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PubMed2004

Flexible N,N,N-chelates as supports for iron and cobalt chloride complexes; synthesis, structures, DFT calculations and ethylene oligomerisation studies.

The aryl-substituted N-picolylethylenediamine and diethylenetriamine ligands, (ArNHCH(2)CH(2))[(2-C(5)H(4)N)CH(2)]NH and (ArNHCH(2)CH(2))(2)NH (Ar = 2,6-Me(2)C(6)H(3), 2,4,6-Me(3)C(6)H(2)), have been prepared by employing palladium-catalysed N-C(aryl) coupling reactions of the corresponding primary amines with aryl bromide. Treatment of MCl(2) with (ArNHCH(2)CH(2))[(2-C(5)H(4)N)CH(2)]NH affords [[(ArNHCH(2)CH(2))((2-C(5)H(4)N)CH(2))NH]CoCl(2)](Ar = 2,6-Me(2)C(6)H(3) 1a; 2,4,6-Me(3)C(6)H(2)) 1b and [[(ArNHCH(2)CH(2))((2-C(5)H(4)N)CH(2))NH]FeCl(2)](n)(n= 1, Ar = 2,6-Me(2)C(6)H(3) 2a; n= 2, 2,4,6-Me(3)C(6)H(2) 2b) in high yield. The X-ray structures of 1a and 1b are isostructural and reveal the metal centres to adopt distorted trigonal bipyramidal geometries with the N,N,N-chelates adopting fac-structures. A facial coordination mode of the ligand is also observed in bimetallic 2b, however, in 2a the N,N,N-chelate adopts a mer-configuration with the metal centre adopting a geometry best described as square pyramidal. Solution studies indicate that mer-fac isomerisation is a facile process for these systems at room temperature. Quantum mechanical calculations (DFT) have been performed on 1a and 2a, in which the ligands employed are identical, and show the fac- to be marginally more stable than the mer-configuration for cobalt (1a) while for iron (2a) the converse is evident. Reaction of (ArNHCH(2)CH(2))(2)NH with CoCl(2) gave the five-coordinate complexes [[(ArNHCH(2)CH(2))(2)NH]CoCl(2)](Ar = 2,6-Me(2)C(6)H(3) 3a, 2,4,6-Me(3)C(6)H(2) 3b), in which the ligand adopts a mer-configuration; no reaction occurred with FeCl(2). All complexes 1-3 act as modest ethylene oligomerisation catalysts on activation with excess methylaluminoxane (MAO); the iron systems giving linear alpha-olefins while the cobalt systems give mixtures of linear and branched products.

Journal Article↗

PubMed2006

Structural characterization of mono-ruthenium substituted Keggin-type silicotungstates.

We have synthesized the mono-ruthenium substituted Keggin-type silicotungstate [SiW(11)O(39)Ru(III)(H(2)O)](5-) (1a) by reaction of the mono-lacunary silicotungstate precursor [SiW(11)O(39)](8-) with Ru(acac)(3) under hydrothermal conditions and isolated as the caesium salt Cs(5)[SiW(11)O(39)Ru(III)(H(2)O)] (1). The DMSO-coordinated complex [SiW(11)O(39)Ru(III)(DMSO)](5-) (2a) was prepared by reaction of 1a with DMSO in aqueous solution at 353 K and isolated as the caesium-potassium mixed salt Cs(4.9)K(0.1)[SiW(11)O(39)Ru(III)(DMSO)] (2). Both compounds 1 and 2 were characterized by single-crystal X-ray structure analysis, powder X-ray structure analysis, UV-Vis spectroscopy, cyclic voltammetry, IR-spectroscopy and elemental analysis. 1 crystallized in the tetragonal space group P4(2)/ncm with a = 20.9299(4), c = 10.3603(4) Angstrom, Z = 4. The ruthenium atom in the Keggin unit could not be distinguished from the tungsten due to disorder. The structural analysis of 2 (monoclinic, P2(1)/c, a = 13.5850(4), b = 20.2764(7), c = 18.1326(4) Angstrom, beta = 90.8730(10) degrees , Z = 4) successfully revealed that the incorporated ruthenium atom is coordinated by DMSO through a Ru-S bond. Polyanion 2a represents the first mono-substituted Keggin ion in which the ruthenium center is not crystallographically disordered. UV-Vis spectroscopy combined with controlled potential electrolysis confirmed that the incorporated rutheniums in 1 and 2 have a valence state of +3. The IR spectra of both 1 and 2 were very similar. All these data indicate that 1 synthesized by reaction of the mono-lacunary silicotungstate K(8)[SiW(11)O(39)] with Ru(acac)(3) under hydrothermal conditions is truly the mono-ruthenium substituted Keggin-type silicotungstate.

