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At least 865 records · Page 48Linked to original sources
PubMed1981

Ground squirrel hepatitis virus DNA: molecular cloning and comparison with hepatitis B virus DNA.

Ground squirrel hepatitis virus (GSHV) shares many ultrastructural antigenic, molecular, and biological features with hepatitis B virus (HBV) of humans, indicating that they are members of the same virus group. Both viruses contain small circular DNA molecules which are partially single stranded. Here, we ligated an endonuclease EcoRI digest of GSHV DNA with EcoRI-cleaved plasmid vector pBR322 and cloned recombinant plasmids in Escherichia coli C600. Two cloned recombinants were characterized. One (pGS2) was found to contain only part of the GSHV genome, and the other (pGS11) was found to contain the entire viral DNA. A restriction endonuclease cleavage map of the GSHV insert in pGS11 and the locations of certain physical features of the virion DNA were determined. The relative positions of the single-stranded region, the unique 5' end of the short DNA strand, and the unique nick in the long DNA strand in GSHV DNA were found to be the same as those previously described for HBV DNA. Hybridization with an HBV [32P]DNA probe containing the apparent coding sequence for the major polypeptide of HBV surface antigen and a probe containing the putative coding sequence for the major polypeptide of the HBV core revealed specific homology with different restriction fragments of GSHV DNA. The two homologous regions had approximately the same locations relative to the single-stranded region, the 5' end of the short strand, and the nick in the long strand in the two viral DNAs. These results suggest that in both viruses the genes for the major HBV surface antigen and core polypeptides have the same locations relative to unique physical features of the viral DNAs.

Animals↗
PubMed1987

DAPI-pUC8 complex: a tool to investigate biological effects of nucleic acid-drug interaction.

A complex consisting of pUC8, a 1.8 Md plasmid, and 4'-6-diamidino-2-phenylindole, a DNA-binding agent, has been performed in vitro under different conditions of ionic strength, and used to transform competent cells. A strong interference with the plasmid-coded activities, related to the P/D ratio where at the DNA-drug complex was formed, was shown to occur. Since the compound does not inhibit the uptake process neither affects plasmid activity once dissociated at high ionic strength, it is likely to be acting from inside the cell while still in the form of a DNA-adduct. This system is proposed as a useful tool to investigate the effects on target genes of drugs endowed with DNA sequence specificity.

Circular Dichroism↗
PubMed1984

Kinetoplast DNA minicircles of trypanosomatids encode for a protein product.

The major constituent of the trypanosomal kinetoplast DNA network are several thousand duplex DNA minicircles whose biological function is still unknown. The coding capacity and expression of these DNA minicircles, was studied in the trypanosomatid Crithidia fasciculata. Kinetoplast DNA minicircle fragments inserted into bacterial plasmid vectors were expressed in the bacterial cell. Sera elicited in rabbits, by immunization with the translational products of kinetoplast DNA minicircles in E. coli, reacted specifically with Crithidia fasciculata cellular antigens. It is inferred that kinetoplast DNA minicircles contain long open reading frames of nucleotides which are expressed in the trypanosomatid cell.

Animals↗
PubMed2003

Ligation of exogenous linear DNA after gene transfer in vitro and in vivo.

BACKGROUND: We have analyzed the physical/topographical state of linear exogenous DNA after gene transfer in vitro and in vivo. METHODS AND RESULTS: Linear DNA carrying a luciferase expression cassette, either intact or corrupted within the coding region, was tested in gene transfer experiments in vitro and in vivo. To this, a plasmid with a CMV-IE1 promoter-driven luciferase gene was rendered non-functional by the insertion of a 1.2 kb EcoRV-EcoRV fragment. After removal of the insert by digestion with EcoRV, the resulting linear DNA fragments were used to transfect HeLa cells. The recovery of luciferase activity from these cells indicated functional reconstitution of the expression cassette. Recovery of low molecular weight DNA from HeLa cells allowed amplification of an intact luciferase gene, confirming accurate ligation of free DNA ends. In the mouse, rapid intravenous injection of plasmid DNA, linearized within the luciferase gene, resulted in significant luciferase activities in liver and lung. Ligation products could be detected by PCR. CONCLUSIONS: These data suggest that linear DNA is efficiently circularized after gene transfer in vitro and in vivo. Secondly, equally high luciferase activities were observed in the mouse after rapid intravenous injection of luciferase expression cassettes, either consisting of linear DNA produced by PCR, or carried by linearized plasmid DNA. These findings encourage the use of linear DNA elements for gene transfer applications in vivo.

Animals↗
PubMed1988

'DNA Strider': a 'C' program for the fast analysis of DNA and protein sequences on the Apple Macintosh family of computers.

