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PubMed1997

[Huntington disease].

The gene responsible for Huntington's disease produces a protein with a molecular weight of about 350k, designated huntingtin. We identified both wild-type and mutant huntingtin in the brain and lymphoblastoid cells. Although the function of huntingtin is still unknown, several associated proteins such as HAP1, Ubiquitin-conjugating enzyme, HIP1 and glyceraldehyde-3-phosphate-dehy dorogenase (GAPDH) were reported. We found the huntingtin can associate in vitro with microtubules. Through the process of assembly and disassembly of microtubules, both wild-type and mutant huntingtin associate with microtubules to almost the same degree. The results suggest that huntingtin may have a role in intracellular organelle transport or axonal transport by its association with microtubules. The functional disturbance by expanded polyglutamine stretch may modify the feature of the disease.

Humans↗
PubMed2005

Process outgrowth in oligodendrocytes is mediated by CNP, a novel microtubule assembly myelin protein.

Oligodendrocytes (OLs) extend arborized processes that are supported by microtubules (MTs) and microfilaments. Little is known about proteins that modulate and interact with the cytoskeleton during myelination. Several lines of evidence suggest a role for 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) in mediating process formation in OLs. In this study, we report that tubulin is a major CNP-interacting protein. In vitro, CNP binds preferentially to tubulin heterodimers compared with MTs and induces MT assembly by copolymerizing with tubulin. CNP overexpression induces dramatic morphology changes in both glial and nonglial cells, resulting in MT and F-actin reorganization and formation of branched processes. These morphological effects are attributed to CNP MT assembly activity; branched process formation is either substantially reduced or abolished with the expression of loss-of-function mutants. Accordingly, cultured OLs from CNP-deficient mice extend smaller outgrowths with less arborized processes. We propose that CNP is an important component of the cytoskeletal machinery that directs process outgrowth in OLs.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1998

A comparison of worker exposure to inhalable and total dust, inorganic arsenic, and borates using two types of particulate sampling assemblies in a borate mining and processing facility.

This study describes a comparison of worker exposure to total and inhalable dust, inorganic As, and borates using two types of particulate sampling assemblies as part of a comprehensive industrial hygiene evaluation in a borate mining and processing facility. Employees were segmented into similar exposure groups (SEG) based on work location within the facility, job classification, and type of chemical agent. Approximately 10% of the employees from each SEG wore two personal sampling devices simultaneously for the purpose of collecting total and inhalable particulate fractions using a closed face, 37-mm mixed cellulose ester matched-weight filters (MMW), and Institute of Occupational Medicine (IOM) sampling assembly. Sample results indicated that the IOM concentrations were consistently higher than the corresponding MMW concentrations for all three agents. An analysis was performed to investigate a relationship between MMW and IOM. The data revealed correlation coefficient values of 0.72, 0.82, and 0.84 for total dust (n = 197), inorganic As (n = 137), and borates (n = 194), respectively. These positive correlation coefficients indicate that the IOM and MMW measurements are consistent with each other, and can be used for predicting exposure levels. The total dust and borate large mean ratios should be considered in developing inhalable fraction-based regulatory standards.

Air Pollutants, Occupational↗
PubMed1989

Isolation of point mutations that affect the folding of the H chain of human ferritin in E.coli.

We have approached the problem of folding and assembly of the heavy (H) chain of human ferritin by isolating point mutations that affect this process. Apoferritin is an ideal model system to approach the problem of protein folding and assembly into multimeric structures. We have developed a recombinant hybrid molecule that allows us to select for ferritin mutants in which the folding-assembly process is altered or completely impaired. The selection procedure is based on a recombinant protein which consists of a fusion between the H chain of human ferritin and the alpha-peptide of beta-galactosidase. In the wild type situation, the alpha-peptide domain is segregated inside the apoferritin shell upon assembly and is unable to interact with the substrate and perform its enzymic function. We show that by selecting for mutations that restore beta-galactosidase activity we are able to identify ferritin mutations that affect the folding-assembly process. The selective procedure was applied to the analysis of the amino acid side chains that are important for the attainment of the correct conformation of the carboxy-terminal E helix in the 4-fold axis.

