SEARCH · PubMed Health

Results for “COPI complex”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

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

At least 1,279 records · Page 71Linked to original sources

PubMed2006

Stress-induced deficits in working memory and visuo-constructive abilities in Special Operations soldiers.

BACKGROUND: Pre-clinical and clinical studies have shown acute stress may impair working memory and visuo-spatial ability. This study was designed to clarify the nature of stress-induced cognitive deficits in soldiers and how such deficits may contribute to operational or battlefield errors. METHODS: One hundred eighty-four Special Operations warfighters enrolled in Survival School completed pre-stress measures of dissociation and trauma exposure. Subjects were randomized to one of three assessment groups (Pre-stress, Stress, Post-stress) and were administered the Rey Ostereith Complex Figure (ROCF). All subjects completed post-stress measures of dissociation. RESULTS: ROCF copy and recall were normal in the Pre- and Post-stress groups. ROCF copy and recall were significantly impaired in the Stress Group. Stress group ROCF copy performance was piecemeal, and ROCF recall was impaired. Symptoms of dissociation were negatively associated with ROCF recall in the Stress group. Baseline dissociation and history of traumatic stress predicted cognitive impairment during stress. CONCLUSIONS: Stress exposure impaired visuo-spatial capacity and working memory. In rats, monkeys, and humans, high dopamine and NE turnover in the PFC induce deficits in cognition and spatial working memory. Improved understanding of stress-induced cognitive deficits may assist in identification of soldiers at risk and lead to the development of better countermeasures.

Adult↗

PubMedDate not supplied

The role of the DNA double-strand break response network in meiosis.

Organisms with sexual reproduction have two homologous copies of each chromosome. Meiosis is characterized by two successive cell divisions that result in four haploid sperms or eggs, each carrying a single copy of homologous chromosome. This process requires a coordinated reorganization of chromatin and a complex network of meiotic-specific signaling cascades. At the beginning of meiosis, each chromosome must recognize its homolog, then the two become intimately aligned along their entire lengths which allows the exchange of DNA strands between homologous sequences to generate genetic diversity. DNA double-strand breaks (DSBs) initiate meiotic recombination in a variety of organisms. Numerous studies have identified both the genomic loci of the initiating DSBs and the proteins involved in their formation. This review will summarize the activation and signaling networks required for the DSB response in meiosis.

Animals↗

PubMed2004

DNA vaccines expressing soluble CD4-envelope proteins fused to C3d elicit cross-reactive neutralizing antibodies to HIV-1.

DNA vaccines expressing the envelope (Env) of the human immunodeficiency virus type 1 (HIV-1) have been relatively ineffective at generating high-titer, long-lasting, neutralizing antibodies in a variety of animal models. In this study, DNA vaccines were constructed to express a fusion protein of the soluble human CD4 (sCD4) and the gp120 subunit of the HIV-1 envelope. To enhance the immunogenicity of the expressed fusion protein, three copies of the murine C3d (mC3d3) were added to the carboxyl terminus of the complex. Monoclonal antibodies that recognize CD4-induced epitopes on gp120 efficiently bound to sCD4-gp120 or sCD4-gp120-mC3d3. In addition, both sCD4-gp120 and sCD4-gp120-mC3d3 bound to cells expressing appropriate coreceptors in the absence of cell surface hCD4. Mice (BALB/c) vaccinated with DNA vaccines expressing either gp120-mC3d3 or sCD4-gp120-mC3d3 elicited antibodies that neutralized homologous virus infection. However, the use of sCD4-gp120-mC3d3-DNA elicited the highest titers of neutralizing antibodies that persisted after depletion of anti-hCD4 antibodies. Interestingly, only mice vaccinated with DNA expressing sCD4-gp120-mC3d3 had antibodies that elicited cross-protective neutralizing antibodies. The fusion of sCD4 to the HIV-1 envelope exposes neutralizing epitopes that elicit broad protective immunity when the fusion complex is coupled with the molecular adjuvant, C3d.

AIDS Vaccines↗

PubMed2002

Shared protein components of SINE RNPs.

