Design, Synthesis, and DNA Binding Properties of Photoisomerizable Azobenzene−Distamycin Conjugates: An Experimental and Computational Study

Here, we present the synthesis, photochemical, and DNA binding properties of three photoisomerizable azobenzene−distamycin conjugates in which two distamycin units were linked via electron-rich alkoxy or electron-withdrawing carboxamido moieties with the azobenzene core. Like parent distamycin A, these molecules also demonstrated AT-specific DNA binding. Duplex DNA binding abilities of these conjugates were found to depend upon the nature and length of the spacer, the location of protonatable residues, and the isomeric state of the conjugate. The changes in the duplex DNA binding efficiency of the individual conjugates in the dark and with their respective photoirradiated forms were examined by circular dichroism, thermal denaturation of DNA, and Hoechst displacement assay with poly[d(A-T).d(T-A)] DNA in 150 mM NaCl buffer. Computational structural analyses of the uncomplexed ligands using ab initio HF and MP2 theory and molecular docking studies involving the conjugates with duplex d[(GC(AT)10CG)]2 DNA were performed to rationalize the nature of binding of these conjugates.

Differential Mechanisms of Binding of Anti-Sigma Factors Escherichia coli Rsd and Bacteriophage T4 AsiA to E. coli RNA Polymerase Lead to Diverse Physiological Consequences

Anti-sigma factors Escherichia coli Rsd and bacteriophage T4 AsiA bind to the essential housekeeping sigma factor, sigma(70), of E. coli. Though both factors are known to interact with the C-terminal region of sigma(70), the physiological consequences of these interactions are very different. This study was undertaken for the purpose of deciphering the mechanisms by which E. coli Rsd and bacteriophage T4 AsiA inhibit or modulate the activity of E. coli RNA polymerase, which leads to the inhibition of E. coli cell growth to different amounts. It was found that AsiA is the more potent inhibitor of in vivo transcription and thus causes higher inhibition of E. coli cell growth. Measurements of affinity constants by surface plasmon resonance experiments showed that Rsd and AsiA bind to or 70 with similar affinity. Data obtained from in vivo and in vitro binding experiments clearly demonstrated that the major difference between AsiA and Rsd is the ability of AsiA to form a stable ternary complex with RNA polymerase. The binding patterns of AsiA and Rsd with sigma(70) studied by using the yeast two-hybrid system revealed that region 4 of sigma(70) is involved in binding to both of these anti-sigma factors; however, Rsd interacts with other regions of sigma(70) as well. Taken together, these results suggest that the higher inhibition of E. coli growth by AsiA expression is probably due to the ability of the AsiA protein to trap the holoenzyme RNA polymerase rather than its higher binding affinity to sigma(70).

Preterm delivery and selected biomarkers : phosphorylated insulin-like growth factor-binding protein-1 and matrix metalloproteinase-8 – in cervical fluid

Premature delivery is a major cause of neonatal morbidity and mortality. The incidence of premature deliveries has increased around the world. In Finland 5.3%, or about 3,000 children per year are born prematurely, before 37 weeks of gestation. The corresponding figure in the United States is about 13%. The morbidity and mortality are highest among infants delivered before 32 weeks of gestation - about 600 children each year in Finland. Approximately 70% of premature deliveries are unexplained. Preterm delivery can be caused by an asympto-matic infection between uterus and the fetal membranes, such can begin already in early pregnancy. It is difficult to predict preterm delivery, and many patients are therefore unnecessarily admitted to hospital for observation and exposed to medical treatments. On the other hand, the high risk women should be identified early for the best treatment of the mother and preterm infant. --- In the prospective study conducted at the Department of Obstetric and Gynecology, Helsinki University Central Hospital two biochemical inflammation related markers were measured in the lower genital tract fluids of asymp-tomatic women in early and mid pregnancy in an order to see whether these markers could identify women with an increased risk of preterm delivery. These biomarkers were phosphorylated insulin-like growth factor binding protein-1 (phIGFBP-1) and matrix metalloproteinase-8 (MMP-8). The study involved 5180 asymptomatic pregnant women, examined during the first and second ultrasound screening visits. The study samples were taken from the vagina and cervicix. In addition, 246 symptomatic women were studied (pregnancy weeks 22 – 34). The study showed that increased phIGFBP-1 concentration in cervical canal fluid in early pregnancy increased the risk for preterm delivery. The risk for very premature birth (before 32 weeks of gestation) was nearly four-fold. Low MMP-8 concentration in mid pregnancy increased the risk of subsequent premature preterm rupture of fetal membranes (PPROM). Significantly high MMP-8 concentrations in the cervical fluid increased the risk for prema-ture delivery initiated by preterm labour with intact membranes. Among women with preterm contractions the shortened cervical length measured by ultrasound and elevated cervical fluid phIGFBP-1 both predicted premature delivery. In summary, because of the relatively low sensitivity of cervical fluid phIGFBP-1 this biomarker is not suitable for routine screening, but provides an additional tool in assessing the risk of preterm delivery. Cervical fluid MMP-8 is not useful in early or mid pregnancy in predicting premature delivery because of its dual role. Further studies on the role of MMP-8 are therefore needed. Our study confirms that phIGFBP-1 testing is useful in predicting pre-term delivery.

