Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Assembly process”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 811 records · Page 45Linked to original sources
PubMed1991

Topoisomer heterogeneity of plasmid chromatin in living cells.

Previous investigations of topoisomer distributions of simian virus 40 (SV40) DNA from monkey cells have revealed that these circular mini-chromosomes, like relaxed, naked, closed circular DNA, exist as a Gaussian distribution of topoisomers. I have extended this comparison by measuring topoisomer distributions for a variety of plasmid episomes that are stably propagated in cells of the yeast Saccharomyces cerevisiae. The breadth of the topoisomer distributions for plasmid chromatin, including SV40, is approximately constant when normalized for DNA length, as is the breadth of distribution for naked DNA. However, the distributions for plasmid chromatin are substantially broader than those for the corresponding relaxed, naked DNAs. The breath is constant for plasmids differing in transcriptional activity, and varies only slightly between synchronized and unsynchronized populations of yeast cells, suggesting that variation in plasmid linking number with transcription or replication does not account for the observed heterogeneity in linking number. Topoisomer heterogeneity for plasmid chromatin in vivo may be due to heterogeneity in the number of nucleosomes on each plasmid, which could reflect either the nature of the assembly process or the dynamics of nucleosomes within the cell.

Cell Cycle↗
PubMed1988

The structure of tubes of bovine rotavirus nucleocapsid protein (VP6) assembled in vitro.

The structure of tubes of reassembled nucleocapsid protein (VP6) from bovine rotavirus (BRV) was determined using optical diffraction of electron micrographs. The tubes consist of a five-start helix of hexagons, with 38 hexagons per helix in a true repeat of three turns. The morphological subunits comprising the hexagons are probably elongated trimers. The structure of naturally occurring tubes (D. Chasey and J. Labram, 1983, J. Gen. Virol. 64, 863-872) was also examined and shown to be similar but not identical to that of tubes assembled in vitro. Considerations of the assembly process are discussed.

Animals↗
PubMed1982

Separable assembly of platelet pseudopodal and contractile cytoskeletons.

Unactivated platelets contain about 69% G actin and less than 10% of the contractile proteins in a cytoskeletal core resistant to extraction with 1% Triton X-100. Activation by thrombin leads, within 1 min, to the formation of pseudopodia and contractile gels, accompanied by the reduction of the G-actin content to about 22% and the development of cytoskeletal cores containing 70%-80% of the total actin and 60%-80% of the total myosin and actin-binding protein. Inhibition of pseudopodal formation by pretreatment with cytochalasin B before thrombin activation results in the loss of most of the actin-binding protein and about one third of the actin from the cytoskeletal core. Myosin incorporation and contractile gel formation are unaltered by this treatment. Conversely, activation with phorbol 12-myristate 13-acetate leads to pseudopodal but not contractile gel formation, with cytoskeletal cores containing mostly actin and actin-binding protein. These results demonstrate that there are separable cytoskeletal assembly processes in platelets for pseudopodal and contractile gel formation.

Actins↗
PubMed1988

Control of the yeast cell cycle is associated with assembly/disassembly of the Cdc28 protein kinase complex.

The Saccharomyces cerevisiae gene CDC28 encodes a protein kinase required for progression from G1 to S phase in the cell cycle. We present evidence that the active form of the Cdc28 protein kinase is a complex of approximately 160 kd containing an endogenous substrate, p40, and possibly other polypeptides. This complex phosphorylates p40 and exogenous histone H1 in vitro. Cell cycle arrest during G1 results in inactivation of the protein kinase accompanied by the disassembly of the complex. Furthermore, assembly of the complex is regulated during the cell cycle, reaching a maximum during G1. Partial complexes thought to be intermediates in the assembly process phosphorylate histone H1 but not p40. Addition of soluble factors to these partial complexes in vitro restores p40 phosphorylation and causes the complex to increase to the mature size. A model is presented in which p40 phosphorylation is required during G1 for cells to initiate a new cell cycle.

Cell Cycle↗
PubMed1992

Dynamic equilibria between subcomponents of C1, the first component of human complement.

