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PubMed1992

In vitro excision and replication of 5' telomeres of minute virus of mice DNA from cloned palindromic concatemer junctions.

HeLa cell extracts containing wild-type copies of the minute virus of mice NS-1 polypeptide produced from a recombinant vaccinia virus vector could support the excision and replication of viral 5' telomeres from cloned concatemer junction fragments. Resolution did not occur if wild-type NS-1 was omitted from the extract or if the substrate DNA contained palindromic sequences without specific viral resolution sites. In the presence of NS-1, [32P]dGTP incorporation into all templates was slightly increased, but if the template contained specific viral resolution sites DNA synthesis was greatly enhanced, and took two distinct forms: (i) generation of a limited number of high-molecular-weight molecules, probably due to a form of rolling-circle replication, and (ii) synthesis of new DNA at the viral telomeres. Resolution of the junction fragment generated two newly synthesized viral telomeres, each of which was covalently associated with NS-1 and contained a duplex copy of the complex palindrome located around the axis of symmetry of the concatemer junction. Cloned junction fragments of 296 bp or more could be resolved efficiently in vitro, and since NS-1 molecules were left covalently attached to the newly resolved termini, the latter could be partially purified by immuno-precipitation with anti-NS-1 serum. Restriction analysis and further fractionation of the precipitated DNA showed that the in vitro resolution sites were in the predicted positions on either side of the axis of symmetry, and that de novo DNA synthesis was associated predominantly with one of the two daughter strands. Telomeres were generated from both arms of the substrate with equal efficiency, and contained the characteristic "flip" and "flop" sequence inversions observed in vivo. Since a high proportion of termini were associated with adjacent viral sequences that retained the bacterial methylation pattern, in vitro resolution was not dependent upon prior DNA replication proceeding through the entire palindromic insert.

Blotting, Southern↗

PubMed1978

Plasmids useable as gene-cloning vectors in an in vitro packaging by coliphage lambda: "cosmids".

A plasmid which contains a cos site of lambda and can be packaged into lambda bacteriophage particles is termed a "cosmid". Such plasmids can be used as gene cloning vectors in conjunction with an in vitro packaging system. The properties of a new series of cosmids based on the ColE1 replicon are described, including small temperature-sensitive plasmids which have lost mobilisation functions and carry no IS sequences. Amongst these plasmids are vectors for XmaI, BglII, BamHI, HindIII, PstI, KpnI, SalI and EcoRI. It is demonstrated that by using cosmids in particular size ranges these plasmids provide a high efficiency cloning system which yields essentially only hybrid clones without resort to a second selection or screening step, and without prior modification (e.g. phosphatase) treatment of the DNA. Attempts were made to optimise the cloning properties of the cosmid system. An Escherichia coli "gene bank" was obtained with an efficiency of 5 . 10(5) clones per microgram of E. coli DNA, and in which any particular unselected marker may be found in about one out of every 400 clones. It was demonstrated that deletion of mobilisation functions leads to loss of ability to form relaxation-complex without affecting copy number or segregation properties of the temperature-sensitive derivatives. The vectors are amplifiable in chloramphenicol to make up about 50% of the total cellular DNA.

Coliphages↗

PubMedDate not supplied

Effects of occupational exposure to organic solvents upon cognitive performance.

Twenty-three individuals exposed to mixed organic solvents were compared with 23 nonexposed controls on a number of cognitive performance tasks. Solvent exposure resulted in a significantly poorer performance on the forward digit span test, copying of a complex figure, and on semantic memory tests which also measure individual's ability to integrate linguistic information into cohesive units. These tasks rely heavily upon short-term memory and its integrative operations in higher cognitive function. Acute exposure effect was also observed for the linguistic integrative task.

Adult↗

PubMed2003

Structure of NFAT bound to DNA as a monomer.

