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PubMedDate not supplied

On the formation of amplified nucleoli during early Xenopus oogenesis.

In Xenopus the genes for ribosomal RNA are selectively amplified during the early stages of oogenesis and give rise to over 1000 extrachromosomal nucleoli. These oocyte nucleoli are unique in that they contain very high copy numbers of rRNA genes, are not attached to chromosomes, and lack nonribosomal DNA. How the amplified rRNA genes induce the formation of multiple nucleoli is as yet poorly understood. To gain some more insight into this assembly process we have studied nucleolar development in early previtellogenic Xenopus oocytes. By using light and electron microscopic immunocytochemistry, in situ hybridization, and the TdT-method for detection of DNA, we have identified three separate structural entities which are related to the three components of the functionally active nucleoli present in midsized oocytes. Besides fibrillar and granular bodies we describe a novel rod-like structure which contains the pol I-specific transcription factor UBF. Notably, rDNA emerges from these rods forming a filamentous layer. These results reinforce UBF's role as an architectural element involved in the spatial organization of the rRNA genes. We consider the rod-like rDNA/UBF complexes as extrachromosomal nucleolar organizers which are first juxtaposed to preformed fibrillar bodies before both elements gradually fuse into a coherent nucleolar structure.

Animals↗
PubMed1999

Intermolecular V(D)J recombination is prohibited specifically at the joining step.

V(D)J recombination, normally an intramolecular process, assembles immunoglobulin and T cell receptor genes from V, D, and J coding segments. Oncogenic chromosome translocations can result from aberrant rearrangements, such as occur in intermolecular V(D)J recombination. How this is normally prevented remains unclear; DNA cleavage, joining, or both could be impaired when the recombination signal sequences (RSS) are located in trans, on separate DNA molecules. Here, we show that both trans cleavage and joining of signal ends occur efficiently in vivo. Unexpectedly, trans joining of coding ends is severely impaired (100-to 1000-fold), indicating that protection against intermolecular V(D)J recombination is established at the joining step. These findings suggest a novel surveillance mechanism for eliminating cells containing aberrant V(D)J rearrangements.

Animals↗
PubMed2003

A two-step scaffolding model for mitotic chromosome assembly.

Topoisomerase IIalpha (topoIIalpha) and 13S condensin are both required for mitotic chromosome assembly. Here we show that they constitute the two main components of the chromosomal scaffold on histone-depleted chromosomes. The structural stability and chromosomal shape of the scaffolding toward harsh extraction procedures are shown to be mediated by ATP or its nonhydrolyzable analogs, but not ADP. TopoIIalpha and 13S condensin components immunolocalize to a radially restricted, longitudinal scaffolding in native-like chromosomes. Double staining for topoIIalpha and condensin generates a barber pole appearance of the scaffolding, where topoIIalpha- and condensin-enriched "beads" alternate; this structure appears to be generated by two juxtaposed, or coiled, chains. Cell cycle studies establish that 13S condensin appears not to be involved in the assembly of prophase chromatids; they lack this complex but contain a topoIIalpha-defined (-mediated?) scaffolding. Condensin associates only during the pro- to metaphase transition. This two-step assembly process is proposed to generate the barber pole appearance of the native-like scaffolding.

Adenosine Diphosphate↗
PubMed2002

Electrochemical studies of blocking properties of solid supported tethered lipid membranes on gold.

The insulating properties of self-assembled thiolipid monolayers and tethered lipid bilayers on polycrystalline gold electrodes were studied by means of cyclic voltammetry (CV). These films were formed by two-step self-assembly processes. Electrochemical measurements of the heterogeneous electron transfer rate constant of different redox couples such as potassium ferrocyanide (K(4)[Fe(CN)(6)]) and dopamine (DP) were used to examine the molecular integrity and structural defects and pinholes within the monolayers. We demonstrate by means of cyclic voltammetry that the bilayer lipid membranes tethered to the gold surface are blocking, stable, yet retaining their dynamic properties and can be used as a model of the cell membrane.

Electrochemistry↗
PubMed1979

The structure and formation of the egg-shell of Syphacia obvelata Rudolphi (Nematoda: Oxyurida).

