BBA - General Subjects (v.1760, #7)

Peroxisome is a reservoir of intracellular calcium by Bikramjit Raychaudhury; Shreedhara Gupta; Shouvik Banerjee; Salil C. Datta (989-992).
We have examined fura 2-loaded purified peroxisomes under confocal microscope to prove that this mammalian organelle is a store of intracellular calcium pool. Presence of calcium channel and vanadate sensitive Ca2+-ATPase in the purified peroxisomal membrane has been demonstrated. We have further observed that machineries to maintain calcium pool in this mammalian organelle are impaired during infection caused by Leishmania donovani. Results reveal that peroxisomes have a merit to play a significant role in the metabolism of intracellular calcium.

Minor groove binding of Co(III)meso-tetrakis(N-methylpyridinium-4-yl)porphyrin to various duplex and triplex polynucleotides by Biao Jin; Jong Sub Shin; Chang Hwan Bae; Jong-Moon Kim; Seog K. Kim (993-1000).
The binding site and the geometry of Co(III)meso-tetrakis(N-methylpyridinium-4-yl)porphyrin (CoTMPyP) complexed with double helical poly(dA)·poly(dT) and poly(dG)·poly(dC), and with triple helical poly(dA)·[poly(dT)]2 and poly(dC)·poly(dG)·poly(dC)+ were investigated by circular and linear dichroism (CD and LD). The appearance of monomeric positive CD at a low [porphyrin]/[DNA] ratio and bisignate CD at a high ratio of the CoTMPyP-poly(dA)·poly(dT) complex is almost identical with its triplex counterpart. Similarity in the CD spectra was also observed for the CoTMPyP-poly(dG)·poly(dC) and -poly(dC)·poly(dG)·poly(dC)+ complex. This observation indicates that both monomeric binding and stacking of CoTMPyP to these polynucleotides occur at the minor groove. However, different binding geometry of CoTMPyP, when bind to AT- and GC-rich polynucleotide, was observed by LD spectrum. The difference in the binding geometry may be attributed to the difference in the interaction between polynucleotides and CoTMPyP: in the GC polynucleotide case, amine group protrude into the minor groove while it is not present in the AT polynucleotide.
Keywords: Metalloporphyrin; Linear dichroism; Circular dichroism; DNA; Triple helical DNA; Binding mode;

Fluorescence and circular dichroism spectroscopic studies were carried out on the galactose-specific lectin from Dolichos lablab seeds (DLL-II). The microenvironment of the tryptophan residues in the lectin under native and denaturing conditions were investigated by quenching of the intrinsic fluorescence of the protein by a neutral quencher (acrylamide), an anionic quencher (iodide ion) and a cationic quencher (cesium ion). The results obtained indicate that the tryptophan residues of DLL-II are largely buried in the hydrophobic core of the protein matrix, with positively charged side chains residing close to at least some of the tryptophan residues under the experimental conditions. Analysis of the far UV CD spectrum of DLL-II revealed that the secondary structure of the lectin consists of 57% α-helix, 21% β-sheet, 7% β-turns and 15% unordered structures. Carbohydrate binding did not significantly alter the secondary and tertiary structures of the lectin. Thermal unfolding of DLL-II, investigated by monitoring CD signals, showed a sharp transition around 75 °C both in the far UV region (205 nm) and the near UV region (289 nm), which shifted to ca. 77–78 °C in the presence of 0.1 M methyl-β-d-galactopyranoside, indicating that ligand binding leads to a moderate stabilization of the lectin structure.
Keywords: Agglutinin; α-helix; β-sheet; Fluorescence quenching; Acrylamide; Iodide ion; Cesium ion;