The nuclear factor of activated T cells (NFAT) is a calcium-dependent transcription factor that cooperates with a myriad of partner transcription factors to regulate distinct transcription programs. Transcription activation by NFAT without the cooperation of co-stimulatory signals in lymphocytes can also impose a genetic program of anergy. Although the ternary NFAT1/Fos-Jun/DNA complex has been structurally characterized, how NFAT1 recognizes DNA in the absence of cooperative partners and how such a binary NFAT/DNA complex may lead to the assembly of distinct high-order NFAT transcription complexes are still poorly understood. We have determined the crystal structure of the entire Rel homology region (RHR) of human NFAT1 (NFATc2) bound to DNA as a monomer. We also present footprinting evidence that corroborates the protein-DNA contacts observed in the crystal structure. Our structural and biochemical studies reveal the mechanism by which the monomeric Rel protein NFAT recognizes its cognate DNA site. A remarkable feature of the binary NFAT/DNA complex is the conformational flexibility exhibited by NFAT1 in the four independent copies of the NFAT/DNA complex in the crystal structure, which may reflect a mechanism by which NFAT1 interacts with a variety of protein partners as it mediates disparate biological responses.

Amino Acid Sequence↗

PubMed1996

Failure to agree on the electrocardiographic diagnosis of ventricular tachycardia.

STUDY OBJECTIVE: To determine the extent of interobserver agreement in the ECG diagnosis of ventricular tachycardia (VT) by using a four-step algorithm and three observers. METHODS: Simulated emergency department setting from records of an urban university teaching hospital. All ECGs taken in the ED during a 2-year period that showed a QRS duration of more than 120 msec and a heart rate faster than 110 beats per minute were reviewed. ECGs were categorized as demonstrating sinus rhythm (SR), irregular broad-complex tachycardia (I-BCT), or regular broad-complex tachycardia (BCT). Copies of the BCT ECGs and short clinical histories were given to each of three emergency physicians, who used a published, four-step algorithm (the Brugada algorithm) to categorize the BCT ECGs as indicating VT, indicating supraventricular tachycardia with aberrancy (SVT-A), or indeterminate. Interobserver agreement was assessed with the K-statistic. RESULTS: The records contained 178 ECGs, 88 of which were SR, 63 I-BCT, and 27 BCT. The 27 BCT ECGs were selected for review. The emergency physicians disagreed with each other 22% of the time in differentiating VT from SVT-A (K = .58). CONCLUSION: Application of the algorithm to actual clinical practice in the ED would probably result in the misdiagnosis of a substantial minority of patients having BCT, with potentially serious adverse consequences.

Adult↗

PubMed2001

Effects of zaleplon or triazolam with or without ethanol on human performance.

Objectives and background: Given that non-selective gamma-aminobutyric acid (GABA) agonist hypnotics impair performance and potentiate the disruptive effects of ethanol, this study was done to determine the performance-impairing and ethanol-potentiating effects of zaleplon, a new selective GABA agonist hypnotic.Methods: Eighteen healthy men (12) and women (six), 31.5+/-5.6 years old, were studied. Each underwent six treatments of 2 days in duration, presented in a Latin square design with 2-12 recovery days between. The treatments were: placebo-placebo; placebo-ethanol; triazolam-placebo; triazolam-ethanol; zaleplon-placebo; and zaleplon-ethanol; with triazolam (0.25 mg) or placebo administered at 08:30 h, zaleplon (10 mg) or placebo at 09:00 h, and ethanol (0.75 g/kg) or placebo consumed from 09:30 h. Performance tests were completed each day at 10:30, 12:00 and 14:30 h.Results: Breath ethanol concentration (BrEC), tested 0.5, 2.0, 4.5 and 6 h post consumption, did not differ among treatments and peaked at 0.052%, declining to 0.037, 0.009 and 0.001%. Triazolam with and without ethanol impaired digit symbol substitution, symbol copying, simple and complex reaction times and divided attention performance relative to placebo-placebo treatment. It did so consistently at 10:30 and 12:00 h, and less consistently at 14:30 h. Zaleplon without ethanol impaired only digit symbol substitution and divided attention tracking, and only at 10:30 h. Zaleplon with ethanol impaired most measures at 10:30 and 12:00 h, but not at 14:30 h. Zaleplon without ethanol consistently differed from triazolam without ethanol in the extent of performance impairment. Zaleplon with ethanol began to differ from triazolam with ethanol in performance impairment on the 12:00 and 14:30 h test sessions. Ethanol itself impaired most measures at 10:30 h, fewer at 12:00 h and none at 14:30 h. All active drug treatments increased self-rated sleepiness compared with placebo-placebo. Triazolam without ethanol produced greater self-rated sleepiness than zaleplon without ethanol. The addition of ethanol to both drugs generally produced comparable levels of self-rated sleepiness.Conclusions: In an absolute sense, zaleplon produced less performance impairment and a shorter period of ethanol potentiation than triazolam.

