endo-Selective (3+2) cycloaddition polymerizations of nitrone monomers with olefins utilising high pressure conditions

A high pressure mediated (3+2) cycloaddition polymerization strategy has been employed to afford linear poly(isoxazolidine) architectures. Under these high pressure conditions this cycloaddition process was found to afford primarily endoheterocycles which when translated to the polymerization should ultimately affect the tacticity and resultant properties of the polymer. The stereoselectivity occurred as a result of a lower volume of activation for the endo-transition state and the application of a 'type-I' regime (HOMODipole-LUMODipolarophile) cycloaddition process that features secondary orbital interactions within the extended molecular orbitals. A variety of linker segments were employed in an attempt to affect the physical properties of the polymeric cycloadducts such as T-g and solubility in order to tailor these materials for use in coating applications. (C) 2007 Elsevier Ltd. All rights reserved.

X-ray crystallographic study of DNA duplex cross-linking: simultaneous binding to two d(CGTACG)(2) molecules by a bis(9-aminoacridine-4-carboxamide) derivative

Acridine-4-carboxamides form a class of known DNA mono-intercalating agents that exhibit cytotoxic activity against tumour cell lines due to their ability to inhibit topoisomerases. Previous studies of bis-acridine derivatives have yielded equivocal results regarding the minimum length of linker necessary between the two acridine chromophores to allow bis-intercalation of duplex DNA. We report here the 1.7 angstrom resolution X-ray crystal structure of a six-carbon-linked bis(acridine-4-carboxamide) ligand bound to d(CGTACG)(2) molecules by non-covalent duplex cross-linking. The asymmetric unit consists of one DNA duplex containing an intercalated acridine-4-carboxamide chromophore at each of the two CG steps. The other half of each ligand is bound to another DNA molecule in a symmetry-related manner, with the alkyl linker threading through the minor grooves. The two crystallographically independent ligand molecules adopt distinct side chain interactions, forming hydrogen bonds to either O6 or N7 on the major groove face of guanine, in contrast to the semi-disordered state of mono-intercalators bound to the same DNA molecule. The complex described here provides the first structural evidence for the non-covalent cross-linking of DNA by a small molecule ligand and suggests a possible explanation for the inconsistent behaviour of six-carbon linked bis-acridines in previous assays of DNA bis-intercalation.

Synthesis, structure and properties of a mononuclear and an end-on double azido-bridged copper(II) complex incorporating an N,N,N,O-coordinating tripodal ligand

The blue coloured complex [Cu(HL)(H2O)(ClO4)]ClO.H2O.MeOH (1.H2O.MeOH) has been synthesised in excellent yields by reacting Cu(ClO4)(2).6H(2)O with N,N-bis(2-methylpyridyl)(3,5-dimethyl-2-hydroxybenzyl)amine (HL) in methanol. The same reaction, when carried out in the presence of sodium azide, afforded a dark-blue complex of formula [Cu-2(HL)(2)(mu-1,1-N-3)(2)](ClO4)(2) (2). The crystal and molecular structures of the complexes have been solved. Variable-temperature magnetic susceptibility data in the range of 2-300 K for 2 reveal the existence of an antiferromagnetic interaction through an end-on azido linker. Temperature-dependent susceptibility studies for 2 were fitted using the Bleaney-Bowers expression, which led to the parameters J = -3.2 cm(-1), g = 2.12 and R = 2.14 x 10(-4). (C) Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004.

Effect of addition of protein fractions extracted from flours of different baking quality on gluten rheology

The effect of change of the rheological properties of gluten with the addition of fractions with specific molecular weight was investigated. Fractions extracted from Hereward, Riband and Soissons flours were added to the dough prior to gluten extraction. Once extracted, the glutens were subjected to temperature sweeps and creep recovery rheological tests. In the temperature sweeps, Hereward fractions containing the larger polypeptides had a strengthening effect on the gluten, indicated by a decrease in tan delta and an increase in elastic creep recovery, while those fractions that comprised monomeric gliadins had a weakening effect. Adding total gluten also had a strengthening effect. For the biscuit-making flour Riband, the results were quite the reverse: all fractions appeared to strengthen the gluten network, while the addition of total gluten did not have a strengthening effect. For Soissons gluten, the addition of total gluten had a strengthening effect while adding any individual fraction weakened the gluten. The results were confirmed with creep-recovery tests.