Biophysical characteristics of neurotensin polyplex for in vitro and in vivo gene transfection by Martha L. Arango-Rodriguez; Ivan Navarro-Quiroga; Juan A. Gonzalez-Barrios; Daniel B. Martinez-Arguelles; Michael J. Bannon; Juan Kouri; Patricia Forgez; William Rostene; Refugio Garcia-Villegas; Ismael Jimenez; Daniel Martinez-Fong (1009-1020).
Previously we improved the neurotensin (NT)-polyplex by the coupling of HA2 fusogenic peptide (FP) and Vp1 SV40 karyophilic peptide (KP). We now report the proportion of [125I]-NT, [3H]-FP, and poly-l-lysine (PLL) in the NT-polyplex, and some of its biophysical properties. We concluded that the most efficient NT-polyplex comprised 1 NT, 4 FP, and 2 PLL molecules. Electrophoresis revealed that high acidity is detrimental for NT-polyplex stability. Electron microscopy and electrophoresis studies showed that 6 μM KP and 1% serum condensed the plasmid DNA (pDNA) before the appearance of toroid structures. Four plasmids were used to evaluate the transfection efficiency. In vitro, maximum expression was produced at molar ratios (pDNA : [125I]-NT-[3H]-FP-PLL conjugate) of 1:34 for pEGFP-N1 and 1:27 for pECFP-Nuc. Cotransfection of those plasmids was attained at their optimum molar ratios. In vivo, maximum expression of the pDAT-BDNF-flag in dopamine neurons was produced at a 1:45 molar ratio, whereas that of pDAT-EGFP was at 1:20. The NT-polyplex in the presence of 1 μM SR-48692, an NT-receptor specific antagonist, and untargeted polyplex did not cause transfection in vivo demonstrating the specificity of gene transfer via NT-receptor endocytosis. This information is essential for synthesizing an efficient NT-polyplex that can provide a useful tool for specific gene transfection.
Keywords: Gene delivery; Receptor-mediated endocytosis; DNA condensation; Fusogenic peptide; Nuclear localization signal; Karyophilic peptide;

The effects of 4th and 5th generation cationic, neutral and anionic polyamidoamine (PAMAM) dendrimers on bilirubin absorbance and fluorescence were studied. Cationic and neutral PAMAM dendrimers shifted the bilirubin absorption maximum from 435 to 442–455 nm, increased the peak absorbance 1.5-fold, shifted the bilirubin fluorescence excitation and emission maxima, increased the fluorescence emission several-fold and significantly protected bilirubin against photodestruction. Using double fluorescence titration technique allowed to receive such constant of binding and the number of binding centers at 20 °C: for PAMAM G4 dendrimer, (2.4 ± 1.4) × 106 (mol/l)−1 and 0.07 ± 0.012; for PAMAM G4-OH dendrimer, (3.1 ± 1.3) × 106 (mol/l)−1 and 0.08 ± 0.014; for PAMAM G5 dendrimer, (7.6 ± 3.6) × 106 (mol/l)−1 and 0.09 ± 0.02; and for PAMAM G5-OH dendrimer, (8.5 ± 3.2) × 106 (mol/l)−1 and 0.09 ± 0.02. These effects can be explained by the formation of bilirubin–PAMAM dendrimer complexes and the formation of bilirubin monomers from tetramers. The formation of complexes sharply increased bilirubin solubility. We conclude that cationic and neutral PAMAM dendrimers bind bilirubin effectively and suggest that such dendrimers may serve as detoxication agents for hydrophobic endogenous toxins.
Keywords: Polyamidoamine dendrimer; Bilirubin; Complex; Solubility; Antioxidant; Photodestruction; Detoxication;

Molecular characterization and localization of Plasmodium falciparum choline kinase by Vinay Choubey; Mithu Guha; Pallab Maity; Sanjay Kumar; Resmi Raghunandan; Prakas R. Maulik; Kalyan Mitra; Umesh C. Halder; Uday Bandyopadhyay (1027-1038).
Generation of phosphocholine by choline kinase is important for phosphatidylcholine biosynthesis via Kennedy pathway and phosphatidylcholine biosynthesis is essential for intraerythrocytic growth of malaria parasite. A putative gene (Gene ID PF14_0020) in chromosome 14, having highest sequence homology with choline kinase, has been identified by BLAST searches from P. falciparum genome sequence database. This gene has been PCR amplified, cloned, over-expressed and characterized. Choline kinase activity of the recombinant protein (PfCK) was validated as it catalyzed the formation of phosphocholine from choline in presence of ATP. The K m values for choline and ATP are found to be 145 ± 20 μM and 2.5 ± 0.3 mM, respectively. PfCK can phosphorylate choline efficiently but not ethanolamine. Southern blotting indicates that PfCK is a single copy gene and it is a cytosolic protein as evidenced by Western immunoblotting and confocal microscopy. A model structure of PfCK was constructed based on the crystal structure of choline kinase of C. elegans to search the structural homology. Consistent with the homology modeling predictions, CD analysis indicates that the α and β content of PfCK are 33% and 14%, respectively. Since choline kinase plays a vital role for growth and multiplication of P. falciparum during intraerythrocytic stages, we can suggest that this well characterized PfCK may be exploited in the screening of new choline kinase inhibitors to evaluate their antimalarial activity.
Keywords: Plasmodium falciparum; Choline kinase; Cloning; Protein characterization; Phosphocholine;