Journal Article↗

PubMedDate not supplied

Fluorescently detectable magnetic resonance imaging agents.

This report describes the synthesis, characterization, and in vivo testing of several bifunctional contrast-enhancing agents for optical and magnetic resonance imaging (MRI) of experimental animals. These new agents integrate the advantages of both techniques since they can be visualized simultaneously by light and MRI microscopy. Employing this strategy allows the same biological structures of a specimen to be studied at dramatically different resolutions and depths. The complexes possess a metal chelator for binding a paramagnetic ion, gadolinium (Gd3+), and a covalently attached fluorescent dye. The first class of complexes are low-molecular weight species that are composed of the macrocyclic tetraamine 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA) as the metal-chelating ligand coupled to tetramethylrhodamine. The second class of MRI-enhancing agents are composed of high-molecular weight polymers that are membrane impermeable and once injected into a cell or cells are trapped inside. These complexes possess multiple copies of both the metal-chelator-diethylenetriaminepentaacetic acid (DTPA) and the tetramethylrhodamine attached to a macromolecular framework of either poly(D-lysine) (pdl) or dextran. Images acquired of single cells after injection with these bifunctional agents enabled us to follow the relative motions and reorganizations of different cell layers during amphibian gastrulation and neurulation in Xenopus laevis embryos.

Animals↗

PubMed2001

Dsl1p, an essential protein required for membrane traffic at the endoplasmic reticulum/Golgi interface in yeast.

To identify novel factors required for ER to Golgi transport in yeast we performed a screen for genes that, when mutated, confer a dependence on a dominant mutant form of the ER to Golgi vesicle docking factor Sly1p, termed Sly1-20p. DSL1, a novel gene isolated in the screen, encodes an essential protein with a predicted molecular mass of 88 kDa. DSL1 is required for transport between the ER and the Golgi because strains bearing mutant alleles of this gene accumulate the pre-Golgi form of transported proteins at the restrictive temperature. Two strains bearing temperature-sensitive alleles of DSL1 display distinct phenotypes as observed by electron microscopy at the restrictive temperature; although both strains accumulate ER membrane, one also accumulates vesicles. Interestingly, the inviability of strains bearing several mutant alleles of DSL1 can be suppressed by expression of either Erv14p (a protein required for the movement of a specific protein from the ER to the Golgi), Sec21p (the gamma-subunit of the COPI coat protein complex coatomer), or Sly1-20p. Because the strongest suppressor is SEC21, we proposed that Dsl1p functions primarily in retrograde Golgi to ER traffic, although it is possible that Dsl1p functions in anterograde traffic as well, perhaps at the docking stage, as suggested by the suppression by SLY1-20.

Amino Acid Sequence↗

PubMed2001

Recruitment of coat-protein-complex proteins on to phagosomal membranes is regulated by a brefeldin A-sensitive ADP-ribosylation factor.