DNA Strider is a new integrated DNA and Protein sequence analysis program written with the C language for the Macintosh Plus, SE and II computers. It has been designed as an easy to learn and use program as well as a fast and efficient tool for the day-to-day sequence analysis work. The program consists of a multi-window sequence editor and of various DNA and Protein analysis functions. The editor may use 4 different types of sequences (DNA, degenerate DNA, RNA and one-letter coded protein) and can handle simultaneously 6 sequences of any type up to 32.5 kB each. Negative numbering of the bases is allowed for DNA sequences. All classical restriction and translation analysis functions are present and can be performed in any order on any open sequence or part of a sequence. The main feature of the program is that the same analysis function can be repeated several times on different sequences, thus generating multiple windows on the screen. Many graphic capabilities have been incorporated such as graphic restriction map, hydrophobicity profile and the CAI plot- codon adaptation index according to Sharp and Li. The restriction sites search uses a newly designed fast hexamer look-ahead algorithm. Typical runtime for the search of all sites with a library of 130 restriction endonucleases is 1 second per 10,000 bases. The circular graphic restriction map of the pBR322 plasmid can be therefore computed from its sequence and displayed on the Macintosh Plus screen within 2 seconds and its multiline restriction map obtained in a scrolling window within 5 seconds.

Amino Acid Sequence↗
PubMed2005

Dissection of human tropoelastin: supramolecular organization of polypeptide sequences coded by particular exons.

Polypeptide sequences encoded by some exons of the human tropoelastin gene (EDP, elastin-derived peptide) have been analysed for their ability to coacervate and to self-assembly. The great majority of them were shown to form organized structures, but only a few were indeed able to coacervate. Negative staining and rotary shadowing transmission electron microscopy showed the polypeptides to adopt a variety of supramolecular organization, from filaments, as those typical of tropoelastin, to amyloid-like fibers. The results obtained gave significant insight to the possible roles played by specific polypeptide sequences of tropoelastin.

Amyloid↗
PubMed1998

Expression, purification, and characterization of recombinant human interleukin-13 from NS-O cells.

Interleukin-13 is a cytokine which is secreted by activated T lymphocytes and primarily impacts monocytes, macrophages, and B cells. A synthetic gene coding for human interleukin-13 has been prepared and cloned into expression vector pEE12. The construct was transfected into NS-O cells, which showed stable expression of the recombinant protein. A four-step purification procedure consisting of S-Sepharose, Q-Sepharose, hydroxyapatite, and Sephacryl-100 chromatographies yielded bioactive interleukin-13 of > 98% purity. The purified protein was structurally characterized. The extinction coefficient at 280 nm was determined to be 5678 M-1 cm-1. Amino acid sequencing confirmed that the N-terminus of the purified protein was intact. Electrospray mass spectrometric analysis, size-exclusion chromatography, and SDS-PAGE revealed that the biologically active protein is monomeric and unglycosylated. Mass spectrometry and a chemical assay for free sulfhydryls indicated that the four cysteine residues of interleukin-13 are involved in two intramolecular disulfide bonds. The circular dichroism spectrum confirms that interleukin-13 belongs to the alpha-helical family of cytokines. A biologically inactive covalent trimer also forms in the cell culture, but can be separated from the monomer by the hydroxyapatite and size-exclusion chromatographies. These data indicate that human interleukin-13 retains many structural similarities to human interleukin-4, from which it arose by a gene duplication event.

Base Sequence↗
PubMed2005

Energetic characterization of short helical polyalanine peptides in water: analysis of 13C=O chemical shift data.

Measured at 2 degrees C in water, NMR chemical shifts of (13)C=O labeled central alanine residues of peptides W-Lys(5)-(t)L(3)-Ala(n)-(t)L(3)-Lys(5)NH(2), n = 9, 11, 13, 15, 19 and W-Lys(5)-(t)L(3)-a-Ala(n)-A-Inp-(t)L(2)-Lys(5)NH(2) (a = D-Ala; (t)L = tert-leucine; Inp = 4-carboxypiperidine) are used to assign jt(L) and ct(L), the N- and C-terminal (t)L capping parameters and length-dependent values for w(Ala)(n), the alanine helical propensity for Ala(n) peptides. These parameters allow Lifson-Roig characterization of the stabilities of Ala(n)() helices in water. To facilitate chemical shift characterization, different (13)C/(12)C ratios are incorporated into specific Ala sites to code up to six residue sites per peptide. Large left/right chemical shift anisotropies are intrinsic to helical polyalanines, and a correcting L-R-based model is introduced. Capping parameters jt(L) = ct(L) lie in the range of 0.3 to 0.5; the (t)L residues are thus moderately helix-destabilizing. For helical conformations of lengths shorter than eight residues, assigned values for w(Ala) approach 1.0 but increase monotonically with length to a value of 1.59 for w(Ala)(19).