Journal Article↗

PubMed2001

Interrogation of heme pocket environment of mammalian peroxidases with diatomic ligands.

Recent studies demonstrate that myeloperoxidase (MPO), eosinophil peroxidase (EPO), and lactoperoxidase (LPO), homologous members of the mammalian peroxidase superfamily, can all serve as catalysts for generating nitric oxide- (nitrogen monoxide, NO) derived oxidants. These enzymes contain heme prosthetic groups that are ligated through a histidine nitrogen and use H(2)O(2) as the electron acceptor in the catalysis of oxidative reactions. Here we show that heme reduction of these peroxidases results in distinct electronic and/or conformational changes in their heme pockets using a combination of rapid kinetics measurements, optical absorbance, and diatomic ligand binding studies. Addition of reducing agent to each peroxidase at ground state [Fe(III) state] causes immediate buildup of the corresponding Fe(II) complexes. Spectral changes indicate that two LPO-Fe(II) species are present in solution at equilibrium. Analyses of stopped-flow traces collected when EPO, MPO, or LPO solutions rapidly mixed with NO were accurately fit by single-exponential functions. Plots of the apparent rate constants as a function of NO concentration for all Fe(III) and Fe(II) forms were linear with positive intercepts, consistent with NO binding to each form in a simple reversible one-step mechanism. Fe(II) forms of MPO and LPO, but not EPO, displayed significantly lower affinity toward NO compared to Fe(III) forms, suggesting that heme reduction causes a dramatic change in the heme pocket electronic environment that alters the affinity and/or accessibility of heme iron toward NO. Optical absorbance spectra indicate that CO binds to the Fe(II) forms of both LPO and EPO, but not with MPO, and generates their respective low-spin six-coordinate complexes. Kinetic analyses indicate that the binding of CO to EPO is monophasic while CO binding to LPO is biphasic. Collectively, these results illustrate for the first time functional differences in the heme pocket environments of Fe(II) forms of EPO, LPO, and MPO toward binding of diatomic ligands. Our results suggest that, upon reduction, the heme pocket of MPO collapses, LPO adopts two spectroscopically and kinetically distinguishable forms (one partially open and the other relatively closed), and EPO remains open.

Anaerobiosis↗

PubMed1995

Asymmetric replication in vitro from a human sequence element is dependent on adeno-associated virus Rep protein.

The DNA of human parvovirus adeno-associated virus type 2 (AAV) integrates preferentially into a defined region of human chromosome 19. Southern blots of genomic DNA from latently infected cell lines revealed that the provirus was not simply inserted into the cellular DNA. Both the proviral and adjoining cellular DNA organization indicated that integration occurred by a complex, coordinated process involving limited DNA replication and rearrangements. However, the mechanism for targeted integration has remained obscure. The two larger nonstructural proteins (Rep68 and Rep78) of AAV bind to a sequence element that is present in both the integration locus (P1) and the AAV inverted terminal repeat. This binding may be important for targeted integration. To investigate the mechanism of targeted integration, we tested the cloned integration site subfragment in a cell-free replication assay in the presence or absence of recombinant Rep proteins. Extensive, asymmetric replication of linear or open-circular template DNA was dependent on the presence of P1 sequence and Rep protein. The activities of Rep on the cloned P1 element are analogous to activities on the AAV inverted terminal repeat. Replication apparently initiates from a 3'-OH generated by the sequence-specific nicking activity of Rep. This results in a covalent attachment between Rep and the 5'-thymidine of the nick. The complexity of proviral structures can be explained by the participation of limited DNA replication facilitated by Rep during integration.

