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At least 1,495 records · Page 83Linked to original sources
PubMed1993

Induction of CD3 delta epsilon omega by phorbol 12-myristate 13-acetate.

The effect of phorbol 12-myristate 13-acetate (PMA) on the synthesis, assembly and processing of the components of the T cell receptor (TcR) was studied with special focus on the CD3 omega chain. Treatment of the human leukemic T cell line Jurkat with PMA increased the synthesis of the Ti alpha, CD3 gamma and CG3 zeta chains two- to threefold and the synthesis of Ti beta and CD3 delta epsilon omega complexes five- to sevenfold as assessed by metabolic labeling, immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by scanning densitometry. The amount of total assembled TcR complexes increased approximately threefold and the maturation of the TcR was not affected as determined by analysis of oligosaccharide side chain processing in the Golgi apparatus. Activation of Jurkat cells with anti-CD3 monoclonal antibody, calcium ionophore, or mitogenic lectins did not affect the synthesis of the TcR components. In other cells studied (the human leukemic T cell line CEM, a panel of variants of the Jurkat T cell line and peripheral blood mononuclear cells) PMA also increased the synthesis of the TcR components. However, for all cell lines studied the amount of TcR complexes expressed on the cell surface was decreased after 16 h of PMA treatment. Based on these results we propose a role of CD3 omega in retention of TcR complexes. From PMA-treated CEM cells more than 50-fold the amount of CD3 delta epsilon omega complexes was immunoprecipitated as compared to the amount obtained from untreated Jurkat cells, and these observations indicate that the CEM cell line may be a qualified candidate for purification of CD3 omega.

Ammonium Chloride↗
PubMed2005

Antibody processing and engineering in plants, and new strategies for vaccine production.

The use of transgenic plants for the production of recombinant proteins is not a universal solution for all proteins. The choice of this expression system depends very much on the type of protein and its applications. Many proteins will best be made by conventional microbial fermentation, similarly, we are already identifying proteins where plants represent the only practical option for one reason or another. It will be important to understand better the cellular mechanisms of protein folding, assembly and processing in plants, in order to maximise the potential of transgenic plants as a protein production system. One of the main advantages that plants offer is that they are higher eukaryotic organisms with an endomembrane system. Therefore, they fold and assemble recombinant proteins using protein chaperones that are homologous to those in mammalian cells, and they perform post-translational modifications. This allows, for example, the expression of monoclonal antibodies, first described in 1989, as well as a range of other types of immunoglobulin molecules and multimeric complexes.

Animals↗
PubMed1983

Inhibition of tubulin assembly by antileprosy drug dapsone.

The effect of dapsone on assembly-disassembly process of bovine brain tubulin was examined. The drug was found to readily bind tubulin dimer and that in its presence colchicine binding to tubulin was enhanced. Although dapsone associated with tubulin at a site other than the colchicine binding site, distinct inhibition of microtubule assembly was detected.

Animals↗
PubMed1995

Termination as a factor in "quality control" during ribosome biogenesis.

In eukaryotes, nascent rDNA and 5 S rRNA gene transcripts undergo 3'-end processing after termination. Mutations in which terminator sequences in these ribosomal RNA genes are deleted completely result in highly unstable transcripts, which are not properly processed and integrated into stable ribosome structure. Mutations that retard RNA processing by extending the 3' external transcribed spacer or by introducing additional secondary structure in the spacers have a similar effect on stable transcript integration. The results indicate that proper termination coupled with efficient rRNA processing acts as a "quality control" process, which helps to ensure that only normal rRNA precursors are effectively processed and assembled into active ribosomes.

Base Sequence↗
PubMed1995

Selective interactions of UPIa and UPIb, two members of the transmembrane 4 superfamily, with distinct single transmembrane-domained proteins in differentiated urothelial cells.

