New nickel(II) and copper(II) complexes with unsymmetrical Schiff bases derived from (1R,2R)(-)cyclohexanediamine and the application of Cu(II) complexes for hybrid thin layers deposition.

New unsymmetrical Schiff bases obtained by condensation of (1R,2R)(-)cyclohexanediamine with 2-hydroxy-3,5-di-tert-butylbenzaldehyde (3,5-(t)bba) and 2-hydroxy-3-methoxybenzaldehyde (3-metoxba) or 2-hydroxy-5-nitrobenzaldehyde (5-nba) and 2-hydroxyacetophenone (hacphen) were used for the synthesis of Cu(ii) and Ni(ii) complexes. The ligands and complexes were characterized by circular dichroism (CD), UV-vis, IR, (1)H (NOE diff) (ligand) and (13)C NMR (ligand) spectra. The X-ray crystal structures solved for Ni(II)(1R,2R)(-)chxn(3,5-(t)bba)(hacphen) exhibit distortion of the coordination sphere towards tetrahedral in the solid phase. The complex crystallized in the orthorhombic non-centrosymmetric P2(1)2(1)2(1) space group. Thin layers of copper(II) complexes were deposited on Si(111) by a spin coating technique and characterized by scanning electron microscopy (SEM/EDS), atomic force microscopy (AFM) and fluorescence spectroscopy. Layer deposition conditions were studied and optimal parameters were found (1500 rpm, time 30 s). For copper(ii) layers the most intensive fluorescence band from intraligand transition at 514 nm was observed. CD spectra of complexes in MeCN suggest the tetrahedral distortion from the square planar geometry of the central ion of the coordination sphere in solution. The (1)H NMR NOE diff. spectra of ligands were measured and the positions of the nearest hydrogen atoms in the cyclohexane and aromatic rings were discussed.

Algunas reflexiones sobre los congresos anuales de la AMEE / Algunas reflexiones sobre los AMEE Annual Conferences

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Phosphorylation of H2AX histone as indirect evidence for double-stranded DNA breaks related to the exchange of nuclear proteins and chromatin remodeling in Chara vulgaris spermiogenesis.

Phosphorylation of H2AX histone results not only from DNA damage (caused by ionizing radiation, UV or chemical substances, e.g. hydroxyurea), but also regularly takes place during spermiogenesis, enabling correct chromatin remodeling. Immunocytochemical analysis using antibodies against H2AX histone phosphorylated at serine 139 indirectly revealed endogenous double-stranded DNA breaks in Chara vulgaris spermatids in mid-spermiogenesis (stages V, VI and VII), when protamine-type proteins appear in the nucleus. Fluorescent foci were not observed in early (stages I-IV) and late (VIII-X) spermiogenesis, after replacement of histones by protamine-type proteins was finished. A similar phenomenon exists in animals. Determination of the localization of fluorescent foci and the ultrastructure of nuclei led to the hypothesis that DNA breaks at stage V, when condensed chromatin adheres to the nuclear envelope. This is transformed into a net-like structure during stage VI, probably allowing chromosome repositioning to specific regions in the mature spermatozoid. However, at stages VI and VII, DNA breaks are necessary for transformation of the nucleosomal structure into a fibrillar and finally the extremely condensed status of sleeping genes at stage X.

Educación médica basada en resultados. II. ¿Fascinación transitoria o nuestro futuro? / Outcome-Based Medical Education II. Transitional fascination or our future?

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Educación médica basada en resultados. I. Principios básicos y ventajas / Outcome-based medical eduaction. I.Basic principal and advantages

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Structure of rat transthyretin (rTTR) complex with thyroxine at 2.5 A resolution: first non-biased insight into thyroxine binding reveals different hormone orientation in two binding sites.

The first observation of the unique environment for thyroxine (T(4)) binding in tetrameric rat transthyretin (rTTR) is reported as determined by X-ray diffraction. These data revealed different modes of hormone binding in the two unique hormone-binding sites in the rat TTR tetramer channel. Differences in the orientation of thyroxine and the position of water molecules in the two binding sites further suggest a mechanism for the docking pathway of the hormone into the channel of TTR. Crystals of the rat transthyretin-thyroxine complex are isomorphous with those reported for apo rTTR and crystallized in the tetragonal space group P4(3)2(1)2 with four independent TTR monomeric subunits in the asymmetric part of the crystal lattice. Data were collected to 2.5 A resolution and the structure was refined to R = 20.9% for 15 384 data in the resolution range 12-2.5 A. Similar to human TTR, the rat protein is also a 54 000 Da tetramer with four identical polypeptide chains of 127 amino-acid residues. Of the 22 amino-acid residues which differ between the human and rat sequences, none are in the thyroxine-binding domains. Analysis of these structural data reveals that the tertiary structure is similar to that of hTTR, with only small differences in the flexible loop regions on the surface of the structure. Conformational changes of the amino acids in the channel result in a hydrogen-bonded network that connects the two binding domains, in contrast to the hydrogen bonds formed along the tetramer interface in the apo transthyretin structure. These changes suggest a mechanism for the signal transmission between thyroxine-binding domains.