The egg of Syphacia obvelata is a flattened elipsoid. The egg-shell consists of 5 layers: external uterine layer, internal uterine layer, vitelline layer, chitinous layer and lipid layer. An operculum is present at one pole of the egg. The opercular groove consists of a break in the uterine layers and the modification of the chitinous layer by the deposition of lipoprotein material. On the curved side of the egg the uterine layers are modified to form alternate ridges and depressions. Discrete spaces are present in the internal uterine layer between the ridges. These are open to the exterior via pores in the external uterine layer. The structure of the uterine layers is quite different on the flattened side of the egg. The morphology of the reproductive system and the formation of the egg-shell is described. It is suggested that the complex structure of the uterine layers of oxyurids forms by a self-assembly process.

Animals↗
PubMed2003

Fabrication of patterned multicomponent protein gradients and gradient arrays using microfluidic depletion.

We demonstrate that depletion effects in the fluids used to fill a poly(dimethylsiloxane) microfluidic device can be used in conjunction with its design rules to generate patterned protein gradients. The linear portions of these structures can be designed to present gradients of bound protein coverage-varying from near-saturation to effectively zero-over distances ranging from a few hundred micrometers to more than 1 cm by design. Such patterns can be developed in a simple, single-channel form as well as in a multichannel gradient array of more complex design. The patterning protocols also support the use of multiple protein sources, and we demonstrate an assembly process mediated by a protein that inhibits adsorption to generate a gradient array in pixel form. We describe examples of multiple protein gradient patterns along with simple immunoassays to illustrate the scope of the methodology, the activity of the patterned proteins, and their recognition in gradient form on a surface. These gradients should prove useful to studies in biosensor and bioassay development and as substrates for cell culture to study growth and motility.

Journal Article↗
PubMed2006

Benefiting from the unique properties of lanthanide ions.

The recent upsurge of interest in contrast agents for magnetic resonance imaging, of luminescent chemosensors for medical diagnostic, and lately, for optical imaging of cells has generated an impressive momentum for the coordination and supramolecular chemistry of trivalent lanthanide ions. We shortly review the synthetic methods allowing the introduction of these spherical ions with fascinating optical and magnetic properties into elaborate mono- and polymetallic edifices. We then illustrate these methods by selected examples describing the use of (i) a coronand to produce luminescent liquid crystals, (ii) derivatized calixarenes for 4f-5f element separation, (iii) podates for the production of nanoparticles with high relaxivity and for sensitizing the near-infrared (NIR) emission, and (iv) self-assembly processes for producing functional bimetallic edifices.

Ions↗
PubMed2005

Ligand design in multimetallic architectures: six lessons learned.

Metallosupramolecular chemistry involves the use of combinations of organic ligands and metals for the construction of both discrete and polymeric aggregates. This Account describes some lessons that we have learned about aspects of ligand design in the course of our work in this area. Specifically, we recommend the incorporation of a diverse range of heterocyclic rings and arene cores within the ligands, as well as attention to symmetry considerations, and offer suggestions for the introduction of chirality and flexibility within the ligands and the exploitation of other weak interactions to assist self-assembly processes.

Journal Article↗
PubMed1995

Functional reconstitution of the human placental transferrin receptor into phospholipid bilayers leads to long tubular structures proceeding from the vesicle surface.

We have reconstituted the human placental transferrin receptor (hTfR) into phospholipid vesicles using either dialysis, dilution, or gel filtration. Several different detergents and phospholipids and a variety of lipid-to-protein weight ratios were tested. Preformed vesicles as well as detergent-solubilized phospholipids were used. Reconstituted mixtures were fractionated by sucrose density gradient centrifugation and screened for ferritransferrin binding activity, and peak fractions were analyzed by electron microscopy. The efficiency of reconstitution was strongly influenced by the choice of the phospholipids used and the reconstitution method. Best results were obtained when hTfR was dissolved with octylpolyoxyethylene, mixed with detergent-solubilized soy bean lecithin, and reconstituted by slow dialysis. The data show that hTfR capable of binding ferritransferrin was reconstituted into vesicles with an irregular surface and many protrusions. In addition the reconstitution of hTfR resulted in the formation of tubular structures proceeding from the vesicle surface, which may serve as an in vitro model for future studies on the relevance of self-assembly processes for cellular endocytosis.

Centrifugation, Density Gradient↗
PubMed2001

Cysteine substitutions in apolipoprotein A-I primary structure modulate paraoxonase activity.