Hyaluronidase and CD44 hyaluronan receptor expression in squamous cell laryngeal carcinoma by Th.A. Christopoulos; N. Papageorgakopoulou; D.A. Theocharis; N.S. Mastronikolis; Th.A. Papadas; D.H. Vynios (1039-1045).
Squamous cell laryngeal carcinoma undergoes significant structural-related modifications of the extracellular matrix components (ECM), the most characteristics being the presence of degraded collagen, aggrecan and hyaluronan. We examined the presence of hyaluronidase and of the cellular hyaluronan receptor CD44 during the various stages of cancer. ECM components were extracted by using PBS, 4 M GdnHCl and 4 M GdnHCl–0.1% Triton-X 100 sequentially and hyaluronidase and CD44 analyzed by zymography and immunochemistry techniques. Total RNA was also extracted and the mRNA of the various hyaluronidases and of CD44 was analyzed after amplification with RT-PCR. Hyaluronidase was detected as a double band of 45 and 55 kDa molecular mass, only in cancer samples. The analysis of mRNA indicated an aberrant expression of PH-20, the testicular-type hyaluronidase, at late stages of cancer and an overexpression of HYAL1 only at stage IV. In addition, CD44 was identified in two protein bands of 80 and 64 kDa in cancer samples. The analysis of mRNA showed that hyaluronan receptor was expressed in a stage-related order. Thus, it could be suggested that in laryngeal squamous cell carcinoma, cancer cells migrated and proliferated under the influence of small molecular mass hyaluronan, by expressing increased amounts of its receptor.
Keywords: Cancer; Extracellular matrix; Enzyme; Hyaluronidase inhibitor; HYAL1; PH-20; CD44; Diagnostic marker;

Cartilage aggrecan undergoes significant compositional and structural alterations during laryngeal cancer by Spyros S. Skandalis; Achilleas D. Theocharis; Demitrios H. Vynios; Nickoletta Papageorgakopoulou; Anders Hjerpe; Nikos K. Karamanos; Dimitrios A. Theocharis (1046-1053).
Aggrecan is a key component of cartilage and is responsible for the integrity and function of the tissue. In this study, the content of aggrecan and its structural modifications in adjacent to cancer apparently normal cartilages (AANCs) from various stages of laryngeal squamous cell carcinoma (LSCC) were investigated. Our data demonstrated a stage-related loss of aggregable aggrecan in AANCs, compared to the healthy laryngeal cartilage (HLC), which was excessive in advanced stages of disease. On aggregable aggrecan level, AANCs were characterized by significant compositional and structural modifications, the extent of which was closely related with the stage of LSCC. Four concrete subpopulations of aggregable molecules with particular physicochemical characteristics were identified with a strong tendency to prevail subpopulations of molecules of lower hydrodynamic sizes with increasing LSCC stage. These findings demonstrated that the cleavage of aggregable aggrecan occurred in concrete peptide bonds within the CS-1 and CS-2 attachment domains. These significant alterations were closely associated with the process of cartilage destruction, indicating the crucial role of aggrecan during LSCC.
Keywords: Laryngeal cancer; Cartilage; Proteoglycans; Aggregable aggrecan; Aggrecan degradation;

Isolation and primary structures of seven serine proteinase inhibitors from Cyclanthera pedata seeds by Jolanta Kowalska; Agnieszka Zabłocka; Tadeusz Wilusz (1054-1063).
Seven new trypsin inhibitors, CyPTI I–VII, were purified from ripe seeds of Cyclanthera pedata by affinity chromatography on immobilized chymotrypsin in the presence of 5 M NaCl followed by preparative native PAGE at pH 8.9. The CyPTIs (Cyclanthera pedata trypsin inhibitors) belong to a well-known squash inhibitor family. They contain 28–30 amino acids and have molecular weights from 3031 to 3367 Da. All the isolated inhibitors strongly inhibit bovine β-trypsin (K a  > 1011 M− 1) and, more weakly, bovine α-chymotrypsin (K a  ≈ 104–106 M− 1). In the presence of 3 M NaCl the association constants of CyPTIs with α-chymotrypsin increased a few hundred fold. Taking advantage of this phenomenon, a high concentration of NaCl was used to isolate the inhibitors by affinity chromatography on immobilized chymotrypsin. It was found that although one of them, CyPTI IV, had split the Asn25–Gly26 peptide bond, its inhibitory activity remained unchanged. The hydrolyzed bond is located downstream of the reactive site. Presumably, the inhibitor is a naturally occurring, double-chain protein arising during posttranslational modifications.
Keywords: Cyclanthera pedata; Double-chain inhibitor; Squash inhibitors;