The production, purification and characterisation of two novel alpha-D-mannosidases from Aspergillus phoenicis

1,6-alpha-D-Mannosidase from Aspergillits phoenicis was purified by anion-exchange chromatography, chromatofocussing and size-exclusion chromatography. The apparent molecular weight was 74 kDa by SDS-PAGE and 81 kDa by native-PAGE. The isoelectric point was 4.6. 1,6-alpha-D-Mannosidase had a temperature optimum of 60 degrees C, a pH optimum of 4.0-4.5. a K-m of 14 mM with alpha-D-Manp-(1 -> 6)-D-Manp as substrate. It was strongly inhibited by Mn2+ and did not need Ca2+ or any other metal cofactor of those tested. The enzyme cleaves specifically (1 -> 6)-linked mannobiose and has no activity towards any other linkages, p-nitrophenyl-alpha-D-mannopyranoside or baker's yeast mannan. 1,3(1,6)-alpha-D-Mannosidase from A. phoenicis was purified by anion-exchange chromatography, chromatofocus sing and size-exclusion chromatography. The apparent molecular weight was 97 kDa by SDS-PAGE and 110 kDa by native-PAGE. The 1,3(1,6)-alpha-D-mannosidase enzyme existed as two charge isomers or isoforms. The isoelectric points of these were 4.3 and 4.8 by isoelectric focussing. It cleaves alpha-D-Manp-(1 -> 3)-D-Manp 10 times faster than alpha-D-Manp-(1 -> 6)-D-Manp, has very low activity towards p-nitrophenyl-alpha-D-mannopyranoside and baker's yeast mannan, and no activity towards alpha-D-Manp-(1 -> 2)-D-Manp. The activity towards (1 -> 3)-linked mannobiose is strongly activated by 1 mM Ca2+ and inhibited by 10 mM EDTA, while (1 -> 6)-activity is unaffected, indicating that the two activities may be associated with different polypeptides. It is also possible that one polypeptide may have two active sites catalysing distinct activities. (c) 2005 Elsevier Ltd. All rights reserved.

Recent highlights in modified oligonucleotide chemistry

The synthesis of modified nucleic acids has been the subject of much study ever since the structure of DNA was elucidated by Watson and Crick at Cambridge and Wilkins and Franklin at King's College over half a century ago. This review describes recent developments in the synthesis and application of these artificial nucleic acids, predominantly the phosphoramidites which allow for easy inclusion into oligonucleotides, and is divided into three separate sections. Firstly, modi. cations to the base portion will be discussed followed secondly by modi. cations to the sugar portion. Finally, changes in the type of nucleic acid linker will be discussed in the third section. Peptide Nucleic Acids ( PNAs) are not discussed in this review as they represent a separate and large area of nucleic acid mimics.