Base Sequence↗

PubMed2005

Self-assembly of sensory neurons into ganglia-like microtissues.

Unraveling intra- and inter-cellular signaling networks managing cell-fate control, coordinating complex differentiation regulatory circuits and shaping tissues and organs in living systems remain major challenges in the post-genomic era. Resting on the laurels of past-century monolayer culture technologies, the cell culture community has only recently begun to appreciate the potential of three-dimensional mammalian cell culture systems to reveal the full scope of mechanisms orchestrating the tissue-like cell quorum in space and time. Capitalizing on gravity-enforced self-assembly of monodispersed primary embryonic mouse cells in hanging drops, we designed and characterized a three-dimensional cell culture model for ganglion-like structures. Within 24h, a mixture of mouse embryonic fibroblasts (MEF) and cells, derived from the dorsal root ganglion (DRG) (sensory neurons and Schwann cells) grown in hanging drops, assembled to coherent spherical microtissues characterized by a MEF feeder core and a peripheral layer of DRG-derived cells. In a time-dependent manner, sensory neurons formed a polar ganglion-like cap structure, which coordinated guided axonal outgrowth and innervation of the distal pole of the MEF feeder spheroid. Schwann cells, present in embryonic DRG isolates, tended to align along axonal structures and myelinate them in an in vivo-like manner. Whenever cultivation exceeded 10 days, DRG:MEF-based microtissues disintegrated due to an as yet unknown mechanism. Using a transgenic MEF feeder spheroid, engineered for gaseous acetaldehyde-inducible interferon-beta (ifn-beta) production by cotransduction of retro-/ lenti-viral particles, a short 6-h ifn-beta induction was sufficient to rescue the integrity of DRG:MEF spheroids and enable long-term cultivation of these microtissues. In hanging drops, such microtissues fused to higher-order macrotissue-like structures, which may pave the way for sophisticated bottom-up tissue engineering strategies. DRG:MEF-based artificial micro- and macrotissue design demonstrated accurate key morphological aspects of ganglions and exemplified the potential of self-assembled scaffold-free multicellular micro-/macrotissues to provide new insight into organogenesis.

Animals↗

PubMed1971

Coordinative binding of divalent cations with ligands related to bacterial spores. Equilibrium studies.

It has been repeatedly postulated that the high heat resistance of bacterial spores is due to stabilization of biopolymers in the spore interior by a solid deposit of protective cement consisting of coordination complexes of ligands with divalent metal ions. This report presents data on metal-binding characteristics of some of the ligands related to spores as determined by means of potentiometric equilibrium measurements under conditions of temperature and ionic strength (t = 25.0 degrees C; mu = 1.0 KNO(3)) identical with those reported earlier by the authors in order to facilitate correlation by using comparable data. The spore ligands investigated in this study included 2,6-pyridinedicarboxylic acid (DPA), alpha,epsilon-diaminopimelic acid, D-glutamic acid, and D-alanine in a ratio of 1:1 with metal ions which are known to play a role in heat resistance of spores. Stability constants of the chelates of these spore ligands with metal ions such as Ca(II), Mg(II), Cu(II), Ni(II), Zn(II), Co(II), and Mn(II) have been determined. In general the metal chelates of DPA exhibited the greatest stability. On the basis of a consideration of the stability data together with the known configurations of the ligand and the coordination requirements of the metal ions, possible structures indicating the coordinate binding of the spore ligands with the metal ions are presented. All the metal chelates except those of Ca(II) were found to undergo hydrolysis and separation of solid phase in the pH range 7-8.5. The relatively greater hydrolytic stability of Ca(II) chelates and the high affinity of DPA for metal ions appear to be of biological significance insofar as these two spore components are more widely associated with the heat resistance of bacterial spores.

Alanine↗

PubMed2000

Conversion of core oxos to water molecules by 4e-/4H+ reductive dehydration of the Mn4O2(6+) core in the manganese-oxo cubane complex Mn4O4(Ph2PO2)6: a partial model for photosynthetic water binding and activation.

