three eNOS polymorphisms (T-786C, VNTR4a/b and Glu298Asp) and their haplotypes on the susceptibility and clinical outcomes in HF outpatients with systolic dysfunction.Methods and resultsWe conducted a case-control and a cohort study in which 316 HF patients and 360 healthy controls were recruited from a tertiary care university hospital. DNA was extracted from peripheral blood and eNOS polymorphisms were detected by PCR or PCR-RFLP. Patients were predominantly men, had a mean left ventricular ejection fraction of 31% and were followed-up for a median of 41 months; there were 96 deaths, including 58 HF-related deaths. Genotype distribution of the eNOS T-786C, VNTR 4a/b and Glu298Asp was similar between HF patients and controls. Haplotype frequencies differed between HF patients and controls only in African–Brazilians (p = 0.043). African–Brazilian patients that carried the haplotype -786C/4b/Asp298 had a better prognosis than patients that carried other haplotypes (log rank p value = 0.016 for all-cause mortality). In a Cox proportional hazard model adjusted for clinical variables of risk, the -786C/4b/Asp298 haplotype remained as an independent genetic predictor of survival (adjusted HR = 0.11; 95% CI = 0.01–0.83; p = 0.03).ConclusionsThe -786C/4b/Asp298 eNOS haplotype had a significant impact on HF susceptibility and prognosis, particularly in African–Brazilian patients.     

Identification,functional characterization,assembly and structure of ToxIN type III toxin–antitoxin complex from E. coli

Toxin–antitoxin (TA) systems are proposed to play crucial roles in bacterial growth under stress conditions such as phage infection. The type III TA systems consist of a protein toxin whose activity is inhibited by a noncoding RNA antitoxin. The toxin is an endoribonuclease, while the antitoxin consists of multiple repeats of RNA. The toxin assembles with the individual antitoxin repeats into a cyclic complex in which the antitoxin forms a pseudoknot structure. While structure and functions of some type III TA systems are characterized, the complex assembly process is not well understood. Using bioinformatics analysis, we have identified type III TA systems belonging to the ToxIN family across different Escherichia coli strains and found them to be clustered into at least five distinct clusters. Furthermore, we report a 2.097 Å resolution crystal structure of the first E. coli ToxIN complex that revealed the overall assembly of the protein-RNA complex. Isothermal titration calorimetry experiments showed that toxin forms a high-affinity complex with antitoxin RNA resulting from two independent (5′ and 3′ sides of RNA) RNA binding sites on the protein. These results further our understanding of the assembly of type III TA complexes in bacteria.     

Molecular architecture of the HerA–NurA DNA double-strand break resection complex

DNA double-strand breaks can be repaired by homologous recombination, during which the DNA ends are long-range resected by helicase–nuclease systems to generate 3′ single strand tails. In archaea, this requires the Mre11–Rad50 complex and the ATP-dependent helicase–nuclease complex HerA–NurA. We report the cryo-EM structure of Sulfolobus solfataricus HerA–NurA at 7.4 Å resolution and present the pseudo-atomic model of the complex. HerA forms an ASCE hexamer that tightly interacts with a NurA dimer, with each NurA protomer binding three adjacent HerA HAS domains. Entry to NurA’s nuclease active sites requires dsDNA to pass through a 23 Å wide channel in the HerA hexamer. The structure suggests that HerA is a dsDNA translocase that feeds DNA into the NurA nuclease sites.     

Structural changes that occur upon photolysis of the Fe(II)a3–CO complex in the cytochrome ba3-oxidase of Thermus thermophilus: A combined X-ray crystallographic and infrared spectral study demonstrates CO binding to CuB

The purpose of the work was to provide a crystallographic demonstration of the venerable idea that CO photolyzed from ferrous heme-a₃ moves to the nearby cuprous ion in the cytochrome c oxidases. Crystal structures of CO-bound cytochrome ba₃-oxidase from Thermus thermophilus, determined at ~ 2.8–3.2 Å resolution, reveal a Fe–C distance of ~ 2.0 Å, a Cu–O distance of 2.4 Å and a Fe–C–O angle of ~ 126°. Upon photodissociation at 100 K, X-ray structures indicate loss of Fe_a3–CO and appearance of Cu_B–CO having a Cu–C distance of ~ 1.9 Å and an O–Fe distance of ~ 2.3 Å. Absolute FTIR spectra recorded from single crystals of reduced ba₃–CO that had not been exposed to X-ray radiation, showed several peaks around 1975 cm^? 1; after photolysis at 100 K, the absolute FTIR spectra also showed a significant peak at 2050 cm^? 1. Analysis of the ‘light’ minus ‘dark’ difference spectra showed four very sharp CO stretching bands at 1970 cm^? 1, 1977 cm^? 1, 1981 cm^? 1, and 1985 cm^? 1, previously assigned to the Fe_a3–CO complex, and a significantly broader CO stretching band centered at ~ 2050 cm^? 1, previously assigned to the CO stretching frequency of Cu_B bound CO. As expected for light propagating along the tetragonal axis of the P4₃2₁2 space group, the single crystal spectra exhibit negligible dichroism. Absolute FTIR spectrometry of a CO-laden ba₃ crystal, exposed to an amount of X-ray radiation required to obtain structural data sets before FTIR characterization, showed a significant signal due to photogenerated CO₂ at 2337 cm^? 1 and one from traces of CO at 2133 cm^? 1; while bands associated with CO bound to either Fe_a3 or to Cu_B in “light” minus “dark” FTIR difference spectra shifted and broadened in response to X-ray exposure. In spite of considerable radiation damage to the crystals, both X-ray analysis at 2.8 and 3.2 Å and FTIR spectra support the long-held position that photolysis of Fe_a3–CO in cytochrome c oxidases leads to significant trapping of the CO on the Cu_B atom; Fe_a3 and Cu_B ligation, at the resolutions reported here, are otherwise unaltered. This article is part of a Special Issue entitled: Respiratory Oxidases.     

