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PubMed1987

Biosynthesis of four rat liver mitochondrial acyl-CoA dehydrogenases: in vitro synthesis, import into mitochondria, and processing of their precursors in a cell-free system and in cultured cells.

The synthesis, translocation, processing, and assembly of rat liver short chain acyl-CoA, medium chain acyl-CoA, long chain acyl-CoA, and isovaleryl-CoA dehydrogenases were studied. These four acyl-CoA dehydrogenases are homotetrameric flavoproteins which are located in the mitochondrial matrix. They were synthesized in a cell-free rabbit reticulocyte lysate system, programmed by rat liver polysomal RNA, as precursor polypeptides which are 2-4 kDa larger than their corresponding mature subunits (Mr 41,000-45,000). When the radiolabeled precursors were incubated with intact rat liver mitochondria, they appeared to bind tightly to the mitochondrial outer membrane. At this stage they were completely susceptible to the action of exogenous trypsin. The precursors bound to mitochondria at 0 degrees C were translocated into the mitochondria and processed when the temperature was raised to 30 degrees C. No reaction occurred when the temperature was kept at 0 degrees C, however, suggesting that the binding of the precursors is temperature independent while the subsequent steps of the pathway are energy dependent. Indeed, the translocation reaction was inhibited by compounds such as dinitrophenol and rhodamine 6G which inhibit mitochondrial energy metabolism. The newly imported (mature) enzymes were inaccessible to the proteolytic action of added trypsin. The processing of the precursors to mature subunits was proteolytically carried out in the mitochondrial matrix, and the processed mature subunits mostly assembled to their respective tetrameric forms. Newly synthesized larger precursors of each of the four acyl-CoA dehydrogenases were recovered from intact, cultured Buffalo rat liver cells in the presence of dinitrophenol. When dinitrophenol was removed in a pulse-chase protocol, the accumulated precursors were rapidly (t1/2 3-5 min) converted to their corresponding mature subunits. On the other hand, when the chase was performed in the presence of the inhibitor, the labeled precursors disappeared with t1/2 of greater than 4 h for long chain acyl-CoA dehydrogenase and 1-2 h for the other three enzyme precursors.

Acyl-CoA Dehydrogenase, Long-Chain↗

PubMed1977

Guanosinetriphosphatase activity of tubulin associated with microtubule assembly.

Tubulin, purified by cycles of assembly followed by phosphocellulose chromatography, exhibits a characteristic GTPase activity that is polymerization dependent and can be attributed to the tubulin itself. This activity has been observed, in a standard reassembly buffer containing low Mg2+, under three conditions that induce microtubule assembly: in the presence of microtubule-associated proteins, in the presence of DEAE-dextran, or after addition of high Mg2+ and glycerol. The phosphocellulose-purified tubulin showed no GTPase activity under the following nonpolymerizing conditions: in buffer with low Mg2+ in the absence of microtubule-associated proteins or DEAE-dextran, in buffer with high Mg2+ and glycerol at tubulin concentrations below the critical concentration, or when microtubule assembly was inhibited by vinblastine. Colchicine, on the other hand, while blocking microtubule assembly, induced a significant GTPase activity in the phosphocellulose-purified tubulin. During the process of assembly, GTP appears to be hydrolyzed as a free tubulin dimer polymerizes into a microtubule. A constant GTPase activity when polymerization equilibrium is reached apparently reflects the cyclic polymerization-depolymerization of tubulin dimers at the ends of the microtubules.

Animals↗

PubMed1974

Intermediates of bacteriophage MS2 assembly in vivo.