Reaction of the Mn4O4(6+) "cubane" core complex, Mn4O4L6 (1) (L = diphenylphosphinate, Ph2PO2-), with a hydrogen atom donor, phenothiazine (pzH), forms the dehydrated cluster Mn4O2L6 (2), which has lost two mu-oxo bridges by reduction to water (H2O). The formation of 2 was established by electrospray mass spectrometry, whereas FTIR spectroscopy confirmed the release of water molecules into solution during the reduction of 1. UV-vis and EPR spectroscopies established the stoichiometry and chemical form of the pzH product by showing the production of 4 equiv of the neutral pz radical. By contrast, the irreversible decomposition of 1 to individual Mn(II) ions occurs if the reduction is performed using electrons provided by various proton-lacking reductants, such as cobaltocene or electrochemical reduction. Thus, cubane 1 undergoes coupled four-electron/four-proton reduction with the release of two water molecules, a reaction formally analogous to the reverse sequence of the steps that occur during photosynthetic water oxidation leading to O2 evolution. 1H NMR of solutions of 2 reveal that all six of the phosphinate ligands exhibit paramagnetic broadening, due to coordination to Mn ions, and are magnetically equivalent. A symmetrical core structure is thus indicated. We hypothesize that this structure is produced by the dynamic averaging of phosphinato ligand coordination or exchange of mu-oxos between vacant mu-oxo sites. The paramagnetic 1H NMR of water molecules in solution shows that they are able to freely exchange with water molecules that are bound to the Mn ion(s) in 2, and this exchange can be inhibited by the addition of coordinating anions, such as chloride. Thus, 2 possesses open or labile coordination sites for water and anions, in contrast to solutions of 1, which reveal no evidence for water coordination. Complex 2 exhibits greater paramagnetism than that of 1, as seen by 1H NMR, and it possesses a broad (440 G wide) EPR absorption, centered at g = 2, that follows a Curie-Weiss temperature dependence (10-40 K) and is visible only at low temperatures, compared to EPR-silent 1. Its comparison to a spin-integration standard reveals that 2 contains 2 equiv of Mn(II), which is in agreement with the formal oxidation state of 2Mn(II)2Mn(III) that was derived from the titration. The EPR and NMR data for 2 are consistent with a loss of two of the intermanganese spin-exchange coupling pathways, versus 1, which results in two "wingtip" Mn(II) S = 5/2 spins that are essentially magnetically uncoupled from the diamagnetic Mn2O2 base. Bond-enthalpy data, which show that O2 evolution via the reaction 1-->2 + O2, is strongly favored thermodynamically but is not observed in the ground state due to an activation barrier, are included. This activation barrier is hypothesized to arise, in part, from the constraining effect of the facially bridging phosphinate ligands.

Electron Spin Resonance Spectroscopy↗

PubMed2005

Cellular and molecular interactions regulating skeletogenesis.

Skeletal development involves complex coordination among multiple cell types and tissues. In long bones, a cartilage template surrounded by the perichondrium is first laid down and is subsequently replaced by bone marrow and bone, during a process named endochondral ossification. Cells in the cartilage template and the surrounding perichondrium are derived from mesenchymal cells, which condense locally. In contrast, many cell types that make up mature bone and in particular the bone marrow are brought in by the vasculature. Three tissues appear to be the main players in the initiation of endochondral ossification: the cartilage, the adjacent perichondrium, and the invading vasculature. Interactions among these tissues are synchronized by a large number of secreted and intracellular factors, many of which have been identified in the past 10 years. Some of these factors primarily control cartilage differentiation, while others regulate bone formation and/or angiogenesis. Understanding how these factors operate during skeletal development through the analyses of genetically altered mice depends on being able to distinguish the effect of these molecules on the different cell types that comprise the skeleton. This review will discuss the complexity of skeletal phenotypes, which arises from the tightly regulated, complex interactions among the three tissues involved in bone development. Specific examples illustrate how gene functions may be further assessed using new approaches including genetic and tissue manipulations.

Animals↗

PubMed2005

A distributed coordination platform for home care: analysis, framework and prototype.