C1r and C1s, the serine protease components of activated C1, form a tetramer in the presence of Ca2+. The stability of this tetramer is sufficient that its association with the third component, C1q, has been successfully treated as a reversible bimolecular equilibrium reaction [Siegel and Schumaker, Molec. Immun. 20, 53-66 (1983)]. We have used the fluorescence anisotropy (A) of fluorescein-labeled C1s (s*) to monitor assembly and subcomponent exchange in 0.15 mol/l NaCl, 0.001 mol/l Ca2+ 0.02 mol/l Tris, pH 7.4. Addition of q to r2s*2 causes a small but measurable delta A of 0.01-0.02. The response is too fast to measure at 37 degrees but can be readily followed at 4 degrees where t 1/2 = 0.6 min when [q] = [r2s*2] = 0.5 mumol/l. The increase in A can be readily reversed by dilution or by addition of unlabeled C1s. Slow incremental addition of q to a solution of r2s*2 produces a dose-dependent delta A from which stoichiometry and dissociation constants can be derived. Measurements of Kd as a function of temperature establish an inverse temperature dependence with delta H = -15 kcal/mol and a value of Kd = 0.031 mumol/l at 37 degrees (delta G = + 11, T delta S = -26 kcal/mol). Thus, the assembly process appears to be entropy-driven presumably due to the exclusion of structured water from protein-protein interfaces in the complex.

Complement C1r↗
PubMed1983

The genetic control of antibody formation.

Studies of the molecular biology of lymphoid cells have markedly increased our understanding of how millions of different antibodies can be synthesized by a single animal. To date, the most detailed understanding has been achieved for the mouse, primarily because of the relatively greater experimental availability of this species. These studies, as well as those involving other species, have shown that the complete genes for antibody polypeptide chains are assembled from disparate genetic elements which are originally widely separated in the genome. The assembly process itself, together with the coding information present in the germ line genetic elements, contributes to the diversity of structure (and thus combining specificities) shown by mature antibody molecules. Specifically, the diversity of structure characteristic of antibody variable regions is due to three distinct mechanisms: innate variability of germ line genes; mismatching of individual gene segments during their somatic rearrangement leading to junctional diversity; and somatic mutation in variable region genetic material during or after the rearrangement. These processes lead to the wide array of combining specificities that permit the humoral immune system of a mature animal to interact with essentially any non-self antigen which it encounters. Complex genetic rearrangements are also responsible for the class switching phenomenon long known to be characteristic of the humoral immune response. A form of homologous recombination between constant region genes, possibly mediated by specific "switching" enzymes, is now believed to be involved in this phenomenon. It is also currently believed that the restriction of gene rearrangement processes to one of the two possible chromosomes of a diploid pair in each cell is responsible for the phenomenon of allelic exclusion that has long been associated with the normal functioning of mammalian B-cells.

Alleles↗
PubMed1991

Fibrillar pattern of self-assembled and cell-assembled collagen: resemblance and analogy.

The geometrical characteristics of fibrillar organizations are studied by electron microscopy in structures obtained in vitro in cell-free assembled collagen gels, and in vivo in dermal tracts of anuran skin. We analyze several characteristics of the fibrils including the diameter, the outline, the curvature and the extrafibrillar space. We analyze also the variation of fibrillar orientation (twist) in longitudinal and transverse thin sections of these structures. The results are compared in the Discussion to determine to what extent these fibrillar patterns are similar to liquid crystalline organizations and to what extent they result from a self-assembly or a cell-assembly process.

Animals↗
PubMed1996

Liposome formation in microgravity.