The transmembrane 4 (TM4) superfamily contains many important leukocyte differentiation-related surface proteins including CD9, CD37, CD53, and CD81; tumor-associated antigens including CD63/ME491, CO-029, and SAS; and a newly identified metastasis suppressor gene R2. Relatively little is known, however, about the structure and aggregation state of these four transmembrane-domained proteins. The asymmetrical unit membrane (AUM), believed to play a major role in stabilizing the apical surface of mammalian urothelium thus preventing it from rupturing during bladder distention, contains two TM4 members, the uroplakins (UPs) Ia and Ib. In association with two other (single transmembrane-domained) membrane proteins, UPII and UPIII, UPIa and UPIb form 16-nm particles that naturally form two-dimensional crystalline arrays, thus providing unique opportunities for studying membrane structure and function. To better understand how these proteins interact to form the 16-nm particles, we analyzed their nearest neighbor relationship by chemical cross-linking. We show here that UPIa and UPIb, which share 39% of their amino acid sequence, are cross-linked to UPII and UPIII, respectively. We also show that UPIa has a propensity to oligomerize, forming complexes that are stable in SDS, and that UPII can be readily cross-linked to form homodimers. The formation of UPII homodimers is sensitive, however, to octyl glucoside that can solubilize the AUMs. These data suggest that there exist two types of 16-nm AUM particles that contain UPIa/UPII or UPIb/UPIII, and support a model in which the UPIa and UPII occupy the inner and outer domains, respectively, of the UPIa/UPII particle. This model can account for the apparent "redundancy" of the uroplakins, as the structurally related UPIa and UPIb, by interacting with different partners, may play different roles in AUM formation. The model also suggests that AUM plaques with different uroplakin compositions may differ in their assembly, and in their abilities to interact with an underlying cytoskeleton. Our data indicate that two closely related TM4 proteins, UPIa and UPIb, can be present in the same cell, interacting with distinct partners. AUM thus provides an excellent model system for studying the targeting, processing, and assembly of TM4 proteins.

Amino Acid Sequence↗
PubMed2005

Epithelial cell shape and Rho small GTPases.

In epithelial cells, morphology is intrinsically related to function. Typically, polarization (i.e. acquisition of a cuboidal cell shape) must occur prior to terminal differentiation and functionality of epithelial sheets. Extensive work has been performed to understand the initial steps that drive cell-cell contact assembly, a process that is essential for polarization. However, not much is known about the subsequent steps that lead to remodelling of actin cytoskeleton and concomitant cell shape changes. Here we review what is known about actin organization during epithelial polarization, and discuss current models for junction assembly, actin reorganization and signalling pathways that may contribute to the generation of a polarized epithelial morphology.

Animals↗
PubMed1990

Total reconstitution of DNA polymerase III holoenzyme reveals dual accessory protein clamps.

DNA polymerase III holoenzyme (holoenzyme) is the 10-subunit replicase of the Escherichia coli chromosome. In this report, pure preparations of delta, delta', and a gamma chi psi complex are resolved from the five protein gamma complex subassembly. Using these subunits and other holoenzyme subunits isolated from overproducing plasmid strains of E. coli, the rapid and highly processive holoenzyme has been reconstituted from only five pure single subunits: alpha, epsilon, gamma, delta, and beta. The preceding report showed that of the three subunits in the core polymerase, only a complex of alpha (DNA polymerase) and epsilon (3'-5' exonuclease) are required to assemble a processive holoenzyme on a template containing a preinitiation complex (Studwell, P.S., and O'Donnell, M. (1990) J. Biol. Chem. 265, 1171-1178). This report shows that of the five proteins in the gamma complex only a heterodimer of gamma and delta is required with the beta subunit to form the ATP-activated preinitiation complex with a primed template. Surprisingly, the delta' subunit does not form an active complex with gamma but forms a fully active heterodimer complex with the tau subunit (as does delta). Hence, the tau delta' and gamma delta heterodimers are fully active in the preinitiation complex reaction with beta and primed DNA. Holoenzymes reconstituted using the alpha epsilon complex, beta subunit, and either gamma delta or tau delta' are fully processive in DNA synthesis, and upon completing the template they rapidly cycle to a new primed template endowed with a preinitiation complex clamp. Since the holoenzyme molecule contains all of these accessory subunits (gamma, delta, tau, delta', and beta) in all likelihood it has the capacity to form two preinitiation complex clamps simultaneously at two primer termini. Two primer binding components within one holoenzyme may mediate its rapid cycling to multiple primers on the lagging strand and also provides functional evidence for the hypothesis of holoenzyme as a dimeric polymerase capable of simultaneous replication of both leading and lagging strands of a replication fork.

Chromatography, Gel↗
PubMed1989

Regulation of desmosome assembly in epithelial cells: kinetics of synthesis, transport, and stabilization of desmoglein I, a major protein of the membrane core domain.