Paraoxonase (PON) is transported primarily on apolipoprotein A-I (apoA-I) -containing high-density lipoprotein (HDL) and is thought to protect against early atherogenic events including low-density lipoprotein (LDL) oxidation and monocyte migration. It has been proposed that apoA-I may be necessary for PON's association with plasma HDL. On the basis of this, we examined the effect of apoA-I on PON's enzymatic activity and its ability to associate with HDL. Additionally, we examined whether changes in apoA-I primary structure (cysteine substitution mutations) could modulate these effects. Chinese hamster ovary cells stably transfected with human PON1A cDNA were incubated in the presence and absence of recombinant wild-type apoA-I (apoA-I(WT)) and specific Cys substitution mutations. Extracellular accumulation of PON activity in the presence of apoA-I(WT) was 0.095 +/- 0.013 unit/mg of cell protein (n = 7) compared to 0.034 +/- 0.010 unit/mg of cell protein in the absence of apoA-I (n = 7), a 2.79-fold increase in activity when apoA-I was incubated with the cells. Lipid-free apoA-I did not increase PON activity, while preformed nascent HDL increased PON activity only 30%, suggesting that maximal PON activity is lipid-dependent and requires coassembly of PON and apoA-I on nascent HDL. The cysteine mutations R10C, R27C, and R61C significantly increased (p < 0.01) PON activity 32.6% +/- 14.7%, 31.6% +/- 18.9%, and 27.4% +/- 20%, respectively, over that of wild type (WT). No changes in PON activity were observed with apoA-I cysteine substitution mutations in the C-terminal portion of the protein. The data suggest that, for optimal PON activity, coassembly of the enzyme onto nascent HDL is required and that the N-terminal region of apoA-I may be important in the assembly process.

Amino Acid Substitution↗
PubMed1994

Self-association and DNA binding of lambda cI repressor N-terminal domains reveal linkage between sequence-specific binding and the C-terminal cooperativity domain.

The effects of temperature, protons, and KCl on self-assembly and site-specific binding of lambda cI N-terminal domains with operator sites OR were studied to assess the roles of these domains in DNA binding and cooperativity of the natural system. Domain self-assembly was studied using sedimentation equilibrium while domain-OR interactions were analyzed by quantitative DNase footprint titration. The self-assembly reactions were modeled best as a monomer-dimer-tetramer stoichiometry. Compared with intact cI, the monomer-dimer assembly is energetically weak and is largely independent of pH and KCl. The van't Hoff enthalpy of dimerization was found to be large and positive (+ 10.8 kcal/mol), in sharp contrast to that of intact cI (i.e., -16.1 kcal/mol; Koblan & Ackers, 1991a), indicating that different driving forces dominate the respective assembly processes. The interactions of OR with N-terminal domains were noncooperative under all conditions studied. Binding at each site is accompanied by a negative enthalpy (large at site 1, small at sites 2 and 3). Identical values for salt release and proton absorption were found for the three sites. Comparisons with the analogous thermodynamic parameters from our previous studies indicate that N-terminal domains exhibit different linkages to pH, KCl, and T from those of intact cI-OR interactions. This implies that the domains do not act independently within the intact repressor. Since the linkage differences are dependent upon which site the proteins are binding, the C-terminal domain must play a role in repressor discrimination between specific sites.

Allosteric Regulation↗
PubMed1984

Physicochemical characterization of the 68 000-dalton protein of bovine neurofilaments.

The 68 000-dalton protein from bovine neurofilaments was purified by a combination of chromatography on DEAE-cellulose and on hydroxylapatite in buffers containing 8 M urea. Although the separation of this protein from the other proteins of the neurofilament appeared to be hampered by a mixed association of the several components, a nearly homogeneous product was obtained for study. Sedimentation equilibrium experiments in buffers containing 8 M urea showed the molecule to be a monomer with a molecular weight of 70 600 +/- 2000. Circular dichroic spectra taken under the same conditions gave no evidence of residual alpha-helix. Molecular sieve chromatography in 8 M urea on controlled-pore glass showed that the molecule eluted at an unexpectedly small volume. The small elution volume did not depend significantly on protein concentration and is unlikely to be the result of intermolecular association. Rather, the monomer probably has a conformation more rigid or extended than a classical random coil. When dialyzed into 0.01 M tris(hydroxymethyl)aminomethane/1 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid/0.1 mM dithioerythritol, pH 8.5, the protein does not assemble into filaments. Sedimentation velocity reveals that under these conditions it consists mainly of a 4.8S molecular species, containing few large particles; sedimentation equilibrium shows that it is composed of oligomers, the smallest present in significant concentration having a molecular weight approximately that of a trimer. Circular dichroism measurements lead to the interpretation that the molecule has refolded in this buffer into a structure that has approximately 55% alpha-helix. Assembly into filamentous particles resembling neurofilaments occurs when the protein is dialyzed against 0.1 M 2-(N-morpholino)ethane-sulfonic acid/0.1% beta-mercaptoethanol/1 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid/0.17 M NaCl, pH 6.5. We suggest that the oligomeric species present in 0.01 M tris(hydroxymethyl)aminomethane may frequently be present in solubilized preparations of intermediate filaments and may represent an intermediate in the assembly process.