Liposomes are artificial vesicles with a phospholipid bilayer membrane. The formation of liposomes is a self-assembly process that is driven by the amphipathic nature of phospholipid molecules and can be observed during the removal of detergent from phospholipids dissolved in detergent micelles. As detergent concentration in the mixed micelles decreases, the non-polar tail regions of phospholipids produce a hydrophobic effect that drives the micelles to fuse and form planar bilayers in which phospholipids orient with tail regions to the center of the bilayer and polar head regions to the external surface. Remaining detergent molecules shield exposed edges of the bilayer sheet from the aqueous environment. Further removal of detergent leads to intramembrane folding and membrane folding and membrane vesiculation, forming liposomes. We have observed that the formation of liposomes is altered in microgravity. Liposomes that were formed at 1-g did not exceed 150 nm in diameter, whereas liposomes that were formed during spaceflight exhibited diameters up to 2000 nm. Using detergent-stabilized planar bilayers, we determined that the stage of liposome formation most influenced by gravity is membrane vesiculation. In addition, we found that small, equipment-induced fluid disturbances increased vesiculation and negated the size-enhancing effects of microgravity. However, these small disturbances had no effect on liposome size at 1-g, likely due to the presence of gravity-induced buoyancy-driven fluid flows (e.g., convection currents). Our results indicate that fluid disturbances, induced by gravity, influence the vesiculation of membranes and limit the diameter of forming liposomes.

Convection↗
PubMed1994

Direct shape determination of ribosomal proteins in solution and within the ribosome by means of neutron scattering.

Following the 'strategy of the glassy ribosome' single protonated ribosomal proteins (r-proteins) were reconstituted into deuterated 50S subunits of Escherichia coli. The deuteration of both rRNA and r-proteins were individually adjusted to such a degree that the ribosomal matrix appeared nearly homogeneous with respect to coherent neutron scattering and had a scattering density equivalent to a D2O solution of about 90%. Neutron scattering of ribosomal subunits was recorded in reconstitution buffer containing three different concentrations of D2O around 90% D2O (contrast variation). The signal-to-noise ratio achieved allowed us to make a direct determination of the radii of gyration of r-proteins within the 50S subunit and thus provides the first information relating to the shape of these proteins in situ. We present the radii of gyration of 11 r-proteins incorporated into 50S subunits and of 9 isolated r-proteins in solution. In addition, the data concerning the overall dimensions of the r-proteins we report on indicate that conformational changes of at least two individual r-proteins occur during the assembly process of the ribosome.

Escherichia coli↗
PubMed1986

Tunicamycin and papulacandin B inhibit incorporation of specific mannoproteins into the wall of Candida albicans regenerating protoplasts.

Regeneration of Candida albicans protoplasts began with the formation of a chitin network which was complemented after a lag of about 60 min by the deposition of beta-glucan. Proteins were incorporated early to the growing structure, beginning with the mannoproteins which are kept in place by non-covalent bonds. Incorporation of covalently linked mannoproteins took place only after deposition of glucan. The incorporation of these mannoproteins did not occur when protoplasts were incubated with papulacandin B which inhibited glucan formation, or with tunicamycin which blocked N-glycosylation of mannoproteins. In the presence of papulacandin B, large amounts of native mannoproteins accumulated in the medium. However, in the presence of tunicamycin, the large mannoprotein material found was of smaller apparent molecular weight, suggesting that it was deficient in glycosylation. Partially regenerated walls were able to incorporate 'in vitro' non-covalently bound mannoproteins, indicating that some components of very large cellular structures such as walls are capable of being articulated by a self-assembly process.

Aminoglycosides↗
PubMed1991

Nucleotide hydrolysis in cytoskeletal assembly.

Two major polymers of the cytoskeleton, actin filaments and microtubules, are assembled with expenditure of energy: the ATP/GTP tightly bound to actin/tubulin is irreversibly hydrolyzed to ADP/GTP during the assembly process, and liberation of Pi in the medium occurs subsequent to the incorporation of subunits in the polymer. Pi release acts as a switch, causing the destabilization of protein-protein interactions in the polymer, therefore regulating the dynamics of these fibres. An understanding of this regulation in vivo requires that progress be made in four areas: the chemistry of the NTPase reaction; the structure of the intermediates in nucleotide hydrolysis and the nature of the conformational switch; the regulation of parameters involved in dynamic instability of microtubules; and the possible involvement of nucleotide hydrolysis in the macroscopic organization of these polymers in highly concentrated solutions, compared with the simple case of a equilibrium polymers. Progress made along these lines will define trends for future investigation.

Actins↗
PubMed1996

Ability of various members of the hsp70 family of chaperones to promote assembly of the glucocorticoid receptor into a functional heterocomplex with hsp90.