Synthesis and biological in vitro evaluation of novel PEG-psoralen conjugates

Psoralens are well-known photosensitizers, and 8- methoxypsoralen and 4,5',8-trimethylpsoralen are widely used in photomedicine as "psoralens plus UVA therapy" (PUVA), in photopheresis, and in sterilization of blood preparations. In an attempt to improve the therapeutic efficiency of PUVA therapy and photopheresis, four poly(ethylene glycol) (PEG)-psoralen conjugates were synthesized to promote tumor targeting by the enhanced permeability and retention (EPR) effect. Peptide linkers were used to exploit specific enzymatic cleavage by lysosomal proteases. A new psoralen, 4-hydroxymethyl-4', 8-dimethylpsoralen (6), suitable for polymer conjugation was synthesized. The hydroxy group allowed exploring different strategies for PEG conjugation, and linkages with different stability such ester or urethanes were obtained. PEG (5 kDa) was covalently conjugated to the new psoralen derivative using four different linkages, namely, (i) direct ester bond (7), (ii) ester linkage with a peptide spacer (8), (iii) a carbamic linker (9), and (iv) a carbamic linker with a peptide spacer (12). The stability of these new conjugates was assessed at different pHs, in plasma and following incubation with cathepsin B. Conjugates 7 and 8 were rapidly hydrolyzed in plasma, while 9 was stable in buffer and in the presence of cathepsin B. As expected, only the conjugates containing the peptide linker released the drug in presence of cathepsin B. In vitro evaluation of the cytotoxic activity in the presence and absence of light was carried out in two cell lines (MCF-7 and A375 cells). Conjugates 7 and 8 displayed a similar activity to the free drug (probably due to the low stability of the ester linkage). Interestingly, the conjugates containing the carbamate linkage (9 and 12) were completely inactive in the dark (IC50 > 100 mu M in both cell lines). However, antiproliferative activity become apparent after UV irradiation. Conjugate 12 appears to be the most promising for future in vivo evaluation, since it was relatively stable in plasma, which should allow tumor targeting and drug release to occur by cathepsin B-mediated hydrolysis.

Self-immolative linkers in polymeric delivery systems

There has been significant interest in the methodologies of controlled release for a diverse range of applications spanning drug delivery, biological and chemical sensors, and diagnostics. The advancement in novel substrate-polymer coupling moieties has led to the discovery of self-immolative linkers. This new class of linker has gained popularity in recent years in polymeric release technology as a result of stable bond formation between protecting and leaving groups, which becomes labile upon activation, leading to the rapid disassembly of the parent polymer. This ability has prompted numerous studies into the design and development of self-immolative linkers and the kinetics surrounding their disassembly. This review details the main concepts that underpin self-immolative linker technologies that feature in polymeric or dendritic conjugate systems and outlines the chemistries of amplified self-immolative elimination.

SARS coronavirus unique domain: three-domain molecular architecture in solution and RNA binding

Nonstructural protein 3 of the severe acute respiratory syndrome (SARS) coronavirus includes a "SARS-unique domain" (SUD) consisting of three globular domains separated by short linker peptide segments. This work reports NMR structure determinations of the C-terminal domain (SUD-C) and a two-domain construct (SUD-MC) containing the middle domain (SUD-M) and the C-terminal domain, and NMR data on the conformational states of the N-terminal domain (SUD-N) and the SUD-NM two-domain construct. Both SUD-N and SUD-NM are monomeric and globular in solution; in SUD-NM, there is high mobility in the two-residue interdomain linking sequence, with no preferred relative orientation of the two domains. SUD-C adopts a frataxin like fold and has structural similarity to DNA-binding domains of DNA-modifying enzymes. The structures of both SUD-M (previously determined) and SUD-C (from the present study) are maintained in SUD-MC, where the two domains are flexibly linked. Gel-shift experiments showed that both SUD-C and SUD-MC bind to single-stranded RNA and recognize purine bases more strongly than pyrimidine bases, whereby SUD-MC binds to a more restricted set of purine-containing RNA sequences than SUD-M. NMR chemical shift perturbation experiments with observations of (15)N-labeled proteins further resulted in delineation of RNA binding sites (i.e., in SUD-M, a positively charged surface area with a pronounced cavity, and in SUD-C, several residues of an anti-parallel beta-sheet). Overall, the present data provide evidence for molecular mechanisms involving the concerted actions of SUD-M and SUD-C, which result in specific RNA binding that might be unique to the SUD and, thus, to the SARS coronavirus.