Animals↗
PubMed1990

Spectropolarimetric analysis of the core histone octamer and its subunits.

The secondary structure of the calf thymus core histone octamer, (H2A-H2B-H3-H4)2, and its two physiological subunits, the H2A-H2B dimer and (H3-H4)2 tetramer, was analyzed by ORD spectropolarimetry as a function of temperature and solvent ionic strength within the ranges of these experimental parameters where assembly of the core histone octamer exhibits pronounced sensitivity. While the secondary structure of the dimer is relatively stable from 0.1 to 2.0 M NaCl, the secondary structure of the tetramer exhibits complex changes over this range of NaCl concentrations. Both complexes exhibit only modest responses to temperature changes. ORD spectra of very high and very low concentrations of stoichiometric mixtures of the core histones revealed no evidence of changes in the ordered structure of the histones as a result of the octamer assembly process at NaCl concentrations above 0.67 M, nor were time-dependent changes detected in the secondary structure of tetramer dissolved in low ionic strength solvent. The secondary structure of the chicken erythrocyte octamer dissolved in high concentrations of ammonium sulfate, including those of our crystallization conditions, was found to be essentially unchanged from that in 2 M NaCl when examined by both ORD and CD spectropolarimetry. The two well-defined cleaved products of the H2A-H2B dimer, cH2A-H2B and cH2A-cH2B, exhibited reduced amounts of ordered structure; in the case of the doubly cleaved moiety cH2A-cH2B, the reductions were so pronounced as to suggest marked structural rearrangements.

Ammonium Sulfate↗
PubMed1990

Intron-exon organization of the active human protein S gene PS alpha and its pseudogene PS beta: duplication and silencing during primate evolution.

The human protein S locus on chromosome 3 consists of two protein S genes, PS alpha and PS beta. Here we report the cloning and characterization of both genes. Fifteen exons of the PS alpha gene were identified that together code for protein S mRNA as derived from the reported protein S cDNAs. Analysis by primer extension of liver protein S mRNA, however, reveals the presence of two mRNA forms that differ in the length of their 5'-noncoding region. Both transcripts contain a 5'-noncoding region longer than found in the protein S cDNAs. The two products may arise from alternative splicing of an additional intron in this region or from the usage of two start sites for transcription. The intron-exon organization of the PS alpha gene fully supports the hypothesis that the protein S gene is the product of an evolutional assembling process in which gene modules coding for structural/functional protein units also found in other coagulation proteins have been put upstream of the ancestral gene of a steroid hormone binding protein. The PS beta gene is identified as a pseudogene. It contains a large variety of detrimental aberrations, viz., the absence of exon I, a splice site mutation, three stop codons, and a frame shift mutation. Overall, the two genes PS alpha and PS beta show between their exonic sequences 96.5% homology. Southern analysis of primate DNA showed that the duplication of the ancestral protein S gene has occurred after the branching of the orangutan from the African apes.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1979

Hemocyanin from the Australian freshwater crayfish Cherax destructor. Electron microscopy of native and reassembled molecules.

Examination and measurement of electron micrographs of negatively stained hemocyanin molecules from Cherax destructor show that the predominant aggregated forms, the 16S and 24S components, are typical structures for arthropod hexamers and dodecamers, respectively. In Cherax hemocyanin the hexamers are formed from the monomeric (Mr congruent to 75,000) subunits, M1 and M2, while the dodecamers contain in addition a dimeric (Mr congruent to 150,000) subunit, M3'. Studies of the composition of solutions of the subunits M1 and m2 to which calcium ions have been added at pH 7.8 show that, under these conditions, reassembly occurs to particles indistinguishable from native hexamers. It is noteworthy that dodecamers are not seen since this confirms the previous suggestion that incorporation of the dimeric subunit in the assembly process is necessary for their formation. The results obtained from Cherax hemocyanin are related to those of previous structural studies of arthropod hemocyanins. In particular, the possible controlling role of certain specific subunits in arthropod hemocyanin oligomers containing more than one kind of subunit is illustrated with a model for the Cherax dodecamer, in which the dimeric subunit is shared between the two halves of the molecule.