The heterogeneous, short RNAs produced from the high, copy, short mobile elements (SINEs) interact with proteins to form RNA-protein (RNP) complexes. In particular, the BC1 RNA, which is transcribed to high levels specifically in brain and testis from one locus of the ID SINE family, exists as a discrete RNP complex. We expressed a series of altered BC1, and other SINE-related RNAs, in several cell lines and tested for the mobility of the resulting RNP complexes in a native PAGE assay to determine which portions of these SINE RNAs contribute to protein binding. When different SINE RNAs were substituted for the BC1 ID sequence, the resulting RNPs exhibited the same mobility as BC1. This indicates that the protein(s) binding to the ID portion of BC1 is not sequence specific and may be more dependent upon the secondary structure of the RNA. It also suggests that all SINE RNAs may bind a similar set of cellular proteins. Deletion of the A-rich region of BC1 RNA has a marked effect on the mobility of the RNP. Rodent cell lines exhibit a slightly different mobility for this shifted complex when compared to human cell lines, reflecting evolutionary differences in one or more of the protein components. On the basis of mobility change observed in RNP complexes when the A-rich region is removed, we decided to examine poly(A) binding protein (PABP) as a candidate member of the RNP. An antibody against the C terminus of PABP is able to immunoprecipitate BC1 RNA, confirming PABP's presence in the BC1 RNP. Given the ubiquitous role of poly(A) regions in the retrotransposition process, these data suggest that PABP may contribute to the SINE retrotransposition process.

Base Sequence↗

PubMed2002

Cell aggregation by scaffolded receptor clusters.

The aggregation of cells by lectins or antibodies is important for biotechnological and therapeutic applications. One strategy to augment the avidity and aggregating properties of these mediators is to maximize the number of their ligand binding sites. The valency of lectins and antibodies, however, is limited by their quaternary structures. To overcome this limitation, we explored the use of polymers generated by ring-opening metathesis polymerization (ROMP) as scaffolds to noncovalently assemble multiple copies of a lectin, the tetravalent protein concanavalin A (Con A). We demonstrate that complexes between Con A and multivalent scaffolds aggregate cells of a T cell leukemia line (Jurkat) more effectively than Con A alone. We anticipate that synthetic scaffolds will offer a new means of facilitating processes that rely on cell aggregation, such as pathogen clearance and immune recognition.

Cell Aggregation↗

PubMed2003

Development of a quantitative real-time RT-PCR assay with internal control for the laboratory detection of tick borne encephalitis virus (TBEV) RNA.

BACKGROUND: Tick borne encephalitis virus (TBEV), is a human flavivirus causing tick borne encephalitis (TBE), a viral infection of the central nervous system endemic in Europe and Asia. OBJECTIVES: To develop a reverse transcription polymerase chain reaction (RT-PCR) assay based on quantitative real-time RT-PCR technology (TaqMan) for detection and quantification of TBEV RNA. The test includes an internal control (IC) to avoid false negative results. STUDY DESIGN: The system was established and validated using wild-type (WT) non-infectious synthetic RNA representing a fragment of the 3' non-coding region of the TBEV genome. In addition, synthetic RNA differing from the WT synthetic RNA by a unique probe binding region was used as IC to monitor the overall efficiency of the RT-PCR. RESULTS: The analytical sensitivity of the assay was at least ten copies of the TBEV synthetic transcript in presence of 50 copies of the IC. Successful amplification was obtained for different strains within the TBEV complex (Hypr, Hochosterwitz, Laibach, Elsass=Alsace, ZZ9, Wladiwostok). Among 14 serum and 21 cerebrospinal fluid (CSF) samples obtained from 28 patients with clinical suspicion of TBEV 1 CSF sample tested positive for TBEV RNA. In addition, no TBEV RNA could be detected in blood samples obtained from three vaccinated people 1 and 3 days post-vaccination. Thus indicating that a positive result is unlikely to be caused by recent vaccination. CONCLUSIONS: A quantitative, highly sensitive and specific real-time RT-PCR assay has been developed for the detection of TBEV RNA. Inclusion of an IC is important to monitor the possible occurrence of false-negative results caused by the presence of inhibitory factors. This assay should be an important asset for the routine laboratory detection of TBEV RNA.

3' Untranslated Regions↗

PubMed2000

Tetrad analysis shows that gene conversion is the major mechanism involved in mutation at the human minisatellite MS1 integrated in Saccharomyces cerevisiae.