Structure of the insecticidal bacterial delta-endotoxin Cry3Bb1 of Bacillus thuringiensis.

The coleopteran-active delta-endotoxin Cry3Bb1 from Bacillus thuringiensis (Bt) strain EG7231 is uniquely toxic to Diabrotica undecimpunctata, the Southern corn rootworm, while retaining activity against Leptinotarsa decemlineata, the Colorado potato beetle. The crystal structure of the delta-endotoxin Cry3Bb1 has been refined using data collected to 2.4 A resolution, with a residual R factor of 17.5% and an R(free) of 25.3%. The structure is made up of three domains: I, a seven-helix bundle (residues 64-294); II, a three-sheet domain (residues 295-502); and III, a beta-sandwich domain (residues 503-652). The monomers in the orthorhombic C222(1) crystal lattice form a dimeric quaternary structure across a crystallographic twofold axis, with a channel formed involving interactions between domains I and III. There are 23 hydrogen bonds between the two monomers conferring structural stability on the dimer. It has been demonstrated that Cry3Bb1 and the similar toxin Cry3A form oligomers in solution. The structural results presented here indicate that the interactions between domains I and III could be responsible for the initial higher order structure and have implications for the biological activity of these toxins. There are seven additional single amino-acid residues in the sequence of Cry3Bb1 compared with that of Cry3A; one in domain I, two in domain II and four in domain III, which also shows the largest conformational difference between the two proteins. These changes can be implicated in the selectivity differences noted for these two delta-endotoxins.

Structure of a new polymorphic monoclinic form of human transthyretin at 3 A resolution reveals a mixed complex between unliganded and T4-bound tetramers of TTR.

The crystal structure of a new polymorphic form of human transthyretin (hTTR) with a lattice containing a unique assembly of apo hTTR and TTR-T(4) complex has been determined to 3 A resolution. The monoclinic form of human TTR reported here crystallizes in space group P2(1), with unit-cell parameters a = 76.7 (6), b = 96.7 (8), c = 81.7 (4) A, beta = 106.8 (4) degrees. The asymmetric unit contains two tetramers of transthyretin related by the non-crystallographic symmetry (NCS) operation of a 90.28 degrees rotation between two hTTR molecules around an axis close to crystallographic z. The r.m.s. difference between the two tetramers calculated from their C(alpha) positions is 0.48 A. The structure was refined using 15.0-3.0 A resolution data to R = 22.9% and R(free) = 28.9% for reflections F > 0.0sigma(F), and R = 19.7% and R(free) = 25.8% for reflections F > 3.0sigma(F). The intermolecular interactions involve the tips of alpha-helices and loops around Arg21, Glu61 and Ser100 of all monomers. The electron-density maps revealed residual thyroxine (T(4)) bound in only one of the two unique tetrameric TTR molecules, with an occupancy of 53%, while the second tetramer is unliganded. One thyroxine ligand is bound in a way similar to the orientations described for the orthorhombic form of the hTTR-T(4) complex. The T(4) bound in the second site is positioned similar to 3',5'-dinitro-N-acetyl-L-thyronine in its hTTR complex. Differences in the size of the central channel defined by the D, A, G and H beta-strands of two monomeric subunits are observed between the apo TTR and T(4)-bound tetramer. The averaged distances between Ala108 C(alpha) and its equivalent measured across each binding site are 12.34 A for the T(4)-bound and 10.96 A for the unliganded TTR tetramer, respectively. The observed differences might reflect the mechanics of the ligand binding in the channel and possibly explain the observed negative cooperativity effect for ligand binding.

Occurrence of ubiquitin during spermiogenesis in Chara vulgaris.

Experiments with an anti-ubiquitin antibody proved the presence of ubiquitin in spermatids at all spermiogenesis stages in Charta vulgaris. Its level increased before marked ultrastructural changes of spermatids correlated with disappearance of somatic proteins (histones) and appearance of protamine-type generative proteins. The obtained results seem to confirm our earlier hypotheses concerning a significant role of ubiquitin-proteasome system in Chara spermatozoid differentiation.