Good cooperation between health care (HC) professionals, patient, and family is indispensable during homecare as mentioned in reports and analyses from different countries. In a French National project named coordination for the quality of care (COQUAS), we aimed to address the problem of improving such cooperation with current tools and techniques. We hypothesized that, as in some other domains, a better integration of use and users in informatics systems could improve the usefulness of the cooperative tool. The first part of this paper is devoted to the cognitive analysis of the homecare process and highlights the requirements which should be met according to this analysis. We describe some specific features of asynchronous cooperation and some communication issues in the cooperation of HC workers. We then detail the analysis of the homecare process: methodology, description of the processes, cognitive activity analyses, and of the requirements which flow from this analysis. The second part of this paper proposes a framework and then describes a modular system prototype, designed to take into account these requirements, including aspects of both cooperation and interoperability. It uses a meta-description of actions and information derived from a cognitive study to build dynamically the interface settings; it respects the current trend in distributed architecture and uses XML communication of messages, manages complex coordination with a workflow and allows mobile work. The last part of the paper presents the evaluation which has been done with the implemented prototype, with actual homecare users.

Communication↗

PubMed2003

Carrier-facilitated bulk liquid membrane transport of iron(III)-siderophore complexes utilizing first coordination sphere recognition.

Carrier-facilitated bulk liquid membrane (BLM) transport from an aqueous source phase through a chloroform membrane phase to an aqueous receiving phase was studied for various hydrophilic synthetic and naturally occurring Fe(III)-siderophore complexes using first coordination sphere recognition. Iron transport systems were designed such that two cis coordination sites on a hydrophilic Fe(III) complex are occupied by labile aquo ligands, while the other four coordination sites are blocked by strong tetradentate ligands (siderophores). The labile aquo coordination sites can be "recognized" by a liquid membrane-bound hydrophobic bidentate ligand, which carries the hydrophilic Fe(III)-siderophore complex across the hydrophobic membrane to an aqueous receiving phase. The system is further designed for uphill transport of Fe(III) against a concentration gradient, driven by anti-port H(+) transport. Three tetradentate siderophore and siderophore mimic ligands were investigated: rhodotorulic acid (H(2)L(RA)), alcaligin (H(2)L(AG)), and N,N'-dihydroxy-N,N'-dimethyldecanediamide (H(2)L(8)). Flux values for the transport of Fe(L(x))(OH(2))(2)(+) (x = RA, AG, 8) facilitated by the hydrophobic lauroyl hydroxamic acid (HLHA) membrane carrier were the highest when x = 8, which is attributed to substrate lipophilicity. Ferrioxamine B (FeHDFB(+)) was also selectively transported through a BLM by HLHA. The process involves partial dechelation of ferrioxamine B to produce the tetradentate form of the complex (Fe(H(2)DFB)(OH(2))(2)(2+)), followed by ternary complex formation with HLHA (Fe(H(2)DFB)(LHA)(+)) and transport across the membrane into the receiving phase. Uphill transport of ferrioxamine B was confirmed by increased flux as [H(+)](source phase) < [H(+)](receiving phase). The membrane flux of ferrioxamine B occurs near neutral pH, as evidence that ternary complex formation and ligand exchange are viable processes at the membrane/receptor surface of microbial cells.

Deferoxamine↗

PubMed2006

Caveolin-1 functions as a novel Cdc42 guanine nucleotide dissociation inhibitor in pancreatic beta-cells.

The cycling of the small Rho family GTPase Cdc42 is required for insulin granule exocytosis, although the regulatory proteins involved in Cdc42 cycling in pancreatic beta-cells are unknown. Here we demonstrate that the caveolar protein caveolin-1 (Cav-1) is a Cdc42-binding protein in beta-cells. Cav-1 associated with Cdc42-VAMP2-bound granules present near the plasma membrane under basal conditions. However, stimulation with glucose induced the dissociation of Cav-1 from Cdc42-VAMP2 complexes, coordinate with the timing of Cdc42 activation. Analyses of the Cav-1 scaffolding domain revealed a motif conserved in guanine nucleotide dissociation inhibitors (GDIs), which suggested a novel role for Cav-1 as a Cdc42 GDI in beta-cells. The novel role was further supported by: 1) in vitro binding analyses that demonstrated a direct interaction between Cav-1 and Cdc42; 2) GST-Cdc42 interaction assays showing preferential Cav-1 binding to GDP-Cdc42 over that of GTP-Cdc42; 3) Cav-1 depletion studies resulting in an inappropriate 40% induction of activated Cdc42 in the absence of stimuli and also a 40% increase in basal insulin release from both MIN6 cells and islets. Expression of wild-type Cav-1 in Cav-1-depleted cells restored basal level secretion to normal, whereas expression of a scaffolding domain mutant of Cav-1 failed to normalize secretion. Taken together, these data suggest that Cav-1 functions as a Cdc42 GDI in beta-cells, maintaining Cdc42 in an inactive state and regulating basal secretion in the absence of stimuli. Through its interaction with the Cdc42-VAMP2-bound insulin granule complex, Cav-1 may contribute to the specific targeting of granules to "active sites" of exocytosis organized by caveolae.