Iron(III) Schiff base complexes of arginine and lysine as netropsin mimics showing AT-selective DNA binding and photonuclease activity

Iron(III) complexes [Fe(L)(2)]Cl (1-3), where L is monoanionic N-salicylidene-arginine (sal-argH for 1), hydroxynaphthylidene-arginine (nap-argH for 2) and N-salicylidene-lysine (sal-lysH for 3), were prepared and their DNA binding and photo-induced DNA cleavage activity studied. Complex 3 as its hexafluorophosphate salt [Fe(sal-lysH)(2)](PF6)center dot 6H(2)O (3a) was structurally characterized by single crystal Xray crystallography. The crystals belonged to the triclinic space group P-1. The complex has two tridentate ligands in FeN2O4 coordination geometry with two pendant cationic amine moieties. Complexes 1 and 2 with two pendant cationic guanidinium moieties are the structural models for the antitumor antibiotics netropsin. The complexes are stable and soluble in water. They showed quasi-reversible Fe(III)/Fe(II) redox couple near 0.6 V in H2O-0.1 M KCl. The high-spin 3d(5)-iron(III) complexes with mu(eff) value of similar to 5.9 mu(B) displayed ligand-to-metal charge transfer electronic band near 500 mm in Tris-HCl buffer. The complexes show binding to Calf Thymus (CT) DNA. Complex 2 showed better binding propensity to the synthetic oligomer poly(dA)center dot poly(dT) than to CT-DNA or poly(dG)center dot poly(dC). All the complexes displayed chemical nuclease activity in the presence of 3-mercaptopropionic acid as a reducing agent and cleaved supercoiled pUC19 DNA to its nicked circular form. They exhibited photo-induced DNA cleavage activity in UV-A light and visible light via a mechanistic pathway that involves the formation of reactive hydroxyl radical species. (C) 2010 Elsevier Inc. All rights reserved.

Identification and thermodynamic characterization of molten globule states of periplasmic binding proteins

Molten globule-like intermediates have been shown to occur during protein folding and are thought to be involved in protein translocation and membrane insertion. However, the determinants of molten globule stability and the extent of specific packing in molten globules is currently unclear. Using far- and near-UV CD and intrinsic and ANS fluorescence, we show that four periplasmic binding proteins (LBP, LIVBP, MBP, and RBP) form molten globules at acidic pH values ranging from 3.0 to 3.4. Only two of these (LBP and LIVBP) have similar sequences, but all four proteins adopt similar three-dimensional structures. We found that each of the four molten globules binds to its corresponding ligand without conversion to the native state. Ligand binding affinity measured by isothermal titration calorimetry for the molten globule state of LIVBP was found to be comparable to that of the corresponding native state, whereas for LBP, MBP, and RBP, the molten globules bound ligand with approximately 5-30-fold lower affinity than the corresponding native states. All four molten globule states exhibited cooperative thermal unfolding assayed by DSC. Estimated values of Delta C-p of unfolding show that these molten globule states contain 28-67% of buried surface area relative to the native states. The data suggest that molten globules of these periplasmic binding proteins retain a considerable degree of long range order. The ability of these sequentially unrelated proteins to form highly ordered molten globules may be related to their large size as well as an intrinsic property of periplasmic binding protein folds.

Epigallocatechin gallate is a slow-tight binding inhibitor of enoyl-ACP reductase from Plasmodium falciparum

Epigallocatechin gallate (EGCG) is known to have numerous pharmacological properties. In the present study, we have shown that EGCG inhibits enoyl–acyl carrier protein reductase of Plasmodium falciparum (PfENR) by following a two-step, slow, tight-binding inhibition mechanism. The association/isomerization rate constant (k5) of the reversible and loose PfENR–EGCG binary complex to a tight [PfENR–EGCG]* or EI* complex was calculated to be 4.0 × 10−2 s−1. The low dissociation rate constant (k6) of the [PfENR–EGCG]* complex confirms the tight-binding nature of EGCG. EGCG inhibited PfENR with the overall inhibition constant (Ki*) of 7.0 ± 0.8 nM. Further, we also studied the effect of triclosan on the inhibitory activity of EGCG. Triclosan lowered the k6 of the EI* complex by 100 times, lowering the overall Ki* of EGCG to 97.5 ± 12.5 pM. The results support EGCG as a promising candidate for the development of tea catechin based antimalarial drugs.