Photochromic dithienylethene derivatives containing Ru(II) or Os(II) metal units. Sensitized photocyclization from a triplet state

Efficient photocyclization from a low-lying triplet state is reported for a photochromic dithienylperfluorocyclopentene with Ru(bpy)(3) units attached via a phenylene linker to the thiophene rings. The ring-closure reaction in the nanosecond domain is sensitized by the metal complexes. Upon photoexcitation into the lowest Ru-to-bpy (MLCT)-M-1 state followed by intersystem crossing to emitting (MLCT)-M-3 states, photoreactive (IL)-I-3 states are populated by an efficient energy-transfer process. The involvement of these (IL)-I-3 states explains the quantum yield of the photocyclization, which is independent of the excitation wavelength but decreases strongly in the presence of dioxygen. This behavior differs substantially from the photocyclization of the nonemissive dithienylperfluorocyclopentene free ligand, which occurs from the lowest (IL)-I-1 state on a picosecond time scale and is insensitive to oxygen quenching. Cyclic voltammetric studies have also been performed to gain further insight into the energetics of the system. The very high photocyclization quantum yields, far above 0.5 in both cases, are ascribed to the strong steric repulsion between the bulky substituents on the dithienylperfluorocyclopentene bridge bearing the chelating bipyridine sites or the Ru(bpy)(3) moieties, forcing the system to adopt nearly exclusively the reactive antiparallel conformation. In contrast, replacement of both Ru(II) centers by Os(II) completely prevents the photocyclization reaction upon light excitation into the low-lying Os-to-bpy (MLCT)-M-1 state. The photoreaction can only be triggered by optical population of the higher lying (IL)-I-1 excited state of the central photochromic unit, but its yield is low due to efficient energy transfer to the luminescent lowest (MLCT)-M-3 state.

Fully automated software solution for protein quantitation by global metabolic labeling with stable isotopes

Metabolic stable isotope labeling is increasingly employed for accurate protein (and metabolite) quantitation using mass spectrometry (MS). It provides sample-specific isotopologues that can be used to facilitate comparative analysis of two or more samples. Stable Isotope Labeling by Amino acids in Cell culture (SILAC) has been used for almost a decade in proteomic research and analytical software solutions have been established that provide an easy and integrated workflow for elucidating sample abundance ratios for most MS data formats. While SILAC is a discrete labeling method using specific amino acids, global metabolic stable isotope labeling using isotopes such as (15)N labels the entire element content of the sample, i.e. for (15)N the entire peptide backbone in addition to all nitrogen-containing side chains. Although global metabolic labeling can deliver advantages with regard to isotope incorporation and costs, the requirements for data analysis are more demanding because, for instance for polypeptides, the mass difference introduced by the label depends on the amino acid composition. Consequently, there has been less progress on the automation of the data processing and mining steps for this type of protein quantitation. Here, we present a new integrated software solution for the quantitative analysis of protein expression in differential samples and show the benefits of high-resolution MS data in quantitative proteomic analyses.

Structural characterisation of bisintercalation in higher-order DNA at a junction-like quadruplex

We report the single-crystal X-ray structure for the complex of the bisacridine bis-(9-aminooctyl(2-(dimethylaminoethyl)acridine-4-carboxamide)) with the oligonucleotide d(CGTACG)2 to a resolution of 2.4 Å. Solution studies with closed circular DNA show this compound to be a bisintercalating threading agent, but so far we have no crystallographic or NMR structural data conforming to the model of contiguous intercalation within the same duplex. Here, with the hexameric duplex d(CGTACG), the DNA is observed to undergo a terminal cytosine base exchange to yield an unusual guanine quadruplex intercalation site through which the bisacridine threads its octamethylene linker to fuse two DNA duplexes. The 4-carboxamide side-chains form anchoring hydrogen-bonding interactions with guanine O6 atoms on each side of the quadruplex. This higher-order DNA structure provides insight into an unexpected property of bisintercalating threading agents, and suggests the idea of targeting such compounds specifically at four-way DNA junctions.