Structural basis of negative cooperativity in transthyretin.

A comparison of the AC and BD binding sites of transthyretin (TTR) was made in terms of the interatomic distances between the Ca atoms of equivalent amino acids, measured across the tetramer channel in each binding site. The comparison of the channel diameter for apo TTR from different sources revealed that in the unliganded transthyretin tetramers the distances between the A, D and H beta-strands are consistently larger, while the distances between the G beta-strands are smaller in one site than in the other. These differences might be described to have a 'wave' character. An analogous analysis performed for transthyretin complexes reveals that the shape of the plot is similar, although the amplitudes of the changes are smaller. The analysis leads us to a model of the changes in the binding sites caused by ligand binding. The sequence of events includes ligand binding in the first site, followed by a slight collapse of this site and concomitant opening of the second site, binding of the second molecule and collapse of the second site. The following opening of the first, already occupied site upon ligand binding in the second site is smaller because of the bridging interactions already formed by the first ligand. This explains the negative cooperativity (NC) effect observed for many ligands in transthyretin.

Complex of rat transthyretin with tetraiodothyroacetic acid refined at 2.1 and 1.8 A resolution.

The crystal structure of rat transthyretin (rTTR) complex with 3,5,3',5'-tetraiodothyroacetic acid (T4Ac) was determined at 1.8 A resolution with low temperature synchrotron data collected at CHESS. The structure was refined to R = 0.207 and Rfree = 0.24 with the use of 8-1.8 A data. The additional 8000 reflections from the incomplete 2.1-1.8 data shell, included in the refinement, reduced the Rfree index by 1.3%. Structure comparison with the model refined against the complete 8-2.1 A data revealed no differences in the ligand orientation and the conformation of the polypeptide chain in the core regions. However, the high-resolution data included in the refinement improved the model in the flexible regions poorly defined with the lower resolution data. Also additional sixteen water molecules were found in the difference map calculated with the extended data. The structure revealed both forward and reverse binding of tetraiodothyroacetic acid in one binding site and two modes of forward ligand binding in the second site, with the phenolic iodine atoms occupying different sets of the halogen binding pockets.

Comparison of binding interactions of dibromoflavonoids with transthyretin.

The crystal structure of rat transthyretin (rTTR) complex with the dibromoflavone EMD21388 was determined to 2.3 A resolution and refined to R = 0.203 and Rfree = 0.288. Two different orientations of EMD21388, which differ in the channel penetration by 1.6 A, were found in the A/C binding site of rTTR. The single ligand position observed in the BID site is intermediate between the two positions found in the A/C site. The position of the dibromoflavone in the B/D site is similar to that reported for dibromoaurone in human TTR. The bromine atoms of EMD21388 form strong interactions in the P3 and P3' pockets of rTTR. Due to the different molecular architectures of both ligands, dibromoflavone forms only one interaction with Lys-15 near the channel entrance, while direct interactions with the pair of Lys-15 were reported for dibromoaurone. The C3* methyl group of EMD21388 mediates the bridging interactions between two TTR subunits in the P2 pockets. The interactions of the O2* hydroxyl group of dibromoaurone with the Thr-119 side chain in the P3 pockets are not matched by similar interactions in EMD21388. Both these alternative interactions can explain the competitive binding of 3',5'-dibromoflavonoids to transthyretin.

Computer modeling studies of the structural role of NADPH binding to active site mutants of human dihydrofolate reductase in complex with piritrexim.

Dihydrofolate reductase (DHFR, EC 1.5.1.3) is one of the enzymes active in the folate cycle which plays an important role in DNA synthesis. Inhibition of DHFR is a key element in the treatment of many diseases, including cancer and AIDS related infections. A search for new selective inhibitors is motivated by the resistance to common drugs observed in the course of treatment. In this paper, results of a detailed computer analysis of human DHFR interactions with the lipophilic inhibitor piritrexim (PTX) are presented. It was found that the NADPH cofactor contributes 30% of the total PTX-enzyme interaction energy. Substitution of the highly conserved Glu30 with alanine does not lead to the release of the inhibitor from the hDHFR pocket. The important L22F point mutation does affect PTX orientation but does not changethe binding energy. Simulations of the dynamics of binary hDHFR-PTX complexes were performed with the use of Extensible Systematic Force Field (ESFF) and the results indicate structural changes in the enzyme induced by NADPH binding.

[Application of Internet technology in public health].