The in vivo process of virion assembly was studied in rifampin-treated, MS2-infected Escherichia coli during late times of infection-after 18 min postinfection. Differential sucrose gradient sedimentation of infected-cell lysates taken at various times after radioactive labeling indicated a definite temporal order of appearance of phage-specific protein in assembly-related structures. Labeled MS2 protein appears first as a low-molecular-weight peak at the tops of gradients, then as a peak at 40S and as a large number of almost unseparable structures between 40 and 80S, and finally as 80S mature phage particles. During the chase of a short labeling period, radioactive phage protein was found to disappear from gradients in the same temporal order as it appeared; the soluble peak disappears first, followed by the 40 to 70S region. The chased label appears quantitatively in the 80S phage peak. Labeled phage RNA was found to appear first in the 40S peak, then in the structures between 40 and 70S, and finally in 80S phage particles. The order of disappearance of labeled phage RNA during a chase is the same as its appearance. Resedimentation of the 40 to 70S region indicated the presence of distinct structures at 60 and 70S and many indistinct ones between 40 and 60S. The smaller intermediates exhibit separable maturation protein-rich and coat protein-rich segments, indicating nonrandom binding of the two proteins during the initial steps of assembly. Larger, discrete intermediates appear at 60 and 70S. Treatment of the various structures with pancreatic RNase results in destruction of those from 40 through 60S; treatment of the 70S structure results in the conversion of some of it to a 45S peak, presumably the complete capsid. A small fraction of the 80S phage peak is also sensitive to RNase, resulting in a similar 45S peak. Pulse-chase experiments indicate that structures from 40 through 60S as well as the RNase-sensitive 70S structure are assembly intermediates, but that the RNase-insensitive 70S and the RNase-sensitive 80S structures are not.

Carbon Radioisotopes↗

PubMed1989

Studies of the mechanisms of action of the antiretroviral agents hypericin and pseudohypericin.

Administration of the aromatic polycyclic dione compounds hypericin or pseudohypericin to experimental animals provides protection from disease induced by retroviruses that give rise to acute, as well as slowly progressive, diseases. For example, survival from Friend virus-induced leukemia is significantly prolonged by both compounds, with hypericin showing the greater potency. Viremia induced by LP-BM5 murine immunodeficiency virus is markedly suppressed after infrequent dosage of either substance. These compounds affect the retroviral infection and replication cycle at least at two different points: (i) Assembly or processing of intact virions from infected cells was shown to be affected by hypericin. Electron microscopy of hypericin-treated, virus-producing cells revealed the production of particles containing immature or abnormally assembled cores, suggesting the compounds may interfere with processing of gag-encoded precursor polyproteins. The released virions contain no detectable activity of reverse transcriptase. (ii) Hypericin and pseudohypericin also directly inactivate mature and properly assembled retroviruses as determined by assays for reverse transcriptase and infectivity. Accumulating data from our laboratories suggest that these compounds inhibit retroviruses by unconventional mechanisms and that the potential therapeutic value of hypericin and pseudohypericin should be explored in diseases such as AIDS.

Animals↗

PubMed1997

Roles of integrins in fibronectin matrix assembly.

Fibronectin (Fn) matrix assembly is a dynamic cellular process in which the soluble dimeric Fn molecules are assembled into insoluble, disulfide bond stabilized fibrillar polymeric matrix. Fn matrix assembly requires specific Fn binding integrins. Several Fn binding integrins that are capable of mediating Fn matrix assembly have been identified. They include alpha5beta1, alphaIIb(beta3) and alpha(v)beta3 integrins. Cells regulate the matrix assembly process not only by controlling cell surface expression level of the Fn binding integrins but also by modulating Fn binding and cytoskeleton binding activities of the integrins. A major challenge of future studies is to delineate the signal transduction pathway that regulates Fn matrix assembly.

Amino Acid Sequence↗

PubMed1987

An integrated system for interstitial 192Ir implants.

An efficient system for preparing, afterloading, and removing interstitial 192Ir strands has been developed. Use of the system reduces the risk of personnel exposure and eliminates some patient discomfort. The system is "integrated" in that all aspects of the implantation process are considered, from source preparation to source removal. Strand preparation is facilitated by an "assembly line" process using shielded equipment. Components include a handling block for measuring and cutting active strands, a mirror, and a transport container. Afterloading and removal techniques use quick release devices and several forms of afterloading tubing and catheters, each terminated by a Luer lock adapter. Both blind-end and through-and-through implants are possible. Each 192Ir strand, threaded through an injection cap that mates with the Luer lock adapter, is quickly inserted into its tubing or catheter and locked into place. No crimping is required and no additional positioning of the sources is needed. Strand removal is easily accomplished by unlocking and removing the injection cap. The strands receive no mechanical damage and can be reused after appropriate cleaning. More than 100 cases have been performed without incident. Applications include head/neck, breast, and template and non-template vaginal wall treatments.