Amino Acid Sequence↗
PubMed1995

A new point of view on the problem of G-protein aggregation. Assembly-disassembly of tubulin as an oscillation process.

Basic principles of nonlinear thermodynamics are used to develop a theory of tubulin assembly. The presence of GTP/GDP switch provides different ways for tubulin aggregation. The GTP-tubulin assembly results in determining the regime of limit cycle favoring growth of tubulin spiral with the formation of microtubules. The GDP-tubulin assembly, on the contrary, results in forming double rings. The difference between the GTP- and GDP-tubulin assemblies is that the former is a dynamical oscillation process while the latter is no more than a transition from a weakly nonequilibrium point to the equilibrium state along a thermodynamical path. Corresponding equations for the GTP- and GDP-tubulin assemblies are proposed.

GTP-Binding Proteins↗
PubMed2000

Involvement of ezrin/moesin in de novo actin assembly on phagosomal membranes.

The current study focuses on the molecular mechanisms responsible for actin assembly on a defined membrane surface: the phagosome. Mature phagosomes were surrounded by filamentous actin in vivo in two different cell types. Fluorescence microscopy was used to study in vitro actin nucleation/polymerization (assembly) on the surface of phagosomes isolated from J774 mouse macrophages. In order to prevent non-specific actin polymerization during the assay, fluorescent G-actin was mixed with thymosin beta4. The cytoplasmic side of phagosomes induced de novo assembly and barbed end growth of actin filaments. This activity varied cyclically with the maturation state of phagosomes, both in vivo and in vitro. Peripheral membrane proteins are crucial components of this actin assembly machinery, and we demonstrate a role for ezrin and/or moesin in this process. We propose that this actin assembly process facilitates phagosome/endosome aggregation prior to membrane fusion.

Actins↗
PubMed1992

Processing of the herpes simplex virus assembly protein ICP35 near its carboxy terminal end requires the product of the whole of the UL26 reading frame.

The herpes simplex virus (HSV) type 1 assembly protein ICP35 consists of a family of polypeptides, ranging in molecular weight from about 45,000-39,000. The lower molecular weight forms of ICP35 are derived from the higher molecular weight species by slow post-translational modification. The reading frame of gene UL26 and the region within this gene which exhibited homology to the cytomegalovirus assembly protein, the analogous protein to ICP35, were expressed separately under immediate-early (IE) gene regulation in a HSV vector containing a temperature-sensitive mutation in the major transcriptional regulator Vmw175. Monoclonal antibody specific for ICP35 immunoprecipitated several polypeptides with molecular weights around 75,000 from extracts of cells infected with a recombinant expressing the IE gene UL26 at the nonpermissive temperature (NPT). These results suggested that the UL26 gene specified a protein distinct from ICP35 but which had some antigenic sites in common with ICP35. In extracts of cells infected at the NPT with a recombinant expressing only the carboxy terminal half of UL26 coding sequences, the monoclonal antibody immunoprecipitated large amounts of the high molecular weight forms of ICP35. The lower molecular weight processed forms of ICP35, however, were not detectable. When cells were coinfected with both recombinants ICP35 was processed to its lower molecular weight forms. This processing step, which occurred near the carboxy terminus of ICP35, was not dependent on capsid formation. The work, together with previous information on the processing of the CMV assembly protein, suggests that UL26 product may be a protease.

Animals↗
PubMed2002

The conserved central domain of yeast U6 snRNA: importance of U2-U6 helix Ia in spliceosome assembly.