Alanine↗
PubMed2001

Nucleotide sequence analysis of a novel circovirus of canaries and its relationship to other members of the genus Circovirus of the family Circoviridae.

The circular, single-stranded DNA genome of a novel circovirus of canaries, tentatively named canary circovirus (CaCV), was cloned and sequenced. Sequence analysis indicated that the genome was 1952 nucleotides (nt) in size and had the potential to encode three viral proteins, including the putative capsid and replication-associated (Rep) proteins. The CaCV genome shared greatest sequence similarity (58.3% nt identity) with the newly characterized columbid circovirus (CoCV) and was more distantly related to the two porcine circovirus strains, PCV1 and PCV2, beak and feather disease virus (BFDV) and a recently isolated goose circovirus (GCV) isolate (46.8-50.9% nt identity). In common with other members of the Circovirus genus, several nt structures and amino acid motifs thought to be implicated in virus replication were identified on the putative viral strand. Phylogenetic analysis of both the capsid and Rep protein-coding regions provided further evidence that CaCV is more closely related to CoCV and BFDV and more distantly related to GCV, PCV1 and PCV2.

Amino Acid Sequence↗
PubMed1994

Recombinant leech-derived tryptase inhibitor: construction, production, protein chemical characterization and inhibition of HIV-1 replication.

A synthetic gene coding for leech-derived tryptase inhibitor, form C (LDTI-C), was designed, cloned and expressed. The gene assembled via 6 oligonucleotides contains linker sequences, stop codons and internal restriction recognition sites for cloning, expression and cassette mutagenesis. Periplasmatic expression products could not be detected in Escherichia coli (E. coli), but strong expression was found using Saccharomyces cerevisiae (S. cerevisiae) ( > 10 mg/l culture broth) if a variant of pVT102U/alpha was used as vector. The secreted material was isolated after cross-flow filtration and purified by cation exchange chromatography. The recombinant material proved to be pure and homogeneous by electrophoretic and chromatographic analyses. Amino acid sequencing and molecular mass determination (4737.6 +/- 0.77 Da) by electrospray ionization mass spectrometry confirmed that rLDTI-C was processed correctly and that it is indistinguishable from LDTI-C. The far UV-CD (circular dichroism) spectrum of the recombinant inhibitor is typical for a small folded protein. rLDTI-C is inhibitorily fully active, its complexes with bovine trypsin and human mast cell tryptase display equilibrium dissociation constants which are nearly identical to those with the natural inhibitor. Remarkably, the inhibitor blocked replication of HIV-1 in HUT-78 cells at a concentration of 20 microM.

Amino Acid Sequence↗
PubMed1999

Mutations altering the moloney murine leukemia virus p12 Gag protein affect virion production and early events of the virus life cycle.

The p12 Gag protein of Moloney murine leukemia virus is a small polypeptide of unknown function, containing two proline-rich motifs. To determine its role in replication, we introduced a series of deletion and alanine-scanning substitution mutations throughout the p12 coding region of a proviral DNA, and characterized the phenotypes of the resulting mutant viruses. Complete deletion of p12 and mutations affecting the PPPY motif caused substantial reduction in the yield of virions and a modest reduction in Gag processing. Proteolytic cleavage of the R-peptide from the cytoplasmic tail of the envelope protein TM was abolished in these mutants, suggesting that the PPPY motif is crucial for the viral protease to access the TM tail. The resulting virions were non-infectious, and unable to initiate DNA synthesis in infected cells. Mutants with alterations in both the N- and C-terminal portions of p12 exhibited a distinct phenotype. The production of virions and processing of Gag, Pol and Env precursors were normal. The viruses were able to direct synthesis of linear viral DNA, but there was almost no detectable circular DNAs or LTR-LTR junction. These data suggest that p12 plays a critical role in the early events of the virus life cycle.

3T3 Cells↗
PubMed1974

Utilization of two distinct modes of replication by a hybrid plasmid constructed in vitro from separate replicons.

A hybrid plasmid, pSC134, that codes for two distinct sets of replication functions has been constructed in vitro by ligation of EcoRI endonuclease-cleaved pSC101 and Col E1 plasmid replicons, and has been introduced into Escherichia coli by transformation. The replication properties of the pSC134 plasmid in DNA polymerase I-defective mutants or in the presence of chloramphenicol indicate that this hybrid plasmid can utilize the functionally distinct modes of replication specified by both of its parent replicons.

Carbon Radioisotopes↗
PubMed1992

Three-dimensional head angular velocity detection from otolith afferent signals.

Afferent signals from the otolith organs can produce compensatory eye position and velocity signals which has been described as linear vestibulo-ocular reflex (LVOR). The afferent otolith signals carry information about head orientation and changes of head orientation relative to gravity