Brachytherapy↗

PubMed1995

Development and application of an in vivo system to study yeast ribosomal RNA biogenesis and function.

We have developed a system for mutational analysis of Saccharomyces cerevisiae ribosomal RNA in vivo in which yeast cells can be made completely dependent on mutant rRNA and ribosomes by a simple switch in carbon source. The system is based on a yeast strain defective in RNA polymerase I (Pol I) transcription [Nogi et al. (1991) Proc. Natl. Acad. Sci. USA 88, 3962-3966]. This normally inviable strain was rescued by integration of multiple copies of the complete 37S pre-rRNA operon under control of the inducible, Pol II-transcribed GAL7 promoter into the rDNA repeat on chromosome XII. The resulting YJV100 strain can only grow on medium containing galactose as the carbon source. A second, episomal vector was constructed in which the rDNA unit was placed under control of the constitutive PGK1 promoter. YJV100 cells transformed with this vector are now also able to grow on glucose-based medium making the cells completely dependent on plasmid-encoded rRNA. We show that the Pol II-transcribed pre-rRNA is processed and assembled similarly to authentic Pol I-synthesised pre-rRNA, making this 'in vivo Pol II system' suitable for the detailed analysis of rRNA mutations, even highly deleterious ones, affecting ribosome biogenesis or function. A clear demonstration of this is our finding that an insertion into variable region V8 in 17S rRNA, previously judged to be neutral with respect to processing of 17S rRNA, its assembly into 40S subunits and the polysomal distribution of these subunits [Musters et al. (1989), Mol. Cell. Biol. 9, 551-559], is in fact a lethal mutation.

Base Sequence↗

PubMed2004

Photochemical competence of assembled photosystem II core complex in cyanobacterial plasma membrane.

Cyanobacterial cells have two autonomous internal membrane systems, plasma membrane and thylakoid membrane. In these oxygenic photosynthetic organisms the assembly of the large membrane protein complex photosystem II (PSII) is an intricate process that requires the recruitment of numerous protein subunits and cofactors involved in excitation and electron transfer processes. Precise control of this assembly process is necessary because electron transfer reactions in partially assembled PSII can lead to oxidative damage and degradation of the protein complex. In this communication we demonstrate that the activation of PSII electron transfer reactions in the cyanobacterium Synechocystis sp. PCC 6803 takes place sequentially. In this organism partially assembled PSII complexes can be detected in the plasma membrane. We have determined that such PSII complexes can undergo light-induced charge separation and contain a functional electron acceptor side but not an assembled donor side. In contrast, PSII complexes in thylakoid membrane are fully assembled and capable of multiple turnovers. We conclude that PSII reaction center cores assembled in the plasma membrane are photochemically competent and can catalyze single turnovers. We propose that upon transfer of such PSII core complexes to the thylakoid membrane, additional proteins are incorporated followed by binding and activation of various donor side cofactors. Such a stepwise process protects cyanobacterial cells from potentially harmful consequences of performing water oxidation in a partially assembled PSII complex before it reaches its final destination in the thylakoid membrane.

Cell Membrane↗

PubMed2005

Efficient chromosome capture requires a bias in the 'search-and-capture' process during mitotic-spindle assembly.