In the pre-mRNA processing machinery of eukaryotic cells, U6 snRNA is located at or near the active site for pre-mRNA splicing catalysis, and U6 is involved in catalyzing the first chemical step of splicing. We have further defined the roles of key features of yeast U6 snRNA in the splicing process. By assaying spliceosome assembly and splicing in yeast extracts, we found that mutations of yeast U6 nt 56 and 57 are similar to previously reported deletions of U2 nt 27 or 28, all within yeast U2-U6 helix Ia. These mutations lead to the accumulation of yeast A1 spliceosomes, which form just prior to the Prp2 ATPase step and the first chemical step of splicing. These results strongly suggest that, at a late stage of spliceosome assembly, the presence of U2-U6 helix Ia is important for promoting the first chemical step of splicing, presumably by bringing together the 5' splice site region of pre-mRNA, which is base paired to U6 snRNA, and the branchsite region of the intron, which is base paired to U2 snRNA, for activation of the first chemical step of splicing, as previously proposed by Madhani and Guthrie [Cell, 1992, 71: 803-817]. In the 3' intramolecular stem-loop of U6, mutation G81C causes an allele-specific accumulation of U6 snRNP. Base pairing of the U6 3' stem-loop in yeast spliceosomes does not extend as far as to include the U6 sequence of U2-U6 helix Ib, in contrast to the human U6 3' stem-loop structure.

Base Sequence↗
PubMed2003

Matchmaking and species marriage: a game-theory model of community assembly.

Theories developed to explain the processes that govern the assembly and composition of natural plant communities can be divided into two broad categories. Niche-based theories propose that coevolutionary changes among species lead to character divergence (displacement), which allows for coexistence by partitioning resources among species. In contrast, ecological-drift theories propose that species diversity results from a balance of migration, speciation, and extinction, with little microevolutionary change. We use a game-theory model to reconcile drift and niche perspectives by developing a theory of species "marriage." Initially, ecological drift may determine which species encounter each other in a competitive arena. Once species come into contact, historical niche development as a result of prior coevolutionary molding of competitive ability determines which species may coexist. The model shows that only species that display the well-known tradeoff between seed size and competitive ability as a result of past competitive histories comply with the requisite for mutual evolutionary stability. Mutual evolutionary stability of competitive ability tends to make reproductive outputs more similar between species, increasing the chances of ecological equilibrium, i.e., the coexistence of species competing for a single resource. Moreover, mutual evolutionary stability guarantees that such an ecological equilibrium will be stable. The species-marriage model predicts that two or more plant species will coexist indefinitely (i.e., "marry") when their difference in seed size, their densities, and the resource availability obey a specific quantitative relation. For example, when resource availability is high, married species should be characterized by a greater asymmetry in seed size than when resource availability is low. Thus, in the species-marriage model, competition can shape the detailed properties of communities without violating the postulates of ecological-drift theory.

Ecology↗
PubMed2005

Histone H4 lysine 91 acetylation a core domain modification associated with chromatin assembly.

The acetylation of the NH2-terminal tail of histone H4 by type B histone acetyltransferases (HATs) is involved in the process of chromatin assembly. Histone H4 associated with a nuclear type B HAT complex contains modifications in its globular core domain as well. In particular, acetylation was found at lysine 91. A mutation that alters this residue, which lies in the interface between histone H3/H4 tetramers and H2A/H2B dimers, confers phenotypes consistent with defects in chromatin assembly such as sensitivity to DNA damaging agents and derepression and alteration of silent chromatin structure. In addition, this mutation destabilizes the histone octamer, leading to defects in chromatin structure. These results indicate an important role for histone modifications outside the NH2-tail domains in the processes of chromatin assembly, DNA repair, and transcriptional silencing.

Acetylation↗
PubMed1997

Viscosity and elasticity during collagen assembly in vitro: relevance to matrix-driven translocation.

In order to better understand the gelation process associated with collagen assembly, and the mechanism of the in vitro morphogenetic phenomenon of "matrix-driven translocation" [S.A. Newman et al. (1985) Science, 228, 885-889], the viscosity and elastic modulus of assembling collagen matrices in the presence and absence of polystyrene latex beads was investigated. Viscosity measurements at very low shear rates (0.016-0.0549 s(-1)) were performed over a range of temperatures (6.9-11.5 degrees C) in a Couette viscometer. A magnetic levitation sphere rheometer was used to measure the shear elastic modulus of the assembling matrices during the late phase of the gelation process. Gelation was detected by the rapid increase in viscosity that occurred after a lag time tL that varied between O and approximately 500 s. After a rise in viscosity that occurred over an additional approximately 500 s, the collagen matrix was characterized by an elastic modulus of the order of several Pa. The lag time of the assembly process was relatively insensitive to differences in shear rate within the variability of the sample preparation, but was inversely proportional to the time the sample spent on ice before being raised to the test temperature, for test temperatures > 9 degrees C. This suggests that structures important for fibrillogenesis are capable of forming at 0 degrees C. The time dependence of the gelation process is well-described by an exponential law with a rate constant K approximately 0.1 s(-1). Significantly, K was consistently larger in collagen preparations that contained cell-sized polystyrene beads. From these results, along with prior information on effective surface tension differences of bead-containing and bead-lacking collagen matrices, we conclude that changes in matrix organization contributing to matrix-driven translocation are initiated during the lag phase of fibrillogenesis when the viscosity is < or = 0.1 Poise. The phenomenon may make use of small differentials in viscosity and/or elasticity, resulting from the interaction of the beads with the assembling matrix. These properties are well described by standard models of concentrated solutions.