Particle internalization in macrophages is followed by a complex maturation process. We have previously observed that proteins bound to phagocytosed particles are sorted from phagosomes into a heterogeneous population of vesicles that fuse with endosomes. However, the mechanism and the protein machinery involved in the formation of these phagosome-derived vesicles are largely unknown. It has been shown that vesicles coated with coat protein complex type I (COPI) have a role in both secretion and endocytosis. To address the possibility that COPI proteins might participate in the formation of phagosome-derived vesicles we studied the recruitment of beta-COP to highly purified phagosomes. The binding of beta-COP to phagosomal membranes was regulated by nucleotides and inhibited by brefeldin A (BFA). An ADP-ribosylation factor 1 (ARF1) mutant defective in GTP hydrolysis supported the binding of beta-COP to phagosomes independently of added nucleotide. AlF(4) and Gbetagamma subunits, agents known to modulate heterotrimeric G-protein activity, were tested in the beta-COP binding assay. AlF(4) increased beta-COP association, whereas binding was inhibited by the addition of Gbetagamma subunits. Our results suggest that COP proteins are recruited to phagosomal membranes by a mechanism that involves heterotrimeric GTP-binding proteins and a BFA-sensitive ARF. In addition, our findings indicate that COPI proteins are involved in the recycling of components from phagosomes to the cell surface.

ADP-Ribosylation Factors↗

PubMed2006

Multimers of the fibroblast growth factor (FGF)-FGF receptor-saccharide complex are formed on long oligomers of heparin.

The minimal signalling unit for tyrosine kinase receptors is two protomers dimerized by one or more ligands. However, it is clear that maximal signalling requires the formation of larger complexes of many receptors at discrete foci on the cell surface. The biological interactions that lead to this are likely to be diverse and have system specific components. In the present study, we demonstrate that, in the FGF (fibroblast growth factor)-FGFR (FGF receptor) system, multimers of the minimal complex composed of two FGF1 and two FGFR2 protomers can form on a single chain of the co-receptor heparin. Using size-exclusion chromatography, we show that two complexes can form on heparin chains as small as 16 saccharide units. We also show by MS that discrete complexes containing exactly two copies of the minimal signalling unit are formed. However, the doublet of complexes appears to be less co-operative than the formation of the 2:2:1 FGF1:FGFR2:heparin complex, suggesting that this mechanism is one of a number of weaker interactions that might be involved in the formation of a focal complex on the cell surface.

Animals↗

PubMed2003

Protein pathway and complex clustering of correlated mRNA and protein expression analyses in Saccharomyces cerevisiae.

The mRNA and protein expression in Saccharomyces cerevisiae cultured in rich or minimal media was analyzed by oligonucleotide arrays and quantitative multidimensional protein identification technology. The overall correlation between mRNA and protein expression was weakly positive with a Spearman rank correlation coefficient of 0.45 for 678 loci. To place the data sets in a proper biological context, a clustering approach based on protein pathways and protein complexes was implemented. Protein expression levels were transcriptionally controlled for not only single loci but for entire protein pathways (e.g., Met, Arg, and Leu biosynthetic pathways). In contrast, the protein expression of loci in several protein complexes (e.g., SPT, COPI, and ribosome) was posttranscriptionally controlled. The coupling of the methods described provided insight into the biology of S. cerevisiae and a clustering strategy by which future studies should be based.

Amino Acid Sequence↗

PubMed1977

Poliovirus-specific primer-dependent RNA polymerase able to copy poly(A).

A template-dependent RNA polymerase has been isolated from poliovirus-infected cells by assaying for the ability of the enzyme to copy poly(A) complexed to an oligo(U) primer. The polymerase was solubilized with detergent, and RNA was removed by precipitation with 2 M LiCl. The solubilized polymerase required both poly(A) and oligo(U) for activity and was stimulated by Mg2+ but was inhibited by Mn2+. Poly(A)-oligo(U)-dependent poly(U) polymerase was not found in extracts of HeLa cells until about 2 hr after poliovirus infection, and then there was a linear increase in activity until about 5 hr. Analysis of the polymerase by glycerol gradient centrifugation showed that the majority of the activity sedimented at about 4 S, indicating that it was no longer complexed with high-molecular-weight RNA or cellular membranes. This poly(A)-oligo(U)-dependent polymerase activity could represent an important component of the poliovirus RNA-dependent RNA polymerase.

