Granulosal and thecal expression of bone morphogenetic protein- and activin-binding protein mRNA transcripts during bovine follicle development and factors modulating their expression in vitro

Evidence supports local roles for TGFβ superfamily members including activins and bone morphogenetic proteins (BMP) in follicle development. Access of these ligands to signaling receptors is likely modulated by extracellular binding proteins (BP). In this study we compared expression of four BPs (chordin, gremlin, noggin, follistatin) in granulosal (GC) and theca interna (TC) compartments of developing bovine antral follicles (1-18mm). Effects of FSH and IGF on BMP and BP expression by cultured GC, and effects of LH and BMPs on BP expression by cultured TC were also examined. Follicular expression of all four BP transcripts was higher in GC than TC compartments (P<0.001) a finding confirmed by immunohistochemistry. Follicle category affected (P<0.01) gremlin and follistatin mRNA abundance, with a significant cell-type x follicle category interaction for chordin, follistatin and noggin. Noggin transcript abundance was lower (P<0.05) in GC of large 'E-active' than 'E-inactive' follicles while follistatin mRNA level was higher (P<0.01). FSH enhanced CYP19, FSHR, INHBA and follistatin by GC without affecting BMP or BMP-BP expression. IGF increased CYP19 and follistatin, reduced BMP4, noggin and gremlin but did not affect chordin or FSHR mRNA levels. LH increased TC androgen secretion but had no effect on BMP or BP expression. BMPs uniformly suppressed TC androgen production whilst increasing chordin, noggin, and gremlin mRNA levels up to 20-fold (P<0.01). These findings support the hypothesis that extracellular BP, mostly from GC, contribute to the regulation of intrafollicular BMP/activin signaling. Enhancement of thecal BP expression by BMP implies an autoregulatory feedback role to prevent excessive signaling.

Nickel(II) and copper(II) complexes of Schiff base ligands containing N4O2 and N4S2 donors with pyrrole terminal binding groups: synthesis, characterization, X-ray structures, DFT and electrochemical studies

A series of hexadentate ligands, H2Lm (m = 1−4), [1H-pyrrol-2-ylmethylene]{2-[2-(2-{[1H-pyrrol-2-ylmethylene]amino}phenoxy)ethoxy]phenyl}amine (H2L1), [1H-pyrrol-2-ylmethylene]{2-[4-(2-{[1H-pyrrol-2-ylmethylene]amino}phenoxy)butoxy]phenyl}amine (H2L2), [1H-pyrrol-2-ylmethylene][2-({2-[(2-{[1H-pyrrol-2-ylmethylene]amino}phenyl)thio]ethyl}thio)phenyl]amine (H2L3) and [1H-pyrrol-2-ylmethylene][2-({4-[(2-{[1H-pyrrol-2-lmethylene]amino}phenyl)thio]butyl}thio) phenyl]amine (H2L4) were prepared by condensation reaction of pyrrol-2-carboxaldehyde with {2-[2-(2-aminophenoxy)ethoxy]phenyl}amine, {2-[4-(2-aminophenoxy)butoxy]phenyl}amine, [2-({2-[(2-aminophenyl)thio]ethyl}thio)phenyl]amine and [2-({4-[(2-aminophenyl)thio]butyl}thio)phenyl]amine respectively. Reaction of these ligands with nickel(II) and copper(II) acetate gave complexes of the form MLm (m = 1−4), and the synthesized ligands and their complexes have been characterized by a variety of physico-chemical techniques. The solid and solution states investigations show that the complexes are neutral. The molecular structures of NiL3 and CuL2, which have been determined by single crystal X-ray diffraction, indicate that the NiL3 complex has a distorted octahedral coordination environment around the metal while the CuL2 complex has a seesaw coordination geometry. DFT calculations were used to analyse the electronic structure and simulation of the electronic absorption spectrum of the CuL2 complex using TDDFT gives results that are consistent with the measured spectroscopic behavior of the complex. Cyclic voltammetry indicates that all copper complexes are electrochemically inactive but the nickel complexes with softer thioethers are more easily oxidized than their oxygen analogs.

Guanine specific binding at a DNA junction formed by d[CG(5-BrU)ACG]2 with a topoisomerase poison in the presence of Co2+Ions†,‡

The structure of the duplex d[CG(5-BrU)ACG]2 bound to 9-bromophenazine-4-carboxamide has been solved through MAD phasing at 2.0 Å resolution. It shows an unexpected and previously unreported intercalation cavity stabilized by the drug and novel binding modes of Co2+ ions at certain guanine N7 sites. For the intercalation cavity the terminal cytosine is rotated to pair with the guanine of a symmetry-related duplex to create a pseudo-Holliday junction geometry, with two such cavities linked through the minor groove interactions of the N2/N3 guanine sites at an angle of 40°, creating a quadruplex-like structure. The mode of binding of the drug is shown to be disordered, with the major conformations showing the side chain bound to the N7 position of adjacent guanines. The other end of the duplex exhibits a terminal base fraying in the presence of Co2+ ions linking symmetry-related guanines, causing the helices to intertwine through the minor groove. The stabilization of the structure by the intercalating drug shows that this class of compound may bind to DNA junctions as well as duplex DNA or to strand-nicked DNA (‘hemi-intercalated'), as in the cleavable complex. This suggests a structural basis for the dual poisoning of topoisomerase I and II enzymes by this family of drugs.