Recent advances in telecommunication technology have been enormous. Application of this technology in public health has the potential to markedly improve global health through better surveillance and information systems. With this assumption the GHNet was established in 1994 by representatives from academia, WHO, Pan American Health Organization, the World Bank, NASA, IBM, and AT & T. The GHNet consists of seven components: 1) promotion of networking with the Internet among people in public health; 2) disease tele-monitoring; 3) distance learning system with the internet; 4) connection of non-governmental health organizations; 5) training cyberdocs who are educated in both public health and telecommunications; 6) establishment of an electronic scientific research server; and 7) a home page on the World Wide Web (WWW). In order to effectively incorporate the Internet into the field, connectivity and knowing how to use it are of critical concern. More and more facilities are connected to the Internet in Japan. However, few courses teaching how to utilize the Internet are provided for people in this field. An Internet training course for people in public health was held as joint venture of the World Health Organization (WHO) and the Global Health Network (GHNet) on October 31, 1996, at the 55th Annual Meeting of Japanese Society of Public Health. Most of the participants for the course were from local public health departments and very few had previous experience with the Internet before the course. During this course participants learned how to use e-mail, how to find health resources on the WWW, how to construct a home page, and how the Internet could be utilized to improve public health, with their computers actually hooked to the Internet. From this experience, we found that this kind of course is feasible and beneficial and hope that this course would serve as a model for training people in public health.

Crystal structure of rat transthyretin at 2.5 A resolution: first report on a unique tetrameric structure.

The first observation of a unique tetrameric molecular structure of transthyretin from rat (rTTR, prealbumin) is reported. The structure has been determined by X-ray diffraction using molecular replacement and the structure of human transthyretin (hTTR) as a starting model. Crystals of native rat transthyretin are tetragonal, space group P4(3)2(1)2, and have four independent monomers in the asymmetric unit of the crystal lattice. Data were collected to 2.5 A resolution and the structure has been refined to R = 18.9% for 13584 data points between 8-2.5 A resolution. Like hTTR, the rat protein is also a 54000 Da tetramer with four identical polypeptide chains of 127 amino-acid residues. Of the 22 amino-acid residues which are different in the human and rat TTR sequences, none are in the thyroxine binding domain. Analysis of these data reveal that the tertiary structure of rTTR is similar to that of hTTR with only small differences in the flexible loop regions on the surface of the protein. As a result of local changes in flexible loop regions near residues 30-41, 60-65 and 102-104, the structure of rTTR monomers is more compact than that of the corresponding hTTR monomers. The loop between residues 30-41 is bound closer to the monomer core in the former as compared with the latter structure and there is a wider opening of the space formed between these loops at two adjacent monomeric subunits. These conformational changes do not affect the interfaces between the monomeric subunits and are not transmitted to the thyroxine binding site so that its topology remains not altered.

Structures of human transthyretin complexed with thyroxine at 2.0 A resolution and 3',5'-dinitro-N-acetyl-L-thyronine at 2.2 A resolution.

The molecular structures of two human transthyretin (hTTR, prealbumin) complexes, co-crystallized with thyroxine (3,5,3',5'-tetraiodo-L-thyronine; T(4)), and with 3',5'-dinitro-N-acetyl-LL-thyronine (DNNAT), were determined by X-ray diffraction methods. Crystals of both structures are orthorhombic, space group P2(1)2(1)2, and have two independent monomers in the asymmetric unit of the crystal lattice. These structures have been refined to 17.0% for 8-2.0 A resolution data for the T(4) complex (I), and to R = 18.4% for 8-2.2 A resolution data for the DNNAT structure (II). This report provides a detailed description of T(4) binding to wild-type hTTR at 2.0 A resolution, as well as DNNAT. In both structures, the two independent hormone-binding sites of the TTR tetramer are occupied by ligand. A 50% statistical disorder model was applied to account for the crystallographic twofold symmetry along the binding channel and the lack of such symmetry for the ligands. Results for the co-crystallized T(4) complex show that T(4) binds deep in the hormone-binding channel and displaces the bound water previously reported for T(4) soaked into a native transthyretin crystal [Blake & Oatley (1977). Nature (London), 268, 115-120]. DNNAT also binds deeper in the channel toward the tetramer center than T(4) with the nitro groups occupying the symmetrical innermost halogen pockets. The N-acetyl moiety does not form polar contacts with the protein side chains as it is oriented toward the center of the channel. The weak binding affinity of DNNAT results from the loss of hydrophobic interactions with the halogen binding pockets as observed in T(4) binding. These data suggest that the halogen-binding sites toward the tetramer center are of primary importance as they are occupied by analogues with weak affinity to TTR, and are therefore selected over the other halogen sites which contribute more strongly to the overall binding affinity.