Importance of tyrosine residues of Bacillus stearothermophilus serine hydroxymethyltransferase in cofactor binding and l-allo-Thr cleavage

Serine hydroxymethyltransferase (SHMT) from Bacillus stearothermophilus (bsSHMT) is a pyridoxal 5'-phosphate-dependent enzyme that catalyses the conversion of l-serine and tetrahydrofolate to glycine and 5,10-methylene tetrahydrofolate. In addition, the enzyme catalyses the tetrahydrofolate-independent cleavage of 3-hydroxy amino acids and transamination. In this article, we have examined the mechanism of the tetrahydrofolate-independent cleavage of 3-hydroxy amino acids by SHMT. The three-dimensional structure and biochemical properties of Y51F and Y61A bsSHMTs and their complexes with substrates, especially l-allo-Thr, show that the cleavage of 3-hydroxy amino acids could proceed via Cα proton abstraction rather than hydroxyl proton removal. Both mutations result in a complete loss of tetrahydrofolate-dependent and tetrahydrofolate-independent activities. The mutation of Y51 to F strongly affects the binding of pyridoxal 5'-phosphate, possibly as a consequence of a change in the orientation of the phenyl ring in Y51F bsSHMT. The mutant enzyme could be completely reconstituted with pyridoxal 5'-phosphate. However, there was an alteration in the λmax value of the internal aldimine (396 nm), a decrease in the rate of reduction with NaCNBH3 and a loss of the intermediate in the interaction with methoxyamine (MA). The mutation of Y61 to A results in the loss of interaction with Cα and Cβ of the substrates. X-Ray structure and visible CD studies show that the mutant is capable of forming an external aldimine. However, the formation of the quinonoid intermediate is hindered. It is suggested that Y61 is involved in the abstraction of the Cα proton from 3-hydroxy amino acids. A new mechanism for the cleavage of 3-hydroxy amino acids via Cα proton abstraction by SHMT is proposed.

Novel flutamide regulated genes in the rat ventral prostate: differential modulation of their expression by castration and flutamide treatments

Aim: To identify flutamide regulated genes in the rat ventral prostate. Methods: Total RNA from ventral prostates control and flutamide treated rats were isolated. Differentially expressed transcripts were identified using display reverse transcriptase polymerase chain reaction. The effect of castration on the expression of regulated transcripts was studied. Results: We have identified beta 2-microglobulin, cytoplasmic FMR1 protein 2 and pumilio 1 as flutamide induced and spermine binding protein and ribophorin II as flutamide targets in the rat ventral prostate. Although flutamide treatment caused an induction of pumilio I mRNA, had no effect. Conclusion: Castration and flutamide treatments exert differential effects on gene expression. might also have direct AR independent effects, which might have implications in the emergence of androgen dent prostate cancer and the failure of flutamide therapy.

The GAF Domain of the cGMP-Binding, cGMP-Specific Phosphodiesterase (PDE5) Is a Sensor and a Sink for cGMP

We describe here a novel sensor for cGMP based on the GAF domain of the cGMP-binding, cGMP-specific phosphodiesterase 5 (PDE5) using bioluminescence resonance energy transfer (BRET). The wild type GAFa domain, capable of binding cGMP with high affinity, and a mutant (GAFaF163A) unable to bind cGMP were cloned as fusions between GFP and Rluc for BRET2 assays. BRET2 ratios of the wild type GAFa fusion protein, but not GAFaF163A, increased in the presence of cGMP but not cAMP. Higher basal BRET2 ratios were observed in cells expressing the wild type GAFa domain than in cells expressing GAFaF163A. This was correlated with elevated basal intracellular levels of cGMP, indicating that the GAF domain could act as a sink for cGMP. The tandem GAF domains in full length PDE5 could also sequester cGMP when the catalytic activity of PDE5 was inhibited. Therefore, these results describe a cGMP sensor utilizing BRET2 technology and experimentally demonstrate the reservoir of cGMP that can be present in cells that express cGMP-binding GAF domain-containing proteins. PDE5 is the target for the anti-impotence drug sildenafil citrate; therefore, this GAF-BRET2 sensor could be used for the identification of novel compounds that inhibit cGMP binding to the GAF domain, thereby regulating PDE5 catalytic activity.

Perpetuation of immunological memory through common MHC-I binding modes of peptidomimic and antigenic peptides

Understanding the molecular mechanisms of immunological memory assumes importance in vaccine design. We had earlier hypothesized a mechanism for the maintenance of immunological memory through the operation of a network of idiotypic and anti-idiotypic antibodies (Ab2). Peptides derived from an internal image carrying anti-idiotypic antibody are hypothesized to facilitate the perpetuation of antigen specific T cell memory through similarity in peptide-MHC binding as that of the antigenic peptide. In the present work, the existence of such peptidomimics of the antigen in the Ab2 variable region and their similarity of MHC-I binding was examined by bioinformatics approaches. The analysis employing three known viral antigens and one tumor-associated antigen shows that peptidomimics from Ab2 variable regions have structurally similar MHC-I binding patterns as compared to antigenic peptides, indicating a structural basis for memory perpetuation. (C)) 2007 Elsevier Inc. All rights reserved.