Amino Acid Sequence↗

PubMed2005

The association between serum ferritin and uric acid in humans.

OBJECTIVE: Urate forms a coordination complex with Fe(3+) which does not support electron transport. The only enzymatic source of urate is xanthine oxidoreductase. If a major purpose of xanthine oxidoreductase is the production of urate to function as an iron chelator and antioxidant, a system for coupling the activity of this enzyme to the availability of catalytically-active metal would be required. We tested the hypothesis that there is an association between iron availability and urate production in healthy humans by correlating serum concentrations of ferritin with uric acid levels. MATERIALS AND METHODS: The study population included 4932 females and 4794 males in the National Health and Nutrition Examination Survey III. They were 20 years of age or older and in good health. RESULTS: Serum concentrations of ferritin correlated positively with uric acid levels in healthy individuals (R(2) = 0.41, p<0.001). This association was independent of an effect of gender, age, race/ethnic group, body mass, and alcohol consumption. CONCLUSIONS: The relationship between serum ferritin and uric acid predicts hyperuricemia and gout in groups with iron accumulation. This elevation in the production of uric acid with increased concentrations of iron could possibly reflect a response of the host to diminish the oxidative stress presented by available metal as the uric acid assumes the empty or loosely bound coordination sites of the iron to diminish electron transport and subsequent oxidant generation.

Adult↗

PubMed1995

Immunocytology of chiasmata and chromosomal disjunction at mouse meiosis.

Immunocytological and in situ hybridization evidence supports the hypothesis that at meiosis of chiasmate organisms, chromosomal disjunction and reductional segregation of sister centromeres are integrated with synaptonemal complex functions. The Mr 125,000 synaptic protein, Syn1, present between cores of paired homologous chromosomes during pachytene of meiotic prophase, is lost from synaptonemal complexes coordinately with homolog separation at diplotene. Separation is constrained by exchanges between non-sister chromatids, the chiasmata. We show that the Mr 30,000 chromosomal core protein, Cor1, associated with sister chromatid pairs, remains an axial component of post-pachytene chromosomes until metaphase I. We demonstrate that at this time the chromatin loops are still attached to their cores. A reciprocal exchange event between two homologous non-sister chromatids is therefore immobilized by anchorage of sister chromatids to their respective cores. Cores thus contribute to the sister chromatid cohesiveness required for maintenance of chiasmata and proper chromosomal disjunction. Cor1 protein accumulates in juxtaposition to pairs of sister centromeres during metaphase I. Presumably, independent movement of sister centromeres at anaphase I is restricted by Cor1 anchorage. That reductional separation of sister centromeres is mediated by Cor1, is supported by the dissociation of Cor1 from separating sister centromeres at anaphase II and by its absence from mitotic anaphases.

Animals↗

PubMed2001

Pulsed-alkylation mass spectrometry for the study of protein folding and dynamics: development and application to the study of a folding/unfolding intermediate of bacterial luciferase.