The mitotic spindle assembles into a bipolar, microtubule-based protein machine during prometaphase. One proposed mechanism for this process is "search-and-capture," in which dynamically unstable microtubules (MTs) search space to capture chromosomes. Although existing theoretical estimates suggest that dynamic instability is efficient enough to allow capture within characteristic mitotic timescales, they are limited in scope and do not address the capture times for realistic numbers of chromosomes. Here we used mathematical modeling to explore this issue. We show that without any bias toward the chromosomes, search-and-capture is not efficient enough to explain the typical observed duration of prometaphase. We further analyze search-and-capture in the presence of a spatial gradient of a stabilizing factor that biases MT dynamics toward the chromosomes. We show theoretically that such biased search-and-capture is efficient enough to account for chromosome capture. We also show that additional factors must contribute to accelerate the spindle assembly for cells with large nuclear volumes. We discuss the possibility that a RanGTP gradient introduces a spatial bias into microtubule dynamics and thus improves the efficiency of search-and-capture as a mechanism for spindle assembly.

Chromosomes, Human↗

PubMed1995

Diffusion and formation of microtubule asters: physical processes versus biochemical regulation.

Microtubule asters forming the mitotic spindle are assembled around two centrosomes through the process of dynamic instability in which microtubules alternate between growing and shrinking states. By modifying the dynamics of this assembly process, cell cycle enzymes, such as cdc2 cyclin kinases, regulate length distributions in the asters. It is believed that the same enzymes control the number of assembled microtubules by changing the "nucleating activity" of the centrosomes. Here we show that assembly of microtubule asters may be strongly altered by effects connected with diffusion of tubulin monomers. Theoretical analysis of a simple model describing assembly of microtubule asters clearly shows the existence of a region surrounding the centrosome depleted in GTP tubulin. The number of assembled microtubules may in some cases be limited by this depletion effect rather than by the number of available nucleation sites on the centrosome.

Animals↗

PubMed1987

Processing of TY1 proteins and formation of Ty1 virus-like particles in Saccharomyces cerevisiae.

We have analysed functional properties of putative proteins encoded by the yeast transposable element, Ty1, by overexpression of TY genes. High-level expression was achieved by appropriate fusion of a Ty sequence, TY9C, to the yeast ADH1 promoter and transformation of yeast cells with this construction. As shown recently by others (Garfinkel et al. 1985; Mellor et al. 1985c) TY overexpression leads to an increase in particle-bound reverse transcriptase activity and to an intracellular accumulation of virus-like particles (Ty-VLPs). We have used a number of deletions in the second open reading frame (TYB) to identify functional domains required for processing and assembly of Ty proteins. Deletions in the TYB region with homology to acid proteases result in overproduction of an unprocessed form of the TYA protein (pro-TYA) which represents the major protein of Ty-VLPs. One particular mutant construction, TY9C-delta 36, led to the accumulation of a particle-bound, 160 kDa protein which cross-reacted with a mouse antiserum raised against purified pro-TYA protein. This supports the hypothesis that TYB is expressed as a TYA/TYB fusion protein which is processed by a TYB-encoded protease activity. Ty-VLPs are formed in the absence of protein processing and even when the TYB gene is not expressed. Thus, we assume that the assembly of Ty particles occurs prior to processing of Ty proteins.

DNA Restriction Enzymes↗

PubMed2006

The early stages of amyloid formation: biophysical and structural characterization of human calcitonin pre-fibrillar assemblies.

Amyloid fibril formation is a nucleation dependent process characterized by a lag-phase prior to the appearance of detectable amyloid fibrils. While the three-dimensional structure of amyloid fibrils at atomic resolution is just beginning to be elucidated, the early process of monomers assembly into oligomers is less understood. Understanding the dynamic processes that lead to the formation of these intermediates is highly important as these assemblies might be the most pathological ones. Here, we investigated the biophysical and structural features characterizing the early stage assemblies formed by the human hormone calcitonin. We calculated the initial nucleus size by experimentally determining the dependence between the lag-time length and the hCT concentrations. We used size exclusion chromatography and dynamic light scattering in order to characterize the dynamic growth process of preliminary intermediates transformed into larger structures. The early structures were visualized using high-resolution transmission electron microscopy. Annular pore-like structures were observed along with protofibrilar structures. This observed morphology is similar to structures revealed during the fibrillization processes of beta-amyloid, alpha-synuclein, and islet amyloid polypeptide, suggesting that these intermediates represent a generic early structure conformation. The results introduced here imply that a variety of intermediate assemblies are formed during the early stages of amyloid fibril formation. The characterizing of their structural features and assembly kinetics will contribute to the rational design of inhibitors directed towards early structure assemblies.