Desmosomes are composed of two morphologically and biochemically distinct domains, a cytoplasmic plaque and membrane core. We have initiated a study of the synthesis and assembly of these domains in Madin-Darby canine kidney (MDCK) epithelial cells to understand the mechanisms involved in the formation of desmosomes. Previously, we reported the kinetics of assembly of two components of the cytoplasmic plaque domain, Desmoplakin I/II (Pasdar, M., and W. J. Nelson. 1988. J. Cell Biol. 106:677-685 and 106:687-699. We have now extended this analysis to include a major glycoprotein component of the membrane core domain, Desmoglein I (DGI; Mr = 150,000). Using metabolic labeling and inhibitors of glycoprotein processing and intracellular transport, we show that DGI biosynthesis is a sequential process with defined stages. In the absence of cell-cell contact, DGI enters a Triton X-100 soluble pool and is core glycosylated. The soluble DGI is then transported to the Golgi complex where it is first complex glycosylated and then titrated into an insoluble pool. The insoluble pool of DGI is subsequently transported to the plasma membrane and is degraded rapidly (t1/2 less than 4 h). Although this biosynthetic pathway occurs independently of cell-cell contact, induction of cell-cell contact results in dramatic increases in the efficiency and rate of titration of DGI from the soluble to the insoluble pool, and its transport to the plasma membrane where DGI becomes metabolically stable (t1/2 greater than 24 h). Taken together with our previous study of DPI/II, we conclude that newly synthesized components of the cytoplasmic plaque and membrane core domains are processed and assembled with different kinetics indicating that, at least initially, each domain is assembled separately in the cell. However, upon induction of cell-cell contact there is a rapid titration of both components into an insoluble and metabolically stable pool at the plasma membrane that is concurrent with desmosome assembly.

Animals↗
PubMed1998

Mitochondrial protein import in plants. Signals, sorting, targeting, processing and regulation.

Mitochondrial biogenesis requires a coordinated expression of both the nuclear and the organellar genomes and specific intracellular protein trafficking, processing and assembly machinery. Most mitochondrial proteins are synthesised as precursor proteins containing an N-terminal extension which functions as a targeting signal, which is proteolytically cleaved off after import into mitochondria. We review our present knowledge on components and mechanisms involved in the mitochondrial protein import process in plants. This encompasses properties of targeting peptides, sorting of precursor proteins between mitochondria and chloroplasts, signal recognition, mechanism of translocation across the mitochondrial membranes and the role of cytosolic and organellar molecular chaperones in this process. The mitochondrial protein processing in plants is catalysed by the mitochondrial processing peptidase (MPP), which in contrast to other sources, is integrated into the bc1 complex of the respiratory chain. This is the most studied component of the plant import machinery characterised to date. What are the biochemical consequences of the integration of the MPP into an oligomeric protein complex and how are several hundred presequences of precursor proteins with no sequence similarities and no consensus for cleavage, specifically cleaved off by MPP? Finally we will address the emerging area of the control of protein import into mitochondria.

Amino Acid Sequence↗
PubMed1993

Missense mutations impair intracellular processing of fibrillin and microfibril assembly in Marfan syndrome.

Dermal fibroblasts from nine Marfan syndrome patients with missense mutations in the fibrillin-1 gene (FBN1) produced nearly normal amounts of fibrillin as determined by quantitative pulse-chase experiments. However, six of the seven mutations involving substitutions of highly conserved cysteine residues exhibited lower rates of intracellular transport and secretion. This effect is likely due to improper folding, since intracellular fibrillin processing was also affected by the reducing agent dithiothreitol. Normal secretion patterns were seen in three mutations that either change the conformation of EGF-like domains or change consensus amino acids required for Ca(++)-binding. In all nine fibroblasts strains, however, the deposition of fibrillin in the extracellular matrix was reduced to 50% of normal in two and to less than 30% in seven of the nine samples studied. The protein alterations caused by these missense mutations are associated with moderate to severe features of Marfan syndrome and a dominant negative mechanism is suggested to play a major role in their pathogenesis.

Amino Acid Sequence↗
PubMed1999

Assembly of very low density lipoprotein: a two-step process of apolipoprotein B core lipidation.

The liver plays a primary role in lipid metabolism. Important functions include the synthesis and incorporation of hydrophobic lipids, triacylglycerols and cholesteryl esters into the core of water-miscible particles called lipoproteins and the secretion of these particles into the circulation for transport to distant tissues. In this article, we present a brief overview of one aspect of the assembly process of very low density lipoproteins, namely, possible mechanisms for combining core lipids with apolipoprotein B. This is a complex process in which apolipoprotein B interacts with core lipids to form very low density lipoproteins by a two-step process that can be dissociated biochemically.

Animals↗
PubMed2026

Microbial succession and assembly shaped by sulfur, spatial partitioning, and water flow in a volcanic acidic river of northern Patagonia.