A new method employing the classical techniques of chemical modification of proteins and the new technology of mass spectrometry, known as pulsed-alkylation mass spectrometry (PA/MS), has been developed to probe the dynamic structure of folding intermediates and folded complexes of proteins under a variety of conditions. This method is fast and simple, and the results are easily interpreted. PA/MS may provide an alternative to H/D exchange monitored either by NMR or by electrospray ionization mass spectrometry for some experiments; for others, it may provide access to questions not readily answered by available methods. The objective of PA/MS is to determine simultaneously the location and the extent of labeling of functional groups in a protein by measuring the reactivity of cysteines with N-ethylmaleimide, within the context of the conformation of the protein under specific conditions. The method can also be applied to chemical modification of other amino acid residues employing any of a vast array of reagents, depending upon the specifics of the protein under investigation. The enormous range of reactivity of the thiol groups of the cysteinyl residues in proteins and the change in reactivity upon denaturation or conformational rearrangement afford a large signal change that can be correlated with changes in accessibility of the thiol group. The information obtained from the correlation of observed thiol reactivity with the local environment of each cysteinyl residue in the structure of the folded protein can be supplemented by results obtained from fluorescence, circular dichroism, or other methods, to develop an understanding of the structure and dynamics of altered conformational states. With bacterial luciferase as a model system, we have applied PA/MS to investigate the structural differences between the native heterodimeric enzyme and a folding intermediate that is well-populated in 2 M urea. The thiol residues at positions 307, 324, and 325 of the alpha subunit were much more reactive with N-ethylmaleimide in the presence of 2 M urea than in the native enzyme, suggesting that the C-terminal region of the alpha subunit was less tightly packed in the folding intermediate. The apparent unfolding of the C-terminal region of the alpha subunit of the alphabeta structure in 2 M urea appears to mimic the unfolding of the C-terminal domain of the free alpha subunit, also in 2 M urea, described by Noland, B. W., Dangott, L. J., and Baldwin, T. O. (1999) Biochemistry 38, 16136-16145. The approach described here should be applicable to a wide array of problems that have in common the need to determine the locations of conformational changes in proteins. Application of PA/MS to the investigation of the relative thermodynamic stability of the coordination complexes of zinc within each of the six zinc-finger domains of MRE-binding transcription factor-1 (Zn(6) MTF-zf) in its free and DNA-bound forms is presented in the companion paper in this issue [Apuy, J. L., Chen, X., Russell, D. H., Baldwin, T. O., and Giedroc, D. P. (2001) Biochemistry 40, 15164-15175].

Alkylation↗

PubMed1997

Differential expression of PSA-NCAM and HNK-1 epitopes in the developing cardiac conduction system of the chick.

Although the critical role of the His-Purkinje system (HPS) in the propagation of cardiac action potentials from the atria to the ventricular myocardium in the mature heart is well appreciated, its functional and anatomical development are not well understood. The embryonic heart begins beating early in development devoid of a mature conduction system, and the HPS cannot be identified by conventional histochemical analysis until the seventh embryonic day of chicken development. Although many biochemical markers have been found that apparently identify HPS precursors, little is known about how these biochemical markers function in the maturation of the cardiac conduction system. Using immunohistological techniques, we demonstrated that the maturation of the HPS may be observed by the expression of two distinct populations of conduction system precursors, identified by the expression of cell surface carbohydrates PSA-NCAM (PSA) and HNK-1, both of which are known to participate in cell-cell and cell-substrate interactions in the development of the nervous system. By stage 25, PSA was detected in ventricular trabeculae and the interventricular septum (IVS) in a pattern that resembles bundle branches and Purkinje fibers. Beginning at stage 28, HNK-1 epitope expression in the IVS was observed in myocardium just superior to the PSA-positive bundles in a pattern that resembles the common His bundle. This junctional region was also positive for atrial myosin heavy chain (alpha MHC), another marker for the HPS. These data suggest that a complex, coordinated process of biochemical and morphological change governs the development of the cardiac conduction system.

Animals↗

PubMed1997

Combining electrospray ionization and the flowing afterglow method.

The design and implementation of a simple electrospray ionization source for a flowing afterglow/triple quadrupole device are described. Ions can be electrosprayed directly into the room temperature flow tube through a heated capillary without the need for differential pumping or ion focusing. Detected ion currents at the detector sampling orifice as high as 3 pA have been achieved, and the mass spectra indicate little or no re-clustering of the desolvated ions with the background solvent vapor in the flow tube. Spatially and temporally resolved ion/molecule reactions of electrosprayed ions can be carried out in the flow reactor under thermal energy conditions. Sufficient ion densities can be achieved for tandem mass spectrometric experiments in the triple quadrupole analyzer, including energy-resolved collision-induced dissociation. Selected chemical applications illustrating these features are described, including proton transfer reactions with aromatic polysulfonate dianions and multiply protonated polypeptides and threshold CID of a doubly charged transition metal coordination complex.