Lipid binding interactions of antimicrobial plant seed defence proteins: puroindoline-a and β-purothionin

The indolines and thionins are basic, amphiphilic and cysteine-rich proteins found in cereals; puroindoline-a (Pin-a) and β-purothionin (β-Pth) are members of these families in wheat (Triticum aestivum). Pin-a and β-Pth have been suggested to play a significant role in seed defence against microbial pathogens, making the interaction of these proteins with model bacterial membranes an area of potential interest. We have examined the binding of these proteins to lipid monolayers composed of 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG) using a combination of neutron reflectometry, Brewster angle microscopy, and infrared spectroscopy. Results showed that both Pin-a and β-Pth interact strongly with condensed phase DPPG monolayers, but the degree of penetration was different. β-Pth was shown to penetrate the lipid acyl chain region of the monolayer and remove lipids from the air/liquid interface during the adsorption process, suggesting this protein may be able to both form membrane spanning ion channels and remove membrane phospholipids in its lytic activity. Conversely, Pin-a was shown to interact mainly with the head-group region of the condensed phase DPPG monolayer and form a 33 Å thick layer below the lipid film. The differences between the interfacial structures formed by these two proteins may be related to the differing composition of the Pin-a and β-Pth hydrophobic regions.

Fluorescence lifetime imaging microscopy (FLIM) to demonstrate the nuclear binding of flavanols and (--epigallocatechin gallate

The use of light microscopy and DMACA staining strongly suggested that plant and animal cell nuclei act as sinks for flavanols [1, 2]. Detailed uv-vis spectroscopic titration experiments indicated that histone proteins are the likely binding sites in the nucleus [2]. Here we report the development of a multi-photon excitation microscopy technique combined with fluorescent lifetime measurements of flavanols. Using this technique, (+) catechin, (-) epicatechin and (-) epigallocatechin gallate (EGCG) showed strikingly different excited state lifetimes in solution. Interaction of histone proteins with flavanols was indicated by the appearance of a significant τ2-component of 1.7 to 4.0ns. Tryptophan interference could be circumvented in the in vivo fluorescence lifetime imaging microscopy (FLIM) experiments with 2-photon excitation at 630nm. This enabled visualisation and semi-quantitative measurements that demonstrated unequivocally the absorption of (+)catechin, (-)epicatechin and EGCG by nuclei of onion cells. 3D FLIM revealed for the first time that externally added EGCG penetrated the whole nucleus in onion cells. The relative proportions of EGCG in cytoplasm: nucleus: nucleoli were ca. 1:10:100. FLIM experiments may therefore facilitate probing the health effects of EGCG, which is the major constituent of green tea.

Flavor-protein binding: disulfide interchange reactions between ovalbumin and volatile disulfides

The irreversible binding of selected sulfur-containing flavor compounds to proteins was investigated in aqueous solutions containing ovalbumin and a mixture of disulfides (diethyl, dipropyl, dibutyl, diallyl, and 2-furfuryl methyl) using solid-phase micro-extraction (SPME). In systems which had not been heated, the recovery of disulfides from the headspace above the protein at the native pH (6.7) was similar to that from an aqueous blank. However, significant losses were observed when the pH of the solution was increased to 8.0. When the protein was denatured by heating, much greater losses were observed and some free thiols were produced. In similar heat-denatured systems at pH 2.0, no losses of disulfides were observed. Disulfides containing allyl or furfuryl groups were more reactive than saturated alkyl disulfides. Interchange reactions between protein sulfhydryl groups and the disulfides are believed to be responsible for the loss of the disulfides.

DC-SIGN increases the affinity of HIV-1 envelope glycoprotein interaction with CD4

Mannose-binding C-type lectin receptors, expressed on Langerhans cells and subepithelial dendritic cells (DCs) of cervico-vaginal tissues, play an important role in HIV-1 capture and subsequent dissemination to lymph nodes. DC-SIGN has been implicated in both productive infection of DCs and the DC-mediated trans infection of CD4(+) T cells that occurs in the absence of replication. However, the molecular events that underlie this efficient transmission have not been fully defined. In this study, we have examined the effect of the extracellular domains of DC-SIGN and Langerin on the stability of the interaction of the HIV-1 envelope glycoprotein with CD4 and also on replication in permissive cells. Surface plasmon resonance analysis showed that DC-SIGN increases the binding affinity of trimeric gp140 envelope glycoproteins to CD4. In contrast, Langerin had no effect on the stability of the gp140:CD4 complex. In vitro infection experiments to compare DC-SIGN enhancement of CD4-dependent and CD4-independent strains demonstrated significantly lower enhancement of the CD4-independent strain. In addition DC-SIGN increased the relative rate of infection of the CD4-dependent strain but had no effect on the CD4-independent strain. DC-SIGN binding to the HIV envelope protein effectively increases exposure of the CD4 binding site, which in turn contributes to enhancement of infection.