Minisatellites are arrays of tandemly repeated DNA sequences which occur at thousands of locations in the human genome. They are frequently hypervariable with respect to allele length as a result of high rates of complex and incompletely understood recombination-based germline mutation events that alter the repeat copy number. MS1 is one of the most variable minisatellites so far isolated from the human genome. We have integrated MS1, flanked by synthetic markers, in the vicinity of a hot spot for meiotic double-strand breaks upstream of the LEU2 locus in chromosome III of Saccharomyces cerevisiae. Here we present the first tetrad analysis of mutations at a human minisatellite locus. The data showed that mutant alleles occur as single mutants in one of the spores in a tetrad, also when the mutant structure was the result of a combination of intra- and inter-allelic rearrangements. The conversional transfer of repeat units from one allele to the other was associated with flanking marker conversion which always involved the same flank of the minisatellite. The results demonstrate that conversion is the predominant mechanism by which minisatellite alleles mutate to new lengths, and also support the assumption that cis-acting elements are involved in the regulation of the mutational process in humans.

Base Sequence↗

PubMed1999

Global conformational changes control the reactivity of methane monooxygenase.

We present here X-ray scattering data that yield new structural information on the multicomponent enzyme methane monooxygenase and its components: a hydroxylase dimer, and two copies each of a reductase and regulatory protein B. Upon formation of the enzyme complex, the hydroxylase undergoes a dramatic conformational change that is observed in the scattering data as a fundamental change in shape of the scattering particle such that one dimension is narrowed (by 25% or 24 A) while the longest dimension increases (by 20% or 25 A). These changes also are reflected in a 13% increase in radius of gyration upon complex formation. Both the reductase and protein B are required for inducing the conformational change. We have modeled the scattering data for the complex by systematically modifying the crystal structure of the hydroxylase and using ellipsoids to represent the reductase and protein B components. Our model indicates that protein B plays a role in optimizing the interaction between the active centers of the reductase and hydroxylase components, thus, facilitating electron transfer between them. In addition, the model suggests reasons why the hydroxylase exists as a dimer and that a possible role for the outlying gamma-subunit may be to stabilize the complex through its interaction with the other components. We further show that proteolysis of protein B to form the inactive B' results in a conformational change and B' does not bind to the hydroxylase. The truncation thus could represent a regulatory mechanism for controlling the enzyme activity.

Computer Simulation↗

PubMed2006

Validated method for quantification of genetically modified organisms in samples of maize flour.

Sensitive and accurate testing for trace amounts of biotechnology-derived DNA from plant material is the prerequisite for detection of 1% or 0.5% genetically modified ingredients in food products or raw materials thereof. Compared to ELISA detection of expressed proteins, real-time PCR (RT-PCR) amplification has easier sample preparation and detection limits are lower. Of the different methods of DNA preparation CTAB method with high flexibility in starting material and generation of sufficient DNA with relevant quality was chosen. Previous RT-PCR data generated with the SYBR green detection method showed that the method is highly sensitive to sample matrices and genomic DNA content influencing the interpretation of results. Therefore, this paper describes a real-time DNA quantification based on the TaqMan probe method, indicating high accuracy and sensitivity with detection limits of lower than 18 copies per sample applicable and comparable to highly purified plasmid standards as well as complex matrices of genomic DNA samples. The results were evaluated with ValiData for homology of variance, linearity, accuracy of the standard curve, and standard deviation.

DNA↗

PubMed2001

The role of sugar residues in molecular recognition by vancomycin.

The sugar residues of the glycopeptide antibiotic vancomycin contribute to the cooperativity of ligand binding, thereby increasing ligand affinity and enhancing antimicrobial activity. To assess the structural basis for these effects, we determined a 0.98 A X-ray crystal structure of the vancomycin aglycon and compared it to structures of several intact vancomycin:ligand complexes. The crystal structure reveals that the aglycon binds acetate anions and forms back-to-back dimeric complexes in a manner similar to that of intact vancomycin. However, the four independent copies of the aglycon in each asymmetric unit of the crystal exhibit a high degree of conformational heterogeneity. These results suggest that the sugar residues, in addition to enlarging and strengthening the dimer interface, provide steric constraints that limit the vancomycin molecule to a relatively small number of productive conformations.