Metabolism of oxidized linoleic acid by glutathione transferases: Peroxidase activity toward 13-hydroperoxyoctadecadienoic acid by Stacy K. Seeley; Julie A. Poposki; John Maksimchuk; Jill Tebbe; Jon Gaudreau; Bengt Mannervik; Arthur W. Bull (1064-1070).
The oxidation of linoleic acid produces several products with biological activity including the hydroperoxy fatty acid 13-hydroperoxyoctadecadienoic acid (13-HPODE), the hydroxy fatty acid 13-hydroxyoctadecadienoic acid (13-HODE), and the 2,4-dienone 13-oxooctadecadienoic acid (13-OXO). In the present work, the peroxidase activity of glutathione transferases (GST) A1-1, M1-1, M2-2, and P1-1(Val 105) toward 13-HPODE has been examined. The alpha class enzyme is the most efficient peroxidase while the two enzymes from the mu class exhibit weak peroxidase activity toward 13-HPODE. It was also determined that the conjugated diene 13-HODE is not a substrate for GST from the alpha and mu classes but that 13-HODE does inhibit the GST-catalyzed conjugation of CDNB by enzymes from the alpha, mu, and pi classes. Finally, both 13-HODE and 13-OXO were shown to be inducers of GST activity in HT-29 and HCT-116 colon tumor cells. These data help to clarify the role of GST in the metabolic disposition of linoleic acid oxidation products.
Keywords: Glutathione transferase; 13-HODE; 13-HPODE; Oxidized linoleic acid metabolism; Enzyme induction;

Esterification of ferulic acid with polyols using a ferulic acid esterase from Aspergillus niger by Moriyasu Tsuchiyama; Tatsuji Sakamoto; Tomoyuki Fujita; Shuichi Murata; Haruhiko Kawasaki (1071-1079).
Commercially available enzyme preparations were screened for enzymes that have a high ability to catalyze direct ester-synthesis of ferulic acid with glycerol. Only a preparation, Pectinase PL “Amano” produced by Aspergillus niger, feruloylated glycerol under the experimental conditions. The enzyme responsible for the esterification was purified and characterized. This enzyme, called FAE-PL, was found to be quite similar to an A. niger ferulic acid esterase (FAE-III) in terms of molecular mass, pH and temperature optima, substrate specificity on synthetic substrates, and the N-terminal amino acid sequence. FAE-PL highly catalyzed direct esterification of ferulic acid and sinapinic acid with glycerol. FAE-PL could feruloylate monomeric sugars including arabinose, fructose, galactose, glucose, and xylose. We determined the suitable conditions for direct esterification of ferulic acid with glycerol to be as follows: 1% ferulic acid in the presence of 85% glycerol and 5% dimethyl sulfoxide at pH 4.0 and 50 °C. Under these conditions, 81% of ferulic acid could be converted to 1-glyceryl ferulate, which was identified by 1H-NMR. The ability of 1-glyceryl ferulate to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals was higher than that of the anti-oxidant butyl hydroxytoluene.
Keywords: Aspergillus niger; Ferulic acid esterase; Esterification; Transesterification; Glyceryl ferulate;