Expression of the collagen receptor glycoprotein VI during megakaryocyte differentiation

This study examined the expression of the platelet collagen receptor glycoprotein VI (GPVI) in megakaryocyte cell lines and primary megakaryocytes by reverse transcriptase-polymerase chain reaction and by flow cytometry and ligand blotting using the snake venom toxin convulxin. Expression of GPVI is increased in the megakaryoblastic cell lines HEL and CMK on differentiation with the phorbol ester phorbol 12-myristate 13-acetate (PMA), along with the Fc receptor gamma-chain (FcR gamma-chain). The increase in GPVI expression is associated with marked potentiation of tyrosine phosphorylation and Ca(++) elevation in response to convulxin. Syk, linker for activated T cells, and phospholipase C gamma 2 (PLC gamma 2) are among the proteins tyrosine phosphorylated on convulxin stimulation in PMA-differentiated HEL cells. Studies on primary murine megakaryocytes grown in vitro confirmed that GPVI is up-regulated in parallel with functional activation, assessed by measurement of [Ca(++)](i), during differentiation. The results demonstrate that expression of GPVI is up-regulated along with the FcR gamma-chain during differentiation of megakaryocytes. (Blood. 2000;96:2740-2745)

The development of a ligase mediated PCR with potential for the differentiation of serovars within Leptospira interrogans

A ligase mediated polymerase chain reaction (LMPCR) was developed to amplify between the repetitive element, IS1533, of Leptospira and adjacent chromosomally located BglII restriction endonuclease enzyme sites. To do this, complimentary oligonucleotide linkers designed to anneal together with an overhanging BglII end were ligated to BglII digested DNA from 35 leptospiral reference strains and field isolates, This ligated DNA was used as template for PCR with oligonucleotide primers specific for the linker and for the repetitive element IS1533. The resultant amplicon profile hybridised a 102 hp region derived from the terminus of IS1533 thus confirming that amplicons generated by LMPCR contained part of IS1533. The number of fragments generated containing IS1533 was significantly fewer than that generated by RFLP but the LMPCR method has the potential to use far less template DNA and be quicker than standard RFLP. Obvious and reproducible interserovar differences were demonstrated by LMPCR whereas for 20 of 21 L. hardjo-bovis isolates tested no intraserovar differences were observed. Of those serovars known to possess IS1533 homologues and tested here by LMPCR, each produced a unique amplicon profile which hybridised the IS1533 terminus probe. The limited heterogeneity amongst hardjo-bovis isolates is discussed as is the potential contribution of this method to diagnosis, differentiation and the phylogenetics of the Leptospires.

Binding properties and adhesion-mediating regions of the major sheath protein of Treponema denticola ATCC 35405

There is growing evidence that a number of oral Treponema species, in particular Treponema denticola, are associated with the progression of human periodontal disease. The major sheath (or surface) protein (Msp) of T. denticola is implicated in adhesion of bacteria to host cells and tissue proteins and is likely to be an important virulence factor. However, the binding regions of the Msp are not known. We have purified from Escherichia coli recombinant Msp (rMsp) polypeptides corresponding to the following: full-length Msp (rMsp) minus 13 N-terminal amino acid (aa) residues, an amino-terminal fragment (rN-Msp, 189 aa residues), a 57-aa residue segment from the central region (rV-Msp), and a C-terminal fragment (rC-Msp, 272 aa residues). rMsp (530 aa residues) bound to immobilized fibronectin, keratin, laminin, collagen type 1, fibrinogen, hyaluronic acid, and heparin. The N- and V-region polypeptides, but not rC-Msp, also bound to these substrates. Binding of rMsp to fibronectin was targeted to the N-terminal heparin I/fibrin I domain. Antibodies to the N-region or V-region polypeptides, but not antibodies to the rC-Msp fragment, blocked adhesion of T. denticola ATCC 35405 cells to a range of host protein molecules. These results suggest that the N-terminal half of Msp carries epitopes that are surface exposed and that are involved in mediating adhesion. Binding of rMsp onto the cell surface of low-level fibronectin-binding Treponema isolates conferred a 10-fold increase in fibronectin binding. This confirms that Msp functions autonomously as an adhesin and raises the possibility that phenotypic complementation of virulence functions might occur within mixed populations of Treponema species.