Metal-ion-mediated tuning of duplex DNA binding by bis(2-(2-pyridyl)-1H-benzimidazole)

Studies of double-stranded-DNA binding have been performed with three isomeric bis)2-(n-pyridyl)-1H-benzimidazole)s (n = 2, 3, 4). Like the well-known Hoechst 33258, which is a bisbenzimidazole compound, these three isomers bind to the minor groove of duplex DNA. DNA binding by the three isomers was investigated in the presence of the divalent metal ions Mg2+, Co2+, Ni2+, Cu2+, and Zn2+. Ligand-DNA interactions were probed with fluorscence and circular dichroism spectroscopy. These studies revealed that the binding of the 2-pyridyl derivative to DNA is dramatically reduced in the presence of Co2+, Ni2+, and Cu2+ ions and is abolished completely at a ligand/metal-cation ratio of 1:1. Control experiments done with the isomeric 3- and 4-pyridyl derivatives showed that their binding to DNA is unaffected by the aforementioned transition-metal ions. The ability of 2-(2-pyridyl)benzimidazole changes of the ligand associated with ion chelation probably ledto such unusual binding results for the ortho isomer. The addition of ethylenediaminetetraacetic acid (EDTA) reversed the effects completely.

Purification, characterization and molecular cloning of a monocot mannose-binding lectin from Remusatia vivipara with nematicidal activity

A mannose-binding lectin (RVL) was purified from the tubers of Remusatia vivipara, a monocot plant by single-step affinity chromatography on asialofetuin-Sepharose 4B. RVL agglutinated only rabbit erythrocytes and was inhibited by mucin, asialomucin, asialofetuin and thyroglobulin. Lectin activity was stable up to 80A degrees C and under wide range of pH (2.0-9.3). SDS-PAGE and gel filtration results showed the lectin is a homotetramer of Mr 49.5 kDa, but MALDI analysis showed two distinct peaks corresponding to subunit mass of 12 kDa and 12.7 kDa. Also the N-terminal sequencing gave two different sequences indicating presence of two polypeptide chains. Cloning of RVL gene indicated posttranslational cleavage of RVL precursor into two mature polypeptides of 116 and 117 amino-acid residues. Dynamic light scattering (DLS) and gel filtration studies together confirmed the homogeneity of the purified lectin and supported RVL as a dimer with Mr 49.5 kDa derived from single polypeptide precursor of 233 amino acids. Purified RVL exerts potent nematicidal activity on Meloidogyne incognita, a root knot nematode. Fluorescent confocal microscopic studies demonstrated the binding of RVL to specific regions of the alimentary-tract and exhibited a potent toxic effect on M. incognita. RVL-mucin complex failed to interact with the gut confirming the receptor mediated lectin interaction. Very high mortality (88%) rate was observed at lectin concentration as low as 30 A mu g/ml, suggesting its potential application in the development of nematode resistant transgenic-crops.

Analysis of the binding of polymyxin B to endotoxic lipid A and core glycolipid using a fluorescent displacement probe

Dansylcadaverine, a cationic fluorescent probe binds to bacterial lipopolysaccharide and lipid A, and is displaced competitively by other compounds which possess affinity toward endotoxins. The binding parameters of dansylcadaverine for lipid A were determined by Scatchard analysis to be two apparently equivalent sites with apparent dissociation constants (Kd) ranging between 16 μM to 26 μM, while that obtained for core glycolipid from Salmonella minnesota Re595 yielded a Kd of 22 μM to 28 μM with three binding sites. The Kd of polymyxin B for lipid A was computed from dansylcadaverine displacement by the method of Horovitz and Levitzki (Horovitz, A., and Levitzki, A. (1987) Proc. Natl. Acad. Sci. USA 84, 6654–6658). The applicability of this method for analyzing fluorescence data was validated by comparing the Kds of melittin for lipid A obtained by direct Scatchard analysis, and by the Horovitz-Levitzki method. The displacement of dansylcadaverine from lipid A by polymyxin B was distinctly biphasic with Kds for polymyxin B-lipid A interactions corresponding to 0.4 μM and 1.5 μM, probably resulting as a consequence of lipid A being a mixture of mono- and di-phosphoryl species. This was not observed with core glycolipid, for which the Kd for polymyxin was estimated to range from 1.1 μM to 5.8 μM. The use of dansylcadaverine as a displacement probe offers a novel and convenient method of quantitating the interactions of a wide variety of substances with lipid A.