Mevalonate 5-diphosphate decarboxylase (MDD) is a peroxisomal enzyme in the cholesterol biosynthetic pathway, which plays an important role in regulating cholesterol biosynthesis. In the present study, rat MDD was cloned and purified to apparent homogeneity. Two fluorinated MDD substrate analogs, P′-geranyl 2-fluoromevalonate 5-diphosphate (4) and 2-fluoromevalonate 5-diphosphate (6), were synthesized, and both were found to be irreversible inhibitors of rat MDD. These two inhibitors were characterized, and mechanisms of the inactivation process were proposed. Kinetic studies indicate both analogs only bind into mevalonate binding-site of MDD. Compound 4 shows competitive inhibition on mevalonate kinase (MVK), and its IC50 value was determined to be comparable with that of geranyl diphosphate. Further kinetic studies indicate compound 4 only bind into ATP binding-site of MVK. These studies provide an example for a single inhibitor to carry out sequential blocking of two enzymes in cholesterol biosynthesis, which may provide useful information for drug discovery for the purpose of treating cardiovascular disease and cancer or for pest control.
Keywords: Mevalonate 5-diphosphate decarboxylase; Fluorinated inhibitor; Mevalonate kinase; Cholesterol; Cardiovascular disease; Cancer;

Downregulation of leukotriene biosynthesis by thymoquinone attenuates airway inflammation in a mouse model of allergic asthma by Mohamed El Gazzar; Rabab El Mezayen; Mark R. Nicolls; John C. Marecki; Stephen C. Dreskin (1088-1095).
Chronic airway inflammation is a key feature of bronchial asthma. Leukotrienes are potent inflammatory mediators that play a role in the pathophysiology of asthma, and their levels are elevated in the airways in response to allergen challenge. We examined the anti-inflammatory effect of thymoquinone (TQ), the active principle in the volatile oil of Nigella sativa seeds, on leukotriene (LT) biosynthesis in a mouse model of allergic asthma. Mice sensitized and challenged with ovalbumin (OVA) antigen had an increased amounts of leukotriene B4 and C4, Th2 cytokines, and eosinophils in bronchoalveolar lavage (BAL) fluid. In addition, there was also a marked increase in lung tissue eosinophilia and goblet cell numbers. Administration of TQ before OVA challenge inhibited 5-lipoxygenase, the main enzyme in leukotriene biosynthesis, expression by lung cells and significantly reduced the levels of LTB4 and LTC4. This was accompanied by a marked decrease in Th2 cytokines and BAL fluid and lung tissue eosinophilia, all of which are characteristics of airway inflammation. These results demonstrate the anti-inflammatory effect of TQ in experimental asthma.
Keywords: Inflammation; Th2 cytokines; Leukotrienes; 5-lipoxygenase; Thymoquinone;

Selective metal binding by Vanabin2 from the vanadium-rich ascidian, Ascidia sydneiensis samea by Norifumi Kawakami; Tatsuya Ueki; Koichi Matsuo; Kunihiko Gekko; Hitoshi Michibata (1096-1101).
Vanadium-binding proteins, or Vanabins, have recently been isolated from the vanadium-rich ascidian, Ascidia sydneiensis samea. Recent reports indicate that Vanabin2 binds twenty V(IV) ions at pH 7.5, and that it has a novel bow-shaped conformation. However, the role of Vanabin2 in vanadium accumulation by the ascidian has not yet been determined. In the present study, the effects of acidic pH on selective metal binding to Vanabin2 and on the secondary structure of Vanabin2 were examined. Vanabin2 selectively bound to V(IV), Fe(III), and Cu(II) ions under acidic conditions. In contrast, Co(II), Ni(II), and Zn(II) ions were bound at pH 6.5 but not at pH 4.5. Changes in pH had no detectable effect on the secondary structure of Vanabin2 under acidic conditions, as determined by circular dichroism spectroscopy, and little variation in the dissociation constant for V(IV) ions was observed in the pH range 4.5–7.5, suggesting that the binding state of the ligands is not affected by acidification. Taken together, these results suggest that the reason for metal ion dissociation upon acidification is attributable not to a change in secondary structure but, rather, that it is caused by protonation of the amino acid ligands that complex with V(IV) ions.
Keywords: Ascidian; Vanadium; Copper; Metal selectivity; Metal-binding protein;