Carbohydrates↗

PubMed1998

Molecular characterization of a novel amplicon at 1q21-q22 frequently observed in human sarcomas.

In a recent comparative genomic hybridization (CGH) study of a panel of sarcomas, we detected recurrent amplification of 1q21-q22 in soft tissue and bone tumours. Amplification of this region had not previously been associated with sarcoma development, but occasional amplification of CACY/S100A6 and MUC1 in 1q21 had been reported for melanoma and breast carcinoma respectively. Initial screening by Southern blot analysis showed amplification of S100A6, FLG and SPRR3 in several sarcomas and, in a first attempt to characterize the 1q21-q22 amplicon in more detail, we have now investigated the amplification status of these and 11 other markers in the region in 35 sarcoma samples. FLG was the most frequently amplified gene, and the markers located in the same 4.5-Mb region as FLG showed a higher incidence of amplification than the more distal ones. However, for most of the 14 markers, amplification levels were low, and only APOA2 and the anonymous marker D1S3620 showed high-level amplifications (> tenfold increases) in one sample each. We used fluorescence in situ hybridization (FISH) to determine the amplification patterns of two overlapping yeast artificial chromosomes (YACs) covering the region between D1S3620 and FLG (789f2 and 764a1), as well as two more distally located YACs in nine selected samples. Six samples had amplification of the YAC containing D1S3620 and, in three, 764a1 was also included. Five of these tumours showed normal copies of the more distal YACs; thus, it seems likely that an important gene may be located within 789f2, or very close. Two samples had high copy numbers of the most distal YACs. Taken together, FISH and molecular analyses indicate complex amplification patterns in 1q21-q22 with at least two amplicons: one located near D1S3620/789f2 and one more distal.

Biomarkers↗

PubMed2005

The genome of the social amoeba Dictyostelium discoideum.

The social amoebae are exceptional in their ability to alternate between unicellular and multicellular forms. Here we describe the genome of the best-studied member of this group, Dictyostelium discoideum. The gene-dense chromosomes of this organism encode approximately 12,500 predicted proteins, a high proportion of which have long, repetitive amino acid tracts. There are many genes for polyketide synthases and ABC transporters, suggesting an extensive secondary metabolism for producing and exporting small molecules. The genome is rich in complex repeats, one class of which is clustered and may serve as centromeres. Partial copies of the extrachromosomal ribosomal DNA (rDNA) element are found at the ends of each chromosome, suggesting a novel telomere structure and the use of a common mechanism to maintain both the rDNA and chromosomal termini. A proteome-based phylogeny shows that the amoebozoa diverged from the animal-fungal lineage after the plant-animal split, but Dictyostelium seems to have retained more of the diversity of the ancestral genome than have plants, animals or fungi.

ATP-Binding Cassette Transporters↗

PubMed1997

Heterologous expression of rat epitope-tagged histamine H2 receptors in insect Sf9 cells.

1. Rat histamine H2 receptors were epitope-tagged with six histidine residues at the C-terminus to allow immunological detection of the receptor. Recombinant baculoviruses containing the epitope-tagged H2 receptor were prepared and were used to infect insect Sf9 cells. 2. The His-tagged H2 receptors expressed in insect Sf9 cells showed typical H2 receptor characteristics as determined with [125I]-aminopotentidine (APT) binding studies. 3. In Sf9 cells expressing the His-tagged H2 receptor histamine was able to stimulate cyclic AMP production 9 fold (EC50=2.1+/-0.1 microM) by use of the endogenous signalling pathway. The classical antagonists cimetidine, ranitidine and tiotidine inhibited histamine induced cyclic AMP production with Ki values of 0.60+/-0.43 microM, 0.25+/-0.15 microM and 28+/-7 nM, respectively (mean+/-s.e.mean, n=3). 4. The expression of the His-tagged H2 receptors in infected Sf9 cells reached functional levels of 6.6+/-0.6 pmol mg(-1) protein (mean+/-s.e.mean, n=3) after 3 days of infection. This represents about 2 x 10(6) copies of receptor/cell. Preincubation of the cells with 0.03 mM cholesterol-beta-cyclodextrin complex resulted in an increase of [125I]-APT binding up to 169+/-5% (mean+/-s.e.mean, n=3). 5. The addition of 0.03 mM cholesterol-beta-cyclodextrin complex did not affect histamine-induced cyclic AMP production. The EC50 value of histamine was 3.1+/-1.7 microM in the absence of cholesterol-beta-cyclodextrin complex and 11.1+/-5.5 microM in the presence of cholesterol-beta-cyclodextrin complex (mean+/-s.e.mean, n=3). Also, the amount of cyclic AMP produced in the presence of 100 microM histamine was identical, 85+/-18 pmol/10(6) cells in the absence and 81+/-11 pmol/10(6) cells in the presence of 0.03 mM cholesterol-beta-cyclodextrin complex (mean+/-s.e.mean, n=3). 6. Immunofluorescence studies with an antibody against the His-tag revealed that the majority of the His-tagged H2 receptors was localized inside the insect Sf9 cells, although plasma membrane labelling could be identified as well. 7. These experiments demonstrate the successful expression of His-tagged histamine H2 receptors in insect Sf9 cells. The H2 receptors couple functionally to the insect cell adenylate cyclase. However, our studies with cholesterol complementation and with immunofluorescent detection of the His-tag reveal that only a limited amount of H2 receptor protein is functional. These functional receptors are targeted to the plasma membrane.

