Synthesis of a negatively charged dibenzofuran-based beta-turn mimetic and its incorporation into the WW miniprotein-enhanced solubility without a loss of thermodynamic stability

A versatile synthesis has been developed to functionalize the 4-(2-aminoethyl)-6-dibenzofuran propionic acid residue (1a) at the 2 and 8 positions with a variety of different substructures. The unfunctionalized version of this peptidomimetic (1a) is known to facilitate beta-hairpin formation in a variety of small peptides and proteins in aqueous solution when incorporated in place of the i + 1 and i + 2 residues of a beta-turn. In this study, we append propionate substituents on 1a at the 2 and 8 positions to successfully overcome solubility problems encountered with the incorporation of 1a in place of the i + 1 and i + 2 residues of the beta-turn in loop 1 of the WW domain. The thermodynamic stability of several WW domain analogues incorporating residues 1a and 1b was compared to that of the wild-type sequence revealing comparable DeltaG(H(2)O) unfolding values at 4 degrees C ranging from 3 to 3.6 kcal/mol. WW domains incorporating residue 1b exhibit improved solubility (exceeding 100 microM) and resistance to aggregation without compromising thermodynamic stability.     

Serum protein immunogenicity: implications for liver xenografting

The objectives of this study were threefold: (i) assess immunogenicity of donor plasma proteins following hepatic xenotransplantation, (ii) identify potential immunogens, and (iii) consider the implications of antibody formation against these plasma proteins in xenograft survival. We studied liver and heart xenografts in a concordant combination, hamster to rat. All grafts were examined at necropsy for evidence of rat immunoglobulin G (IgG) deposition. Cardiac xenografts were placed in recipients who had, or had not, been sensitized with hamster serum. Hepatic xenografts were placed in naive recipients to see if antibodies to hamster serum proteins could be eluted from the rejecting organ. Sera of immunized rats were examined for the presence of anti-hamster antibodies by immunoelectrophoresis and by Western blotting following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation of hamster serum. Antibodies in sera of immunized rats were compared with those eluted from rejecting livers. Candidate antigens were identified by tandem mass spectrometry, sequence analysis, and reference to protein databases. Results showed that sera of immunized rats recognized a minimum of four different antigens in hamster serum by immunoelectrophoresis, and a minimum of seven by the more sensitive SDS-PAGE Western blot. IgG eluted from rejecting livers bound three of seven candidate antigens recognized by sera of the immunized animals. Sequence analysis searches revealed proteinase inhibitors in each of the three SDS-PAGE bands common to the above samples. All of these candidate proteinase inhibitor immunogens share a common catabolic fate, uptake via the lipoprotein-related protein (LRP/alpha 2-macroglobulin receptor (CD91). Sensitization to hamster serum proteins hastened cardiac xenograft rejection in 30-50% of recipients (depending on sensitization protocol). Vascular deposition of rat IgG occurred in all rejecting xenografts. Antibody binding to proteinase inhibitors could disturb their functional activity and contribute to the pathogenesis of delayed xenograft rejection.     

Light Dosimetry for Intraperitoneal Photodynamic Therapy in a Murine Xenograft Model of Human Epithelial Ovarian Carcinoma

Few studies have been published to date measuring spatially resolved fluence rates in complex tissue geometries. Here the light distributions of three different intraperitoneal light delivery geometries in a murine ovarian cancer model were investigated to assess their influence on the tumorcidal efficacy of photodynamic therapy (PDT). In vivo fluence rate measurements in the peritoneal cavities of mice, with the light intensity being mapped in three transverse planes, were performed using fiber-optic detectors. Three different source fiber designs and placements were tested for their ability to provide uniform irradiation of the peritoneal cavity. The biological response to a PDT protocol comprising three separate treatments administered at 72 h intervals, each consisting of a 0.25 mg kg intraperitoneal injection of benzopor-phyrin derivative-mono acid ring A followed 90 min later by delivery of 15 J of 690 nm light, was measured. The tissue response was evaluated by measuring the number of remaining visible lesions and the total residual tumor mass. Fluence rate measurements showed large variations in the fluence rate distribution for similar intended treatments. The most uniform and reproducible illumination was achieved using two 18 mm long cylindrical emitting optical fibers. The biological response was comparable to that produced when a flat-cleaved end optical fiber is used to illuminate the four quadrants of the abdomen sequentially. While a good reproducibility in tumor induction in this animal model exists, no correlation was found between the fluence rate distribution measured in one group of animals and the biological response in a separate group of similarly treated animals. Due to the large intra-animal variability in fluence rate distribution, representative fluence rate mapping in complex tissue geometries is of limited value when applied to an individual PDT treatment. Thus, surveillance of the fluence rate during individual treatments will be required for acceptable PDT dosimetry. To improve the versatility of this particular animal model for PDT research, a large number of extended sources are required to increase uniformity of the illumination in order to reduce unwanted cytotoxic side effects resulting from foci of high fluence rates. In this way, subsequent increase of the total energy delivered to the tumor may be possible.     