Animals↗
PubMed2025

The role of stochasticity in fungal community assembly: explaining apparent stochasticity with field experiments.

Stochasticity is a main process in community assembly. However, experimental studies rarely target stochasticity in natural communities, and hence experimental validation of stochasticity estimates in observational studies is lacking. Here, we combine experimental and observational data to unravel the role of stochasticity in the assembly of wood-inhabiting fungi. We carried out a replicated field experiment where the natural colonization of a focal fungal species was simulated through inoculation, and the local fungal communities were monitored through DNA metabarcoding before and after the inoculations. The amount of stochasticity in fungal colonization was less pronounced than expected from the amount of unpredictability in observational data, suggesting that stochasticity may play a smaller role in fungal occurrence than previously anticipated, or that it may be a stronger influence in the dispersal and establishment phases than in colonization per se. Stochasticity was more prominent in the initial phase of community succession, with the earliest successional stage involving a higher level of stochasticity than the later stage after 2 years. We conclude that experimentally measuring the role of stochasticity in community assembly is feasible for species-rich communities under natural conditions and highlight the importance of experimentally testing the accuracy of stochasticity estimates based on observational data.

Stochastic Processes↗
PubMed2002

Cell-cell interactions in primordial follicle assembly and development.

Two critical processes in ovarian biology are the assembly of the primordial follicles early in development and then the subsequent development and transition of the primordial follicle to the primary follicle. These processes directly effect the number of oocytes available to a female throughout her reproductive life. Once the pool of primordial follicles is depleted a series of physiological changes known as menopause begins in humans. The inappropriate coordination of these processes contributes to ovarian pathologies such as premature ovarian failure. Studies demonstrate primordial follicle assembly and development are coordinated by locally produced paracrine and autocrine factors. Factors have been identified that influence follicular assembly such as neurotropins. Several local factors that promote the primordial to primary follicle transition have also recently been identified. These include growth factors such as kit-ligand, leukemia inhibitory factor and basic fibroblast growth factor. Interestingly, recent studies demonstrate Müllerian inhibitory substance appears to inhibit the primordial to primary follicle transition. Therefore, observations suggest a mechanism for both positive and negative control of the primordial to primary follicle transition. The studies reviewed regarding the control of primordial follicle assembly and the primordial to primary follicle transition help elucidate these poorly understood aspects of ovarian biology.

Animals↗
PubMed2005

Human mitochondrial complex I assembly is mediated by NDUFAF1.

Complex I (NADH:ubiquinone oxidoreductase) is the largest multiprotein enzyme of the oxidative phosphorylation system. Its assembly in human cells is poorly understood and no proteins assisting this process have yet been described. A good candidate is NDUFAF1, the human homologue of Neurospora crassa complex I chaperone CIA30. Here, we demonstrate that NDUFAF1 is a mitochondrial protein that is involved in the complex I assembly process. Modulating the intramitochondrial amount of NDUFAF1 by knocking down its expression using RNA interference leads to a reduced amount and activity of complex I. NDUFAF1 is associated to two complexes of 600 and 700 kDa in size of which the relative distribution is altered in two complex I deficient patients. Analysis of NDUFAF1 expression in a conditional complex I assembly system shows that the 700 kDa complex may represent a key step in the complex I assembly process. Based on these data, we propose that NDUFAF1 is an important protein for the assembly/stability of complex I.