To be in a conformation that binds steroid, the hormone-binding domain of the glucocorticoid receptor (GR) must be bound to the 90 kDa heat shock protein (hsp90). Rabbit reticulocyte lysate contains a protein chaperone system that assembles the receptor into a heterocomplex with hsp90 and converts it from a non-steroid-binding to a steroid-binding form. Assembly of the GR-hsp90 heterocomplex requires hsp70, and in this work we examine the activities of four members of the hsp70 protein family in GR-hsp90 heterocomplex assembly. Rabbit reticulocyte lysate was depleted of hsp70 by passing it through a column of ATP agarose, resulting in the inactivation of its GR-hsp90 heterocomplex assembly activity. Addition of purified animal (mouse) or plant (wheat germ) hsp70 to the hsp70-depleted lysate permits assembly of a GR-hsp90 heterocomplex with a high affinity steroid binding site. However, purified hsp70 homologues from bacteria (DnaK) or the endoplasmic reticulum (BiP) do not promote heterocomplex formation, despite the fact that both DnaK and BiP bind to the GR in the assay system. When added to whole (i.e. hsp70-containing) reticulocyte lysate, DnaK and BiP inhibit GR-hsp90 heterocomplex assembly. Wheat germ lysate forms a heterocomplex between mouse GR and plant hsp90, but the addition of purified rabbit hsp70 to the wheat germ lysate does not increase the amount of receptor-wheat hsp90 complex produced, despite the fact that the rabbit hsp70 binds to the GR when it is added to the wheat chaperone system. The conclusion is that binding of hsp70 to receptors does not necessarily reflect a physiologically meaningful interaction. When native receptor heterocomplexes isolated from cytosols contain hsp70, it is likely that the hsp70-bound receptors represent a minority of receptors that have not yet proceeded fully through the receptor heterocomplex assembly process, which includes the dissociation of hsp70 after the binding of hsp90.

Animals↗
PubMed1996

Destroying retroviruses from within.

One strategy for neutralizing retroviral infectivity is to induce the incorporation of lethal fusion proteins, such as capsid protein-nuclease fusions, into the virion during the normal viral assembly process. Genes encoding such antiviral fusion proteins must be nontoxic to the host, lethal to the virus, and must be efficiently delivered to, and expressed in, appropriate target cells.

Antiviral Agents↗
PubMed1993

Beat the clock: paradigms for NTPases in the maintenance of biological fidelity.

Kinetic proofreading is a strategy used by cells to reduce the number of errors made when replicating and expressing genetic information. Recent advances in mRNA splicing suggest a variation on the theme of previously described kinetic proofreading mechanisms, which may apply to other multicomponent assembly processes in the cell.

Acid Anhydride Hydrolases↗
PubMed2005

Spectrins and the Golgi.

Several isoforms of spectrin membrane skeleton proteins have been localized to the Golgi complex. Golgi-specific membrane skeleton proteins associate with the Golgi as a detergent-resistant cytoskeletal structure that likely undergoes a dynamic assembly process that accommodates Golgi membrane dynamics. This review discusses the potential roles for this molecule in Golgi functions. In particular, it will focus on a recently identified distant cousin to conventional erythroid spectrin variously named Syne-1, Nesprin, myne, Enaptin, MSP-300, and Ank-1. Syne-1 has the novel ability to bind to both the Golgi and the nuclear envelope, a property that raises several intriguing and novel insights into Golgi structure and function. These include (1) the facilitation of interactions between Golgi and transitional ER sites on the nuclear envelope of muscle cells, and (2) an ability to impart localized specificity to the secretory pathway within large multinucleate syncytia such as skeletal muscle fibers.

Animals↗
PubMed2005

Probing the pressure-temperature stability of amyloid fibrils provides new insights into their molecular properties.