Extreme acidic environments represent natural laboratories for investigating the mechanisms of microbial community assembly, yet the ecological processes structuring these communities remain incompletely understood. Here, we investigate how spatial partitioning, hydrodynamics, and colonization history shape microbial succession in a unique sulfur-rich, acidic river of volcanic origin in northern Patagonia. We combined 16S rRNA gene profiling and shotgun metagenomics with a multi-scale experimental framework encompassing water column fractionation and colonization assays under native and controlled conditions. Microbial diversity was strongly influenced by spatial fractionation, with free-living communities exhibiting higher richness and temporal variability than particle-associated assemblages. Water flow modulated community structure, increasing evenness in free-living fractions under high-flow conditions, but had limited impact on particle-attached communities. Colonization of sulfur-beads followed a structured successional trajectory, with autotrophic sulfur oxidizers dominating early stages and heterotrophs adapted to biofilm lifestyles increasing over time. Ex situ recolonization assays revealed strong priority effects, with initial colonizers determining successional trajectories. Turnover analyses revealed that the balance among stochastic and deterministic assembly processes shifted across communities with pronounced stochasticity in the water column and flow-dependent effects in free-living communities, while biofilm associated communities on sulfur-beads exhibited stronger contribution of deterministic selection. These ecological patterns were mirrored by functional differentiation, with gene enrichment analyses revealing adaptive signatures of substrate attachment and resource acquisition. By integrating fine-scale environmental variation with colonization dynamics, this study reveals how microscale habitat structure and temporal fluxes jointly modulate microbial community assembly rules, offering a nuanced framework to dissect ecological processes in extreme systems.

Sulfur↗
PubMed1989

Collagen processing, crosslinking, and fibril bundle assembly in matrix produced by fibroblasts in long-term cultures supplemented with ascorbic acid.

Human foreskin fibroblasts were cultured for up to 6 weeks in medium supplemented with ascorbic acid. During this time, the cells produced an extensive new connective tissue matrix in which the accumulated collagen (mostly type I) amounted to about 0.25 mg/10(6) cells. The matrix was highly differentiated as shown by complete processing of procollagen to collagen alpha-chains and covalent crosslinking of the collagen. Alignment of collagen fibrils occurred as the fibrils were deposited between cells, and binding of adjacent fibrils to the cell surface appeared to hold the fibrils in register. Groups of aligned fibrils were subdivided into bundles by cell-surface folds. If beta-aminopropionitrile was added to the medium, collagen crosslinking was inhibited, but not collagen synthesis or fibril bundle organization. If ascorbic acid was omitted from the culture medium, the extensive new connective tissue matrix was not produced. Our results indicate that fibroblasts in long-term cultures supplemented with ascorbic acid produce a connective tissue matrix with many in vivo-like properties including supermolecular organization of collagen.

Aminopropionitrile↗
PubMed1990

Dihydropyridine receptor regulation of acetylcholinesterase biosynthesis.

The dihydropyridine calcium channel antagonist nifedipine causes marked reductions in the amounts of acetylcholinesterase (AchE) molecular forms in primary tissue cultures of avian pectoral muscle. These reductions are time-dependent, requiring passage of 3 h prior to any observable response, dose-dependent, with principal actions occurring in the 1-100 nM range, are greater on the 7 S and 19 S forms than on the 11.4 S form, and, based on susceptibility of AchE to irreversible inhibition by a cationic inhibitor, occur almost exclusively with intracellular AchE coincident with a 2-fold reduction in the rate of secretion. The effects are markedly more pronounced in skeletal muscle than in neurons and differ from those observed for verapamil, diltiazem, and the calcium ionophore A23187. These reductions are incompatible with accelerated protein degradation, alterations in posttranslational processing and assembly in the Golgi complex, or enhanced loss of enzyme to the medium, but instead indicate that nifedipine causes a reduction in AchE biosynthesis. Since AchE forms are thought to arise from a single gene, these findings imply a linkage in skeletal muscle between transcription and posttranscriptional processing of mRNA and ligand occupation of the dihydropyridine receptor.

Acetylcholine↗
PubMed2006

New softwares for automated microsatellite marker development.