Cell Fractionation↗
PubMed2005

Synthetic micelle sensitive to IR light via a two-photon process.

A micellar assembly of molecules constituted of poly(ethylene glycol) as the hydrophilic component and 2-diazo-1,2-naphthoquinone as the hydrophobic component was shown to be destroyed in a two-photon photoreaction triggered by infrared light with release of an encapsulated fluorescent probe molecule.

Drug Delivery Systems↗
PubMedDate not supplied

Centrosome inheritance in insects: fertilization and parthenogenesis.

Centrosome biogenesis is unclear, although much structural and biochemical research has been performed in several experimental systems. An alternative model to study the assembly of functional centrosomes could be the process of zygotic centrosome formation at the beginning of embryonic development. Although it seems obvious that the sperm cell provides the centrosome at fertilization, some pieces of evidence are not in line with this point of view and give controversial results. Such an analysis could provide useful information if applied to a large variety of organisms. Since insects are a highly diverse group of organisms they provide a variety of models in which to study the process of centrosome reconstitution during fertilization. Moreover, many insect species reproduce by parthenogenesis, a special mode of reproduction in which embryonic development occurs without male contribution. Studies of unfertilized parthenogenetic eggs may therefore teach us much about the process of centrosome assembly in the absence of preexisting centrioles.

Animals↗
PubMed2003

Assembly of amelogenin proteolytic products and control of octacalcium phosphate crystal morphology.

The formation of enamel apatite crystals involves extracellular molecular events among which matrix assembly, interactions with growing crystals, and protein processing and removal are the subject of numerous investigations. Following the description of amelogenin nanospheres and the evidence for their presence in vivo as the principal structural component of developing dental enamel, we have focused our studies on investigating at the molecular level the process of nanosphere assembly and evaluating the effects of amelogenin on crystal growth and morphology. This paper is a short review of our recent studies with a focus on the assembly of amelogenin proteolytic products and their modulating effect on octacalcium phosphate (OCP) crystal morphology. In addition, we report that incorporation of amelogenins into 10% gelatin gel does not affect diffusion of calcium. This remarkable finding indicates that the observed modulation effect by amelogenin on OCP crystal morphology is not due to alteration of calcium diffusion into the gels but is the result of direct amelogenin-mineral interactions.

Amelogenin↗
PubMed1982

A bacteriophage T4 DNA packaging related DNA-dependent ATPase-endonuclease.

A new bacteriophage T4-induced DNA-dependent ATPase-endonuclease was purified to essential homogeneity from an extract of late infected Escherichia coli. Both DNA-dependent ATPase and endonuclease activities co-chromatograph, co-sediment, and have been renatured from a single 43-kilodalton protein eluted following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that both activities are exerted by one multifunctional protein. Duplex, single-stranded, and supercoiled DNAs are all effective activators of the high specific activity ATPase which produces ADP and inorganic PO4. The enzyme displays a broad specificity towards the nucleoside and deoxynucleoside triphosphates, and the ATPase activity is strongly inhibited by DNA-intercalating compounds. The endonuclease appears to be most active on supercoiled DNA, producing double-stranded breaks in duplex DNA, and does not require nucleoside triphosphates. An antiserum against the purified enzyme immunoprecipitated it, inhibited its ATPase activity, and also precipitated from extracts a T4-induced protein of Mr = 43,000. This antigen was not found in uninfected E. coli, or following a gene 55am mutant (late protein synthesis defective) infection, and was not detected following infection with T4 amber mutants of any early capsid protein gene which blocks T4 head protein cleavage in vivo. In a pulse-chase experiment, the radioactive antigen was not found following a pulse of radioactive amino acids, but appeared after a chase with excess nonradioactive amino acids. The enzyme-related antigen is apparently produced by cleavage of a precursor by the T4 head assembly proteinase which processes a number of prohead proteins. These processing reactions are dependent in vivo upon assembly of the prohead and are required for its maturation. The evidence suggests that this enzyme functions in head assembly and DNA packaging, and originates as the cleavage product of a prohead precursor protein.

DNA, Viral↗