Animals↗
PubMed1975

Cooperative interactions in hybrids of aspartate transcarbamylase containing succinylated regulatory polypeptide chains.

Succinylated derivatives of the regulatory subunit of aspartate transcarbamylase of Escherichia coli were prepared by treating the intact enzyme with succinic anhydride followed by dissociation of the modified protein into catalytic and regulatory subunits which were separated by ion exchange chromatography. The succinylated regulatory subunits were used in hybridization experiments with native subunits to study the organization of the six regulatory polypeptide chains in the intact enzyme. Rapid mixing of succinylated and native regulatory subunits with native catalytic subunits yielded a four-membered hybrid set of reconstituted enzyme-like molecules; hence, the assembly process involves three regulatory combining units and the six regulatory polypeptide chains in the intact enzyme must be arranged as three dimeric subunits. When the modified and native regulatory subunits were incubated together for only brief periods (less than 1 min) followed by the addition of catalytic subunits, the resulting hybrid set was complex with no resolution of discrete species. Apparently the isolated regulatory dimers dissociate readily and reversibly into single polypeptide chains due to relatively weak intra-subunit bonding domains. In contrast, after reconstitution of enzyme-like molecules, the incorporated succinylated regulatory subunits did not exchange with free subunits. Enzyme-like molecules containing three extensively succinylated regulatory subunits show reduced binding of the inhibitor, CTP, and lack both the homotropic and heterotropic effects characteristic of native aspartate transcarbamylase. Preparations containing only slightly succinylated regulatory subunits showed only little inhibition by CTP and considerable cooperativity. The decrease in homotropic effects in these reconstituted molecules correlated with the reduction in the succinate-promoted change in the sedimentation coefficient. Reconstituted enzyme-like molecules containing regulatory subunits which had been extensively succinylated in the presence of CTP retained their binding capacity even though they were only slightly inhibited by CTP and exhibited reduced cooperativity. Hybrid molecules containing both native and succinylated regulatory subunits also possessed reduced allosteric behavior.

Aspartate Carbamoyltransferase↗
PubMed2003

Highly asymmetric interactions between globin chains during hemoglobin assembly revealed by electrospray ionization mass spectrometry.

Dynamics of bovine hemoglobin assembly was investigated by monitoring monomers/oligomers equilibria in solution with electrospray ionization mass spectrometry and circular dichroism spectroscopy. Intensities of ionic signals corresponding to various protein species (tetramers, dimers, heme-deficient dimers, as well as apo- and holo-monomers) were used to estimate relative fractions of these species in solution as a function of pH. The fraction of folded protein for each observed species was estimated based on charge-state distributions of corresponding ionic species in the mass spectra. The cumulative numbers (averaged across the entire protein population) were in good agreement with circular dichroism data at the Soret band and in the far-UV region, respectively. The mass spectral data confirm that hemoglobin dissociation involves a step where heme is first lost from the beta-chain of the alpha beta-dimer to form a heme-deficient dimeric species. This dimer dissociates further to produce a holo-alpha-chain and an apo-beta-chain. The former is tightly folded into a comparatively compact structure at neutral pH, while the latter always exhibits significant backbone disorder. Acidification of the protein solution to pH 4 leads to partial heme dissociation and significant increase of the backbone flexibility in the alpha-chains as well. Complete dissociation of the heme from the alpha-chains at a pH below 4 coincides with the total disappearance of the dimeric and tetrameric hemoglobin species from the mass spectra. The experimental data provide strong evidence that binding of a partially unstructured apo-beta-chain to a tightly folded holo-alpha-chain to form a heme-deficient dimer is the initial step of hemoglobin assembly. Such binding locks the beta-chain in a highly ordered conformation, which allows for an efficient heme acquisition, followed by docking of two hemoglobin dimers to form a tetrameric form of the protein. The asymmetry of the roles of the two chains in the assembly process is surprising, given a rather high sequence homology (ca. 43%) and highlights functional importance of intrinsic protein disorder. The study also demonstrates a tremendous potential of mass spectrometry as an analytical tool capable of elucidating protein interaction mechanisms in highly heterogeneous systems.

Animals↗