The role of myeloid receptors on murine plasmacytoid dendritic cells in induction of type I interferon

This study tested the hypothesis that a set of predominantly myeloid restricted receptors (F4/80, CD36, Dectin-1, CD200 receptor and mannan binding lectins) and the broadly expressed CD200 played a role in a key function of plasmacytoid DC (pDC), virally induced type I interferon (IFN) production. The Dectin-1 ligands zymosan, glucan phosphate and the anti-Dectin-1 monoclonal antibody (mAb) 2A11 had no effect on influenza virus induced IFNα/β production by murine splenic pDC. However, mannan, a broad blocking reagent against mannose specific receptors, inhibited IFNα/β production by pDC in response to inactivated influenza virus. Moreover, viral glycoproteins (influenza virus haemagglutinin and HIV-1 gp120) stimulated IFNα/β production by splenocytes in a mannan-inhibitable manner, implicating the function of a lectin in glycoprotein induced IFN production. Lastly, the effect of CD200 on IFN induction was investigated. CD200 knock-out macrophages produced more IFNα than wild-type macrophages in response to polyI:C, a MyD88-independent stimulus, consistent with CD200's known inhibitory effect on myeloid cells. In contrast, blocking CD200 with an anti-CD200 mAb resulted in reduced IFNα production by pDC-containing splenocytes in response to CpG and influenza virus (MyD88-dependent stimuli). This suggests there could be a differential effect of CD200 on MyD88 dependent and independent IFN induction pathways in pDC and macrophages. This study supports the hypothesis that a mannan-inhibitable lectin and CD200 are involved in virally induced type I IFN induction.

Molecular modelling studies of binding of DACD derivatives into G-Quadruplex DNA: comparison of force field and quantum polarized ligand docking methods

The DNA G-qadruplexes are one of the targets being actively explored for anti-cancer therapy by inhibiting them through small molecules. This computational study was conducted to predict the binding strengths and orientations of a set of novel dimethyl-amino-ethyl-acridine (DACA) analogues that are designed and synthesized in our laboratory, but did not diffract in Synchrotron light.Thecrystal structure of DNA G-Quadruplex(TGGGGT)4(PDB: 1O0K) was used as target for their binding properties in our studies.We used both the force field (FF) and QM/MM derived atomic charge schemes simultaneously for comparing the predictions of drug binding modes and their energetics. This study evaluates the comparative performance of fixed point charge based Glide XP docking and the quantum polarized ligand docking schemes. These results will provide insights on the effects of including or ignoring the drug-receptor interfacial polarization events in molecular docking simulations, which in turn, will aid the rational selection of computational methods at different levels of theory in future drug design programs. Plenty of molecular modelling tools and methods currently exist for modelling drug-receptor or protein-protein, or DNA-protein interactionssat different levels of complexities.Yet, the capasity of such tools to describevarious physico-chemical propertiesmore accuratelyis the next step ahead in currentresearch.Especially, the usage of most accurate methods in quantum mechanics(QM) is severely restricted by theirtedious nature. Though the usage of massively parallel super computing environments resulted in a tremendous improvement in molecular mechanics (MM) calculations like molecular dynamics,they are still capable of dealing with only a couple of tens to hundreds of atoms for QM methods. One such efficient strategy that utilizes thepowers of both MM and QM are the QM/MM hybrid methods. Lately, attempts have been directed towards the goal of deploying several different QM methods for betterment of force field based simulations, but with practical restrictions in place. One of such methods utilizes the inclusion of charge polarization events at the drug-receptor interface, that is not explicitly present in the MM FF.

Predicting metal-binding site residues in low-resolution structural models

The accurate prediction of the biochemical function of a protein is becoming increasingly important, given the unprecedented growth of both structural and sequence databanks. Consequently, computational methods are required to analyse such data in an automated manner to ensure genomes are annotated accurately. Protein structure prediction methods, for example, are capable of generating approximate structural models on a genome-wide scale. However, the detection of functionally important regions in such crude models, as well as structural genomics targets, remains an extremely important problem. The method described in the current study, MetSite, represents a fully automatic approach for the detection of metal-binding residue clusters applicable to protein models of moderate quality. The method involves using sequence profile information in combination with approximate structural data. Several neural network classifiers are shown to be able to distinguish metal sites from non-sites with a mean accuracy of 94.5%. The method was demonstrated to identify metal-binding sites correctly in LiveBench targets where no obvious metal-binding sequence motifs were detectable using InterPro. Accurate detection of metal sites was shown to be feasible for low-resolution predicted structures generated using mGenTHREADER where no side-chain information was available. High-scoring predictions were observed for a recently solved hypothetical protein from Haemophilus influenzae, indicating a putative metal-binding site.