A number of medical disorders, including Alzheimer's disease and type II diabetes, is characterised by the deposition of amyloid fibrils in tissue. The insolubility and size of the fibrils has largely precluded the determination of their structures at high resolution. Studies probing the stability of amyloid fibrils can reveal which non-covalent interactions are important in the formation and maintenance of the fibril structure. In particular, we review here the use of high hydrostatic pressure and high temperature as perturbation techniques. In general, small aggregates formed early in the assembly process can be dissociated by high pressure, but mature amyloid fibrils are highly pressure stable. This finding suggests that a temporal transition occurs during which side chain packing and hydrogen bond formation are optimised, whereas the hydrophobic effect and electrostatic interactions play a dominant role in the early stages of the aggregation. High temperatures, however, can disrupt most aggregates. Though the observed stability of amyloid fibrils is not unique to these structures, the notion that amyloid fibrils can represent the global minimum in free energy is supported by this type of investigations. Some implications regarding the nature of toxic species, associated with at least many of the amyloid disorders, and recently proposed structural models are discussed.

Alzheimer Disease↗
PubMed2005

Covalent attachment of glucose oxidase to an Au electrode modified with gold nanoparticles for use as glucose biosensor.

A feasible method to fabricate glucose biosensor was developed by covalent attachment of glucose oxidase (GOx) to a gold nanoparticle monolayer modified Au electrode. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) of ferrocyanide followed and confirmed the assemble process of biosensor, and indicated that the gold nanoparticles in the biosensing interface efficiently improved the electron transfer between analyte and electrode surface. CV performed in the presence of excess glucose and artificial redox mediator, ferrocenemethanol, allowed to quantify the surface concentration of electrically wired enzyme (Gamma(E)(0)) on the basis of kinetic models reported in literature. The Gamma(E)(0) on proposed electrode was high to 4.1 x 10(-12) mol.cm(-2), which was more than four times of that on electrode direct immobilization of enzyme by cystamine without intermediate layer of gold nanoparticles and 2.4 times of a saturated monolayer of GOx on electrode surface. The analytical performance of this biosensor was investigated by amperometry. The sensor provided a linear response to glucose over the concentration range of 2.0 x 10(-5)-5.7 x 10(-3) M with a sensitivity of 8.8 microA.mM(-1).cm(-2) and a detection limit of 8.2 microM. The apparent Michaelis-Menten constant (K(m)(app)) for the sensor was found to be 4.3 mM. In addition, the sensor has good reproducibility, and can remain stable over 30 days.

Biosensing Techniques↗
PubMed2005

Size-controlled hydroxyapatite nanoparticles as self-organized organic-inorganic composite materials.

This paper presents some results concerning the size-controlled hydroxyapatite nanoparticles obtained in aqueous media in a biopolymer matrix from soluble precursors salts. Taking the inspiration from nature, where composite materials made of a polymer matrix and inorganic fillers are often found, e.g. bone, shell of crustaceans, shell of eggs, etc., the feasibility on making composite materials containing chitosan and nanosized hydroxyapatite was investigated. A stepwise co-precipitation approach was used to obtain different types of composites by means of different ratio between components. The synthesis of hydroxyapatite was carried out in the chitosan matrix from calcium chloride and sodium dihydrogenphosphate in alkaline solutions at moderate pH of 10-11 for 24 h. Our research is focused on studying and understanding the structure of this class of composites, aiming at the development of novel materials, controlled at the nanolevel scale. The X-ray diffraction technique was employed in order to study the kinetic of hydroxyapatite formation in the chitosan matrix as well as to determine the HAp crystallite sizes in the composite samples. The hydroxyapatite synthesized using this route was found to be nano-sized (15-50 nm). Moreover, applying an original approach to analyze the (002) XRD diffraction peak profile of hydroxyapatite by using a sum of two Gauss functions, the bimodal distribution of nanosized hydroxyapatite within the chitosan matrix was revealed. Two types of size distribution domains such as cluster-like (between 200 and 400 nm), which are the habitat of ''small'' hydroxyapatite nanocrystallites and scattered-like, which are the habitat of ''large'' hydroxyapatite nanocrystallites was probed by TEM and CSLM. The structural features of composites suggest that self-assembly processes might be involved. The composites contain nanosized hydroxyapatite with structural features close to those of biological apatites that make them attractive for bone tissue engineering applications.

Biocompatible Materials↗