Adenosine 2b receptor (A2bR) signals through adenylate cyclase (AC) 6 isoform in the intestinal epithelial cells by Vasantha L. Kolachala; Tracy Sitler Obertone; Lixin Wang; Didier Merlin; Shanthi V. Sitaraman (1102-1108).
Adenosine 2b receptor (A2bR), a G-protein coupled receptor positively coupled to adenylate cyclase, mediates key events such as chloride, IL-6 and fibronectin secretion in intestinal epithelial cells and is upregulated during intestinal inflammation. In order to gain insight into the overall mechanism of A2bR activation, in this study, we sought to characterize the AC isoform associated with A2bR signaling. The colonic epithelial cell line T84, expressing only the A2b subtype of adenosine receptor, and Chinese hamster ovary (CHO) cells, were used in these studies. cAMP was measured by luminometric assay and AC isoform expression was determined by Western blot, RT-PCR, isoform-specific stealth RNAi and Quantigene. T84 and CHO cells express all nine known AC isoforms. In order to characterize which AC isoform(s) are associated with A2bR, we used the differential inhibition of specific AC isoforms by calcium and nitric oxide. Pretreatment of cells with carbachol or nitric oxide donors such as S-Nitroso-N-acetylpencillamine (SNAP) and PAPANANOATE inhibited A2bR mediated increase in cAMP. Further, overexpression of AC-5 or AC-6 potentiated A2bR-mediated increases in cAMP levels. Finally, transfection with AC isoform-specific RNAi demonstrated that AC-6 but not AC-5 RNAi inhibited adenosine-induced cAMP levels. Taken together, these results suggest that A2bR mediates signaling through AC-6 isoform. Since pro-inflammatory cytokines such as interferon-gamma (IFN-γ) modulate the expression of specific AC isoforms in the intestinal epithelia, our observation may have therapeutic implications for intestinal inflammation or diarrhea wherein aA2bR is upregulated.
Keywords: Adenosine 2b receptor; Adenosine; Adenylate cylase isoform; cAMP; Nitric oxide; T84 cell; CHO cell;

Homocysteine and related amino thiols, homocysteic acid, cysteic acid, homocysteine sulphinic acid and cysteine sulphinic acid have been labelled as neurotoxins. Homocysteine thiolactone, a metabolic derivative of homocysteine, is cytotoxic to endothelial cells and other cell lineages. Since pancreatic beta cells share many phenotypic similarities with neuronal cells, the present study uses clonal pancreatic BRIN-BD11 cells to investigate possible detrimental effects of these amino thiols on insulin secretion and pancreatic beta cell function. Insulin secretion was concentration-dependently inhibited at both basal (1.1 mM) and stimulatory (16.7 mM) glucose by homocysteine, homocysteine thiolactone and homocysteine sulphinic acid. Cysteic acid concentration-dependently inhibited insulin secretion at 16.7 mM glucose. Cell viability was not compromised by any of the amino thiols. Insulin secretory responses to alanine were inhibited by homocysteine, homocysteine thiolactone, homocysteic acid and cysteic acid. Insulin secretion in the presence of elevated Ca2+ and forskolin were lowered by all amino thiols, except homocysteic acid. The secretory responsiveness to PMA, GLP-1 and KCl were only impaired in the presence of homocysteine and homocysteine thiolactone. These findings indicate that homocysteine, homocysteine thiolactone and, to a lesser extent, other amino thiols cause dysfunctional insulin secretion from pancreatic beta cells.
Keywords: Homocysteine; Amino thiol; Insulin secretion; BRIN-BD11 cell;

Sequential O-methylation of tricetin by a single gene product in wheat by Jian-Min Zhou; Nicholas D. Gold; Vincent J.J. Martin; Eckhard Wollenweber; Ragai K. Ibrahim (1115-1124).
Flavonoid compounds are ubiquitous in nature. They constitute an important part of the human diet and act as active principles of many medicinal plants. Their O-methylation increases their lipophilicity and hence, their compartmentation and functional diversity. We have isolated and characterized a full-length flavonoid O-methyltransferase cDNA (TaOMT2) from a wheat leaf cDNA library. The recombinant TaOMT2 protein was purified to near homogeneity and tested for its substrate preference against a number of phenolic compounds. Enzyme assays and kinetic analyses indicate that TaOMT2 exhibits a pronounced preference for the flavone, tricetin and gives rise to three methylated enzyme reaction products that were identified by TLC, HPLC and ESI-MS/MS as its mono-, di- and trimethyl ether derivatives. The sequential order of tricetin methylation by TaOMT2 is envisaged to proceed via its 3′-mono- → 3′,5′-di- → 3′,4′,5′-trimethyl ether derivatives. To our knowledge, this is the first report of a gene product that catalyzes three sequential O-methylations of a flavonoid substrate.
Keywords: Wheat (Triticum aestivum L.); O-methyltransferase; cDNA clone; Sequential methylation; Tricetin methyl ethers;