Adenylyl Cyclases↗

PubMed2002

Ceramide-induced apoptosis of D283 medulloblastoma cells requires mitochondrial respiratory chain activity but occurs independently of caspases and is not sensitive to Bcl-xL overexpression.

Ceramides are potent lipid second messengers that are involved in apoptotic and hypoxic/ischaemic neurone death. We investigated the role of mitochondria and the mitochondrial apoptosis pathway in ceramide-induced cell death using human D283 medulloblastoma cells with a reduced mitochondrial DNA copy number (rho- cells) and a corresponding defect in mitochondrial respiration. Treatment with the complex I inhibitor rotenone, C2- or C8-ceramide induced cell death in D283 control cells, while rho- cells were significantly protected. In contrast, activation of the mitochondrial apoptosis pathway by transient overexpression of the pro-apoptotic Bax protein or exposure to the kinase inhibitor staurosporine induced apoptosis to a similar extent in control and rho- cells. Overexpression of the antiapoptotic protein Bcl-xL failed to inhibit the toxic effect of C2-ceramide in D283 control cells, and no significant increase in caspase-3-like protease activity could be detected during the death process. Despite this, C2-ceramide induced significant chromatin condensation and cell shrinkage in D283 control cells, reminiscent of apoptosis. These morphological alterations were associated with the activation of calpains. Both apoptotic morphology and calpain activation were attenuated in rho- cells. Our data indicate that the apoptosis-inducing effect of C2-ceramide may require mitochondrial respiratory chain activity and can occur independently of the mitochondrial apoptosis pathway, but involves the activation of calpains.

Apoptosis↗

PubMed2006

Distinguishing features of delta-proteobacterial genomes.

We analyzed several features of five currently available delta-proteobacterial genomes, including two aerobic bacteria exhibiting predatory behavior and three anaerobic sulfate-reducing bacteria. The delta genomes are distinguished from other bacteria by several properties: (i) The delta genomes contain two "giant" S1 ribosomal protein genes in contrast to all other bacterial types, which encode a single or no S1; (ii) in most delta-proteobacterial genomes the major ribosomal protein (RP) gene cluster is near the replication terminus whereas most bacterial genomes place the major RP cluster near the origin of replication; (iii) the delta genomes possess the rare combination of discriminating asparaginyl and glutaminyl tRNA synthetase (AARS) together with the amido-transferase complex (Gat CAB) genes that modify Asp-tRNA(Asn) into Asn-tRNA(Asn) and Glu-tRNA(Gln) into Gln-tRNA(Gln); (iv) the TonB receptors and ferric siderophore receptors that facilitate uptake and removal of complex metals are common among delta genomes; (v) the anaerobic delta genomes encode multiple copies of the anaerobic detoxification protein rubrerythrin that can neutralize hydrogen peroxide; and (vi) sigma(54) activators play a more important role in the delta genomes than in other bacteria. delta genomes have a plethora of enhancer binding proteins that respond to environmental and intracellular cues, often as part of two-component systems; (vii) delta genomes encode multiple copies of metallo-beta-lactamase enzymes; (viii) a host of secretion proteins emphasizing SecA, SecB, and SecY may be especially useful in the predatory activities of Myxococcus xanthus; (ix) delta proteobacteria drive many multiprotein machines in their periplasms and outer membrane, including chaperone-feeding machines, jets for slime secretion, and type IV pili. Bdellovibrio replicates in the periplasm of prey cells. The sulfate-reducing delta proteobacteria metabolize hydrogen and generate a proton gradient by electron transport. The predicted highly expressed genes from delta genomes reflect their different ecologies, metabolic strategies, and adaptations.