Amyloid↗

PubMed1998

Devoted to the lagging strand-the subunit of DNA polymerase III holoenzyme contacts SSB to promote processive elongation and sliding clamp assembly.

Escherichia coli DNA polymerase III holoenzyme contains 10 different subunits which assort into three functional components: a core catalytic unit containing DNA polymerase activity, the beta sliding clamp that encircles DNA for processive replication, and a multisubunit clamp loader apparatus called gamma complex that uses ATP to assemble the beta clamp onto DNA. We examine here the function of the psi subunit of the gamma complex clamp loader. Omission of psi from the holoenzyme prevents contact with single-stranded DNA-binding protein (SSB) and lowers the efficiency of clamp loading and chain elongation under conditions of elevated salt. We also show that the product of a classic point mutant of SSB, SSB-113, lacks strong affinity for psi and is defective in promoting clamp loading and processive replication at elevated ionic strength. SSB-113 carries a single amino acid replacement at the penultimate residue of the C-terminus, indicating the C-terminus as a site of interaction with psi. Indeed, a peptide of the 15 C-terminal residues of SSB is sufficient to bind to psi. These results establish a role for the psi subunit in contacting SSB, thus enhancing the clamp loading and processivity of synthesis of the holoenzyme, presumably by helping to localize the holoenzyme to sites of SSB-coated ssDNA.

Binding Sites↗

PubMed1990

Complementation of the Aspergillus nidulans arg B1 mutation by ornithine transcarbamylase cDNA from rat liver.

An Aspergillus nidulans strain which is deficient in ornithine transcarbamylase due to the arg B1 mutation was transformed with a plasmid containing the ornithine transcarbamylase cDNA from rat liver under the control of the amd S promoter. Stable transformants were obtained by selection on arginine free medium indicating complementation of the arg B mutation. Proof of expression of the rat enzyme in transformants was obtained by immunoprecipitation of all ornithine transcarbamylase activity from cell extracts with antibodies specific for the rat enzyme. The presence of catalytically active rat ornithine transcarbamylase in the transformants indicated that it is capable of being imported into mitochondria in A. nidulans, proteolytically processed and assembled into its homotrimeric form. In vitro uptake experiments using isolated A. nidulans mitochondria demonstrate that processing of the precursor of rat ornithine transcarbamylase occurs in two temporally separated steps as it does in rat liver mitochondria suggesting evolutionary conservation of the processing machinery. Up to 560 ng of active rat enzyme was produced per gm wet weight mycelia. Use of beta-D-alanine, an inducer of amd S, as sole N-source resulted in increased levels of active rat ornithine transcarbamylase relative to uninduced cultures.

Animals↗

PubMed1993

Assembly of envelope structure with vesicles associated with Ku-homologous protein in Xenopus egg extract in the absence of chromatin.

To study the process of nuclear envelope assembly at the end of mitosis, we developed a chromatin-free in vitro system for assembly of envelope structures in Xenopus interphase egg extract, and examined the participation of Ku-homologous protein in the assembly. The envelope structure assembled spontaneously in the absence of chromatin or DNA between glass plates under a condition that minimized generation of flow of the extract. Morphological study using an electron microscopy has revealed that the membrane surrounding the envelope structure is a double membrane that contains gaps resembling nuclear pore complex. Their assembly was dependent on ATP and was inhibited by the addition of GTP-gamma-S or N-ethylmaleimide. Depletion of a pre-nuclear vesicle by preincubating the interphase egg extracts with large excess of sperm head chromatins impaired the assembly. The membrane vesicle, which was associated with Ku-homologous protein of Xenopus, participated in the assembly as proven by reaction with monoclonal antibody made specific for Ku p70 protein. However, the assembly process of the envelope structure was inhibited only slightly by the antibody, suggesting that the Ku-homologous protein does not participate in the fusion process of vesicles to form the envelope structure.