The solvatochromic fluorescent dye 8-anilino-1-naphthalenesulfonate (ANS) is one of the popular probes of protein folding. Folding kinetics is tracked with ANS fluorescence intensity, usually interpreted as a reflection of protein structure—the hydrophobicity of the binding environments. Such simplistic view overlooks the complicated nature of ANS–protein complexes: the fluorescence characteristics are convoluted results of the ground state populational distribution of the probe–protein complex, the structural changes in the protein and the excited state photophysics of the probe. Understanding of the interplay of these aspects is crucial in accurate interpretation of the protein dynamics. In this work, the fluorescence decay of ANS complexed with apomyoglobin at different conformations denatured by pH is modeled. The fluorescence decay of the ANS–apomyoglobin complex contains information on not only apomyoglobin structure but also molecular populational distributions. The challenge in modeling fluorescence decay profiles originates from the convolution of heterogeneous binding and excited-state relaxation of the fluorescent probe. We analyzed frequency-domain fluorescence lifetime data of ANS-apomyoglobin with both maximum entropy methods (MEM) and nonlinear least squares methods (NLLS). MEM recovers a model of two expanding-and-merging lifetime distributions for ANS–apomyoglobin in the equilibrium transition from the native (N) through an intermediate (I-1) to the acid-unfolded state UA. At pH 6.5 and above, when apomyoglobin is mostly populated at the N-state, ANS–apomyoglobin emits a predominant long-lifetime fluorescence from a relaxed charge transfer state S1,CT of ANS, and a short-lifetime fluorescence that is mainly from a nascent excited-state S1,np of ANS stabilized by the strong ANS–apomyoglobin interaction. Lowering the pH diminishes the contribution from the S1,np state. Meanwhile, more protein molecules become populated at the UA state, which exhibits a short lifetime that is not distinguishable from the S1,np state. At pH 3.4, when the population of the UA becomes significant, the short-lifetime fluorescence comes predominantly from ANS binding to the UA. Further lowering the pH leads to more exposure of the bound ANS. The long lifetime shifts toward and finally merges with the short lifetime and becomes one broad distribution that stands for ANS binding to the UA below pH 2.4. The above expanding-and-merging model is consistent with F-statistic analysis of NLLS models. The consistency of this model with the knowledge from the literature, as well as the continuity of the decay parameters changing upon experimental conditions are also crucial in drawing the conclusions.
Keywords: Fluorescence lifetime; Nonlinear least squares; Maximum entropy method; 1,8-ANS; Apomyoglobin; Folding intermediates;

Structural differences between TSEs strains investigated by FT-IR spectroscopy by Sashko Spassov; Michael Beekes; Dieter Naumann (1138-1149).
Strain diversity in transmissible spongiform encephalopathies (TSEs) has been suggested to be “enciphered” in the structure of the misfolded prion protein isoform PrPSc. We have recently demonstrated the strain typing potential of the FT-IR spectroscopy technique, analyzing four different TSE agents adapted to Syrian hamsters [A. Thomzig, S. Spassov, M. Friedrich, D. Naumann and M. Beekes, Discriminating scrapie and BSE isolates by infrared spectroscopy of pathological prion protein J. Biol. Chem. 279 (2004) 33847-33854.] [1]. In the present paper, we have extended the FT-IR study, exploring the secondary structure, temperature stability, and hydrogen–deuterium exchange characteristics of PrP27–30, from the TSE agents 263K, ME7-H, 22A-H, and BSE-H. The strain differentiation capacity of the FT-IR approach was objectively proven for the first time by multivariate cluster analysis. The second derivative FT-IR spectra obtained from dried protein films or samples hydrated in H2O or D2O consistently exhibited strain-specific infrared characteristics in the secondary structure sensitive amide I region, complemented by strain dependent spectral traits in the amide II and amide A absorption regions, and the different H/D-exchange behaviour of the various PrP27-30 samples. FT-IR spectra of PrP27–30 samples from 263K, ME7-H and 22A-H exposed to increasing temperature (up to 90 °C) showed that a strain-specific response to heat treatment is associated with strain specific thermostability of distinct secondary structure elements, providing additional means for TSEs strain discrimination.
Keywords: FT-IR; Prion; Strains; Secondary structure; Thermal stability; Cluster analysis;