Small molecule tolfenamic acid and dietary spice curcumin treatment enhances antiproliferative effect in pancreatic cancer cells via suppressing Sp1, disrupting NF-kB translocation to nucleus and cell cycle phase distribution

Combination of dietary/herbal spice curcumin (Cur) and COX inhibitors has been tested for improving therapeutic efficacy in pancreatic cancer (PC). The objective of this study was to identify agent with low toxicity and COX-independent mechanism to induce PC cell growth inhibition when used along with Cur. Anticancer NSAID, tolfenamic acid (TA) and Cur combination were evaluated using PC cell lines. L3.6pl and MIA PaCa-2 cells were treated with Cur (5–25 μM) or TA (25–100 μM) or combination of Cur (7.5 μM) and TA (50 μM). Cell viability was measured at 24–72 h posttreatment using CellTiter-Glo kit. While both agents showed a steady/consistent effect, Cur + TA caused higher growth inhibition. Antiproliferative effect was compared with COX inhibitors, Ibuprofen and Celebrex. Cardiotoxicity was assessed using cordiomyocytes (H9C2). The expression of Sp proteins, survivin and apoptotic markers (western blot), caspase 3/7 (caspase-Glo kit), Annexin-V staining (flow cytometry), reactive oxygen species (ROS) and cell cycle phase distribution (flow cytometry) was measured. Cells were treated with TNF-α, and NF-kB translocation from cytoplasm to nucleus was evaluated (immunofluorescence). When compared to individual agents, combination of Cur + TA caused significant increase in apoptotic markers, ROS levels and inhibited NF-kB translocation to nucleus. TA caused cell cycle arrest in G₀/G₁, and the combination treatment showed mostly DNA synthesis phase arrest. These results suggest that combination of Cur + TA is less toxic and effectively enhance the therapeutic efficacy in PC cells via COX-independent mechanisms.     

The crystallographic structure of Panicum Mosaic Virus (PMV)

The structure of Panicum Mosaic Virus (PMV) was determined by X-ray diffraction analysis to 2.9 Å resolution. The crystals were of pseudo symmetry F23; the true crystallographic unit cell was of space group P2₁ with a = 411.7 Å, b = 403.9 Å and c = 412.5 Å, with β = 89.7°. The asymmetric unit was two entire T = 3 virus particles, or 360 protein subunits. The structure was solved by conventional molecular replacement from two distant homologues, Cocksfoot Mottle Virus (CfMV) and Tobacco Necrosis Virus (TNV), of ～20% sequence identity followed by phase extension. The model was initially refined with exact icosahedral constraints and then with icosahedral restraints. The virus has Ca⁺⁺ ions octahedrally coordinated by six aspartic acid residues on quasi threefold axes, which is completely different than for either CfMV or TNV. Amino terminal residues 1–53, 1–49 and 1–21 of the A, B and C subunits, respectively, and the four C-terminal residues (239–242) are not visible in electron density maps. The additional ordered residues of the C chain form a prominent “arm” that intertwines with symmetry equivalent “arms” at icosahedral threefold axes, as was seen in both CfMV and TNV. A 17 nucleotide hairpin segment of genomic RNA is icosahedrally ordered and bound at 60 equivalent sites at quasi twofold A–B subunit interfaces at the interior surface of the capsid. This segment of RNA may serve as a conformational switch for coat protein subunits, as has been proposed for similar RNA segments in other viruses.     

Spectroscopic characterization of the inclusion complexes of luteolin with native and derivatized β-cyclodextrin

The inclusion complexes of Luteolin (LU) with cyclodextrins (CDs) including β-cyclodextrin (βCD), hydroxypropyl-β-cyclodextrin (HPβCD) and dimethyl-β-cyclodextrin (DMβCD), Scheme 1, have been investigated using the method of steady-state fluorescence. The stoichiometric ratio of the three complexes was found to be 1:1 and the stability constants (K) were estimated from spectrofluorometric titrations, as well as the thermodynamic parameters. Maximum inclusion ability was obtained in the case of HPβCD followed by DMβCD and βCD. Moreover, ¹H NMR and 2D NMR were carried out, revealing that LU has different form of inclusion which is in agreement with molecular modeling studies. These models confirm that when LU–βCD and LU–DMβCD complexes are formed, the B-ring is oriented toward the primary rim; however, for LU–HPβCD complex this ring is oriented toward the secondary rim. The ESR results showed that the antioxidant activity of luteolin was the order LU–HPβCD > LU–DMβCD > LU–βCD > LU, hence the LU-complexes behave are better antioxidants than luteolin free.