Adenosine Triphosphate↗

PubMed2003

Dissociations of visual recognition in a developmental agnosic: evidence for separate developmental processes.

We report the results of tests investigating the recognition of faces, places, and objects in a developmental agnosic, because dissociations of visual recognition in developmental agnosics provide insight into the separable procedures performing recognition and the developmental origins of these procedures. TA is a software engineer in his early 40s with developmental prosopagnosia. He performs normally on tests of low-level vision, and he names objects at the basic level normally. In order to compare his recognition abilities for different classes, we have presented him with a famous landmarks test, a famous faces test, and old/new discriminations involving unfamiliar faces, houses, natural landscapes, cars, horses, guns, sunglasses, and tools. He was impaired on the face recognition tests, but performed normally on the place recognition tests. He also showed severe impairments with horses and cars, borderline impairments with guns and sunglasses, and normal performance with tools. These results indicate that the developmental processes that assemble the procedures used for face recognition and certain types of object recognition are separate from those processes that produce the procedures used for place recognition.

Adult↗

PubMed1996

The presence of direct repeats does not influence coding joint formation during V(D)J recombination.

During the recombination process that assembles immunoglobulin and T-cell receptor gene segments, the coding ends to be joined are extensively processed. Contradictory reports have been made in the past about the existence of homology directed mechanisms in V(D)J recombination. In this study we analyse coding end processing and the influence of the presence of homology stretches on coding joint formation using artificial substrates in which short sequence changes creating direct repeats have been introduced. These changes were monitored 3 bp away from the termini in order to avoid any differences due to the initiation steps of V(D)J recombination. Our results show that the sequence of the coding ends influences joint formation, but no evidence was found for a mechanistic bias due to the presence of direct repeats.

3T3 Cells↗

PubMed2007

Amperometric glucose biosensor based on multilayer films via layer-by-layer self-assembly of multi-wall carbon nanotubes, gold nanoparticles and glucose oxidase on the Pt electrode.

A novel amperometric glucose biosensor based on the nine layers of multilayer films composed of multi-wall carbon nanotubes (MWCNTs), gold nanoparticles (GNp) and glucose oxidase (GOD) was developed for the specific detection of glucose. MWCNTs were chemically modified with the H(2)SO(4)-HNO(3) pretreatment to introduce carboxyl groups which were used to interact with the amino groups of poly(allylamine) (PAA) and cysteamine via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide cross-linking reaction, respectively. A cleaned Pt electrode was immersed in PAA, MWCNTs, cysteamine and GNp, respectively, followed by the adsorption of GOD, assembling the one layer of multilayer films on the surface of Pt electrode (GOD/GNp/MWCNTs/Pt electrode). Repeating the above process could assemble different layers of multilayer films on the Pt electrode. PBS washing was applied at the end of each assembly deposition for dissociating the weak adsorption. Film assembling and characterization were studied by transmission electron microscopy and quartz crystal microbalance, and properties of the resulting glucose biosensors were measured by electrochemical measurements. The marked electrocatalytic activity of Pt electrode based on multilayer films toward H(2)O(2) produced during GOD enzymatic reactions with glucose permitted effective low-potential amperometric measurement of glucose. Taking the sensitivity and selectivity into consideration, the applied potential of 0.35 V versus Ag/AgCl was chosen for the oxidation detection of H(2)O(2) in this work. Among the resulting glucose biosensors, the biosensor based on nine layers of multilayer films was best. It showed a wide linear range of 0.1-10mM glucose, with a remarkable sensitivity of 2.527 microA/mM, a detection limit of 6.7 microM estimated at a signal-to-noise ratio of 3 and fast response time (within 7s). Moreover, it exhibited good reproducibility, long-term stability and the negligible interferences of ascorbic acid, uric acid and acetaminophen. The study can provide a feasible approach on developing new kinds of oxidase-based amperometric biosensors, and can be used as an illustration for constructing various hybrid structures.

Biosensing Techniques↗