Microsatellites are repeated small sequence motifs that are highly polymorphic and abundant in the genomes of eukaryotes. Often they are the molecular markers of choice. To aid the development of microsatellite markers we have developed a module that integrates a program for the detection of microsatellites (TROLL), with the sequence assembly and analysis software, the Staden Package. The module has easily adjustable parameters for microsatellite lengths and base pair quality control. Starting with large datasets of unassembled sequence data in the form of chromatograms and/or text data, it enables the creation of a compact database consisting of the processed and assembled microsatellite containing sequences. For the final phase of primer design, we developed a program that accepts the multi-sequence 'experiment file' format as input and produces a list of primer pairs for amplification of microsatellite markers. The program can take into account the quality values of consensus bases, improving success rate of primer pairs in PCR. The software is freely available and simple to install in both Windows and Unix-based operating systems. Here we demonstrate the software by developing primer pairs for 427 new candidate markers for peanut.

Arachis↗
PubMed1988

Biosynthesis of human von Willebrand factor.

Endothelium forms the inner lining of all blood vessels and, as a consequence, is in direct contact with the blood. Because of this and the synthesis and secretion of hemostatic components, the endothelium is able to modulate coagulation and fibrinolysis. An important hemostatic factor synthesized by endothelial cells is the von Willebrand factor (vWF). vWF is a large plasma glycoprotein which promotes the adhesion of platelets to the vessel wall after a vascular injury. vWF is initially synthesized as a pre-pro-polypeptide. During its transport to the outside of the cell, the single-chain polypeptides are assembled into multimers. The pro-polypeptide can be cleaved and also be secreted. Free pro-polypeptide is identified as von Willebrand antigen II, a plasma glycoprotein of unknown function. Plasma vWF consists of a heterogenous series of multimers, composed of an apparently single-type glycoprotein subunit, linked together by disulfide bonds. The hemostatic potency of vWF was shown to increase with increasing multimer size. Therefore, the multimeric assembly of vWF is a crucial aspect in vWF biosynthesis. Furthermore, vWF synthesized by endothelial cells can either be secreted constitutively or stored and released upon stimulation of the endothelial cell. In this review, data are presented which contribute to the understanding of the biosynthetic pathway and complex processing which vWF has to undergo before it is secreted by the endothelial cell. These data have allowed a prediction of the sequential events underlying vWF biosynthesis, processing, multimer assembly, and secretion.

Endothelium↗
PubMed1997

Assembly of a nucleoprotein complex required for DNA packaging by bacteriophage lambda.

A critical step in the assembly of bacteriophage lambda is the excision of a single genome from a concatemeric DNA precursor and insertion of genomic DNA into an empty viral capsid. DNA packaging is mediated by the lambda proteins gpNu1 and gpA, which form an enzyme complex known as terminase. Initiation of the packaging process requires assembly of the terminase subunits onto cos, the lambda DNA packaging sequence, and nicking of the duplex, thus forming the 12-base-pair "sticky" ends of the mature genome. We have utilized gel-retardation techniques to examine the interaction of gpNu1, gpA, and terminase holoenzyme with DNA. Our data demonstrate that gpNu1 interacts specifically with cos-containing DNA, forming three gel-retarded complexes. Similarly, the larger gpA subunit binds to DNA, forming two complexes; however, this subunit forms similar complexes with DNA substrates of random sequence. All of the nucleoprotein complexes examined are disrupted by elevated concentrations of NaCl and we suggest that altered DNA binding is responsible for the extreme salt sensitivity of the endonuclease activity of the enzyme [Tomka, M. A., & Catalano, C. E. (1993) J. Biol. Chem. 268, 3056-3065]. DNA binding by each subunit is strongly affected by the presence of the other, with 10- and 3-fold increases in the affinity of gpNu1 and gpA, respectively, for DNA. Moreover, our data suggest that the terminase subunits interact in solution prior to DNA binding. Finally, we provide evidence that complex I, the first stable intermediate in the packaging pathway, is composed of the mature left genome end bound to the terminase subunits and demonstrate that dissociation of the complex is quite slow (t1/2 > 8 h). The significance of these data with respect to terminase-mediated genome packaging is discussed.

Adenosine Triphosphate↗
PubMed1990

Role of the major capsid protein in herpes simplex virus type-1 capsid assembly.

Two herpes simplex virus type 1 (HSV-1) temperature-sensitive (ts) mutants with defects in the gene for the major capsid protein, ICP5, were examined for their effects on virion capsid assembly. Polyacrylamide gel electrophoresis revealed that both mutants were able to synthesize wild-type (WT) levels of ICP5 at the nonpermissive temperature. However, the 53 kD capsid protein disappeared concomitant with the appearance of a new, 51 kD species. These results, taken together with ultrastructural and immunological analyses indicate that the processing and assembly of capsid proteins, DNA packaging and thermal stability of HSV-1 virions are dependent upon functional ICP5.

Animals↗