Spirobicyclic diamines. Part 2: Synthesis of homochiral diastereoisomeric proline derived [4.4]-spirolactams

l-Proline derived diastereoisomeric [4.4]-spirolactams have been prepared by a reductive-amination reaction of (R)- or (S)-alanine methyl ester, followed by thermal cyclisation of the resulting amine onto the proline ester group in refluxing toluene. Under similar conditions (R)- or (S)-phenylalanine methyl ester gave no cyclisation products, while R- or S-α-methylbenzylamine required treatment with NaNH₂ in refluxing toluene to induce cyclisation giving diastereoisomeric [4.4]-spirolactams.     

Trends in Preconcentration Procedures for Metal Determination Using Atomic Spectrometry Techniques

 Methods for metal preconcentration are often described in the literature. However, purposes are often different, depending on whether the methods are applied in environmental, clinical or technological fields. The respective method needs to be efficient, give high sensitivity, and ideally also is selective which is useful when used in combination with atomic spectroscopy. This review presents the actual tendencies in metal preconcentration using techniques such as Flame Atomic Absorption Spectrometry (FAAS), Electrothermal Atomic Absorption Spectrometry (ETAAS), Hydride Generation Atomic Absorption Spectrometry (HGAAS), Inductively Coupled Plasma Optical Emission Spectrometry (ICP OES) and Inductively Coupled Plasma Mass Spectrometry (ICP-MS). Procedures based on related to electrochemical, coprecipitation/precipitation, liquid–liquid and solid–liquid extraction and atom trapping mechanisms are presented. Correspondence: Department of Analytical Chemistry, Universidade Estadual de Campinas – UNICAMP, P.O. Box 6154, 13083-970 Campinas, Brazil. e-mail: zezzi@iqm.unicamp.br Received December 20, 2001; accepted October 11, 2002     

The ligand, the metal and the 'Holey'-host: Synthesis, structural and magnetic characterisation of Co(II), Ni(II) and Mn(II) metal-organic frameworks incorporating 4,4'-dicarboxy-2,2'-bipyridine

We report herein the single crystal structures of four metal-organic framework complexes incorporating the 4,4'-dicarboxy-2,2'-bipyridine ligand, H(2)dcbp: alpha-[Co(dcbp)(H(2)O)(2)], 1; beta-[Co(dcbp)(H(2)O)(2)], 2, [Ni(dcbp)(H(2)O)(2)], and [[Mn(dcbp)].1/2DEF], 4 (DEF = diethylformamide). In each complex the ligand is deprotonated giving neutral species with 1:1 stoichiometry that form three-dimensional coordination polymers. Supramolecular isomerism (polymorphism) in 1 and 2 arises from the different ligand connectivity around the octahedral Co(II) centres. The two coordinated water molecules in 1 occupy cis positions, which are trans to the chelating bipyridine nitrogen atoms, leaving the carboxylate oxygen atoms in axial trans positions. In 2 all like donors occupy cis positions. Different modes of carboxylate coordination in 1 and 2 give dissimilar network topologies. A rare example of two interpenetrating 6(4)8(2)-b (quartz-like) chiral networks in 1 results from both dcbp carboxylate groups coordinating in a monodentate fashion to adjacent Co(II) centres, whereas in 2 only one carboxylate group bridges between adjacent Co(II) centres giving rise to a single chiral (10,3)-a net. In 1 and 2 the coordinated water molecules hydrogen bond to the non-coordinated carboxylate oxygen atoms. These interactions give rise to water-carboxylate double helices in , and support the coordination network in 2. Strikingly for a pair of dimorphs the crystal densities of 1 and 2 differ by ca. 0.3 g cm(-3)(1.654 vs. 1.940 g cm(-3), respectively). Compound 3 is isomorphous with 1 and likewise features two chiral interpenetrating nets of quartz topology. In 4, chelating bipyridine nitrogen atoms and four carboxylate oxygen atoms from a total of five adjacent dcbp ligands provide distorted octahedral geometry around Mn(II). The carboxylate groups bridge adjacent Mn(II) centres to produce bis-carboxylato chains which cross-link and generate a 3D network that is perforated with channels. The channels are occupied with disordered DEF molecules. The network topology in 4 is quite different to 1-3 and has a (4.6(2))(4(2).6)(4(3).6(6).8(6)) Schlafli notation. Magnetic susceptibility studies performed on 1, 2, [[Mn(dcbp)].1/2DMF] 5 (DMF = dimethylformamide) and [[Mn(dcbp)].2H(2)O] 6 reveal very weak antiferromagnetic coupling between the metal centres in each case.