HeLa Cells↗

PubMed1995

Altered coding for a strictly conserved di-glycine in the major bilirubin UDP-glucuronosyltransferase of a Crigler-Najjar type I patient.

The characterization (Ritter, J.K., Chen, F., Sheen, Y. Y., Tran, H.M., Kimura, S., Yeatman, M.T., and Owens, I. S. (1992) J. Biol. Chem. 267, 3257-3261) of the single-copy UGT1 gene complex locus encoding both bilirubin and phenol UDP-glucuronosyltransferases (transferase) has been critical to the determination of genetic defects in Crigler-Najjar patients. The complex (UGT1A-UGT1M) codes for at least two bilirubin, three bilirubin-like, and eight phenol transferase isozymes. In the 5' region, a minimum of 13 different exons 1, each with an upstream promoter, are arrayed in series with 4 common exons in the 3' region of the locus. Each exon 1 encodes the amino terminus of a transferase, and the common exons encode the common carboxyl terminus of each isoform. Although a deleterious mutation in a common exon inactivates the entire locus, a deleterious mutation in an exon 1, as we report here for the UGT1A gene in a Crigler-Najjar Type I patient, affects the amino terminus of that single isoform. Recessively inherited mutant alleles for the predominant bilirubin isozyme, the HUG-Br1 protein, substituted Arg for Gly at codon 276 (G276R) in exon 1 of UGT1A abolishing a conserved di-glycine. The mutant HUG-Br1-G276R protein expressed in COS-1 cells had no detectable bilirubin glucuronidating activity at either pH 7.6 or 6.4. Although each of the bilirubin-type isozymes contains a conserved peptide between residues 270 and 288, all UDP-glucuronosyltransferases contain a di-glycine at approximately position 276-277, making it strictly conserved. Structure-function relationship was studied by site-directed mutations of the HUG-Br1 cDNA; G276A, G276Q, G276E, G276I, and P270G mutants were inactive, and V2751- and P285G-altered transferases expressed normal activity. Conservation of residues between the related baculoviral ecdysone UDP-glucosyltransferase and the UDP-glucuronosyltransferases confirms the critical role of the Gly-276 as well as other residues.

Amino Acid Sequence↗

PubMed1996

A novel factor required for the assembly of the DnaK and DnaJ chaperones of Thermus thermophilus.

We previously reported the isolation of T.DnaK.DnaJ chaperone complex from Thermus thermophilus. Here, we show that a novel factor is necessary for the assembly of T.DnaK and T.DnaJ into the complex. A dnaK gene cluster of T. thermophilus contained five genes, dnaK-grpE-dnaJ-orf4-clpB. Interestingly, T.DnaJ lacks the whole "cysteine-rich region" that has been postulated to be necessary to bind unfolded proteins. The orf4 gene encodes a novel 78-amino acid protein. Curiously, T.DnaK and T.DnaJ expressed in Escherichia coli did not form the complex. Careful reexamination of the T.DnaK.DnaJ complex revealed the presence of a small protein in the complex, which turned out to be a product of orf4. As expected, expression of three genes, dnaK-dnaJ-orf4, resulted in production of a T.DnaK.DnaJ complex in E. coli that was indistinguishable from the authentic complex in its ability to interact with nucleotide and denatured protein. The product of orf4 was also required for in vitro reconstitution of the complex and named T.DafA (T.DnaK.DnaJ assembly factor A). The complex comprises three copies each of T.DnaK, T.DnaJ, and T.DafA. Even though a definite homolog of T.DafA has not been found in the data base, this finding raises a possibility that interaction between DnaK and DnaJ chaperones in other organisms is also mediated by a small protein yet unnoticed.

Amino Acid Sequence↗

PubMed1997

Different domains of mammalian ADP-ribosylation factor 1 mediate interaction with selected target proteins.