Amino Acid Sequence↗

PubMedDate not supplied

Intermolecular chiral recognition probed by enantiodifferential excited-state quenching kinetics.

Time-resolved chiroptical luminescence (TR-CL) measurements are used to study the kinetics of chirality-dependent excited-state quenching processes in aqueous solution. Experiments are carried out on samples that contain a racemic mixture of chiral luminophore molecules (L) in solution with a small, optically active concentration of chiral quencher molecules (Q). The luminophores are excited with a pulse of unpolarized light to create an initially racemic excited-state population of lambda L* and delta L* enantiomers, and TR-CL measurements are then used to monitor the differential decay kinetics of the lambda L* and delta L* subpopulations. Observed differences between the lambda L* and delta L* decay kinetics reflect differential rate processes and efficiencies for lambda L*-Q versus delta L*-Q quenching actions, and they are diagnostic of chiral discriminatory interactions between the luminophore and quencher molecules. In this study, the luminophores are either Eu(dpa)3(3-) or Tb(dpa)3(3-) coordination complexes (where dpa denotes a dipicolinate dianion ligand), and the quenchers are diastereomeric structures of Cr(H2O)4(ATP), Rh(H2O)4(ATP) and Rh(H2O)3(ATP) (where ATP identical to adenosine triphosphate). The Ln(dpa)3(3-) (Ln identical to En3+ or Tb3+) complexes have three-bladed propeller-like structures of D3 symmetry, and in aqueous solution they exist as a racemic mixture of left-handed (lambda) and right-handed (delta) configurational isomers (enantiomers). The results show that the chiral quencher molecules can distinguish between the lambda and delta enantiomeric structures of the luminophores in their excited-state quenching actions. The degree and sense of enantiomeric preference in these quenching actions are governed by the electronic and stereochemical properties of the quencher molecules. Twenty-one different luminophore-quencher systems are examined in this study, and they exhibit interestingly diverse enantiodifferential quenching kinetics. The results reflect the extraordinary sensitivity of chiral recognition and discrimination processes to relatively small, and sometimes subtle, changes in molecular electronic and stereochemical structure.

Adenosine Triphosphate↗

PubMed1997

Functional Domains within the Nucleus of a Cell Infected with HSV-1.

HSV-1 is a nuclear replicating DNA virus capable of establishing both lytic and latent infections in mammalian cells. Expression of the more than 80 HSV genes (the majority of which do not contain introns) requires complex coordination of viral and cellular factors both temporally, at appropriate points during the infectious cycle, and spatially as the virus transcription, replication and DNA packaging factories develop in the cell nucleus. Whilst the HSV genome encodes sufficient proteins to sustain viral DNA replication, it is reliant upon its host cell for RNA polymerase II and RNA processing machinery, in addition to an increasing number of cellular cofactors, for gene expression. As HSV establishes a lytic infection, cellular gene expression and splicing are inhibited as cellular chromatin is displaced and a dramatic reorganisation of the host cell nucleus occurs. The formation of large protein-rich factories synthesising viral RNA and replicating and packaging the viral genomes is the most striking alteration. In addition to the synthetic factories, large clumps of cellular and viral intron-containing RNAs accumulate in the nucleus as a result of the inhibition of splicing, at locations which colocalise with splicing factors, but are separate from transcription sites. An essential HSV protein IE63, discussed here, has been identified with a role in the organisation of the nucleus at many levels including replication and transcription site formation, splicing factor organisation and the transport of RNA. This review is a summary of our present understanding of the organisation of the HSV infected cell nucleus, relating viral genomes, RNA, DNA and proteins in the context of the nucleus. However this is a rapidly evolving field and new factors (both viral and cellular) involved in the regulation of these functional domains are constantly being identified. Copyright 1997 by John Wiley & Sons, Ltd.

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