Amino Acyl-tRNA Synthetases↗

PubMed2006

A singular enzymatic megacomplex from Bacillus subtilis.

Nonribosomal peptide synthetases (NRPS), polyketide synthases (PKS), and hybrid NRPS/PKS are of particular interest, because they produce numerous therapeutic agents, have great potential for engineering novel compounds, and are the largest enzymes known. The predicted masses of known enzymatic assembly lines can reach almost 5 megadaltons, dwarfing even the ribosome (approximately 2.6 megadaltons). Despite their uniqueness and importance, little is known about the organization of these enzymes within the native producer cells. Here we report that an 80-kb gene cluster, which occupies approximately 2% of the Bacillus subtilis genome, encodes the subunits of approximately 2.5 megadalton active hybrid NRPS/PKS. Many copies of the NRPS/PKS assemble into a single organelle-like membrane-associated complex of tens to hundreds of megadaltons. Such an enzymatic megacomplex is unprecedented in bacterial subcellular organization and has important implications for engineering novel NRPS/PKSs.

Amino Acid Sequence↗

PubMed1972

The thiol group in the catalytic chains of aspartate transcarbamoylase.

The allosteric enzyme aspartate transcarbamoylase (EC 2.1.3.2) was previously shown to consist of two functionally distinct types of polypeptide chains. X-ray diffraction and chemical studies showed that there are six copies of both catalytic (C) and regulatory (R) chains, and that the intact molecular complex (C(6)R(6)) has D(3) symmetry. Organomercurials react preferentially with the four thiol groups on each R chain, dissociating the molecular complex. We show that 2-chloromercuri-4-nitrophenol reacts specifically and rapidly with the single C-chain thiol, which is believed to be near the catalytic site. This reaction inactivates the enzyme in solution and does not dissociate the molecular complex. Spectrophotometric titration and mercury analysis indicates that six molecules of this mercurial are firmly bound to the enzyme (R(6)C(6)), and crystallographic studies establish that only six sites, related by D(3) symmetry, are modified. The known low reactivity of this C-chain thiol with other sulfhydryl reagents, the unusual structural requirements in the reaction with 2-chloromercuri-4-nitrophenol, and the spectral properties of the resulting derivative provide insight into the environment of this thiol. Probably, at least one positively charged group of the enzyme is nearby, and the environment of this thiol is at least partially hydrophobic.

Allosteric Regulation↗

PubMed1993

The ATP-binding component of a prokaryotic traffic ATPase is exposed to the periplasmic (external) surface.

The membrane-bound complex of bacterial periplasmic permeases consists of two hydrophobic integral membrane proteins and two copies of a hydrophilic ATP-binding protein. The ATP-binding proteins from all periplasmic permeases display a high level of sequence similarity and are referred to as "conserved components." The conserved component from the histidine permease, HisP, has been postulated on the basis of genetic evidence to be accessible at the exterior membrane surface, in contrast to the commonly postulated association with the interior membrane surface as peripheral membrane proteins. We have used proteolysis and biotinylation of membrane vesicles to show that HisP is accessible to these reagents at the external surface and that this orientation depends on the presence of the two hydrophobic components, HisQ and HisM. Several binding-protein-independent hisP mutants are shown to produce HisP proteins that are more susceptible to proteases from the external membrane surface. Since the hydrophilic component is well conserved also in a group of eukaryotic transporters, which together with many prokaryotic systems form the superfamily of traffic ATPases, this insight about its membrane topology has general implications for understanding the molecular mechanism of action of this large superfamily, which includes the cystic fibrosis transmembrane conductance regulator and multidrug-resistance proteins.

ATP-Binding Cassette Transporters↗