Mammalian ADP-ribosylation factor 1 (mARF1) is a small GTP-binding protein that is activated by a Golgi guanine nucleotide exchange factor. Once bound to the Golgi membranes in the GTP form, mARF1 initiates the recruitment of the adaptor protein 1 (AP-1) complex and coatomer (COPI) onto these membranes and activates phospholipase D1 (PLD1). To map the domains of mARF1 that are important for these activities, we constructed chimeras between mARF1 and Saccharomyces cerevisiae ARF2, which functions poorly in all of these processes except COPI recruitment. The carboxyl half of mARF1 (amino acids 95-181) was essential for activation by the Golgi guanine nucleotide exchange factor, whereas a separate domain (residues 35-94) was required to effectively activate PLD1 and to promote efficient AP-1 recruitment. Since residues 35-94 of mARF1 are critical for optimal activity in both PLD1 activation and AP-1 recruitment, we hypothesize that this region of ARF contains residues that interact with effector molecules.

ADP-Ribosylation Factors↗

PubMed1994

Visuomotor organization in the right-handed and the left-handed child: a comparative neuropsychological approach.

The present study was to assess differences between left- and right-handed children on the variable of visual-motor organization, as demonstrated by copying of a complex figure. The sample consisted of 420 right-handed and 389 left-handed children aged 5.5 to 12.5 years, who were group-matched on the basis of age, sex and hand-preference. Analysis indicated that girls show significantly better performance than boys during the ages 7.5 to 9.5, while both sex and hand-preference create a handicap for right-handed boys during ages 9.5 to 12.5. The differences in performance can be attributed to differential neuropsychological strategies or functional differences between sexes and between right and left-handed children.

Journal Article↗

PubMed1999

Recombinant major vault protein is targeted to neuritic tips of PC12 cells.

The major vault protein (MVP) is the predominant constituent of ubiquitous, evolutionarily conserved large cytoplasmic ribonucleoprotein particles of unknown function. Vaults are multimeric protein complexes with several copies of an untranslated RNA. Double labeling employing laser-assisted confocal microscopy and indirect immunofluorescence demonstrates partial colocalization of vaults with cytoskeletal elements in Chinese hamster ovary (CHO) and nerve growth factor (NGF)-treated neuronlike PC12 cells. Transfection of CHO and PC12 cells with a cDNA encoding the rat major vault protein containing a vesicular stomatitis virus glycoprotein epitope tag demonstrates that the recombinant protein is sorted into vault particles and targeted like endogenous MVPs. In neuritic extensions of differentiated PC12 cells, there is an almost complete overlap of the distribution of microtubules and vaults. A pronounced colocalization of vaults with filamentous actin can be seen in the tips of neurites. Moreover, in NGF-treated PC12 cells the location of vaults partially coincides with vesicular markers. Within the terminal tips of neurites vaults are located near secretory organelles. Our observations suggest that the vault particles are transported along cytoskeletal-based cellular tracks.

Actins↗

PubMed2003

Manipulation of Rubisco: the amount, activity, function and regulation.

Genetic modification to increase the specificity of Rubisco for CO(2) relative to O(2) and to increase the catalytic rate of Rubisco in crop plants would have great agronomic importance. The availability of three-dimensional structures of Rubisco at atomic resolution and the characterization of site-directed mutants have greatly enhanced the understanding of the catalytic mechanism of Rubisco. Considerable progress has been made in identifying natural variation in the catalytic properties of Rubisco from different species and in developing the tools for introducing both novel and foreign Rubisco genes into plants. The additional complexities of assembling copies of the two distinct polypeptide subunits of Rubisco into a functional holoenzyme in vivo (requiring sufficient expression, post-translational modification, interaction with chaperonins, and interaction with Rubisco activase) remain a major challenge. The consequences of changing the amount of Rubisco present in leaves have been investigated by the use of antisense constructs. The manipulation of genes encoding Rubisco activase has provided a means to investigate the regulation of Rubisco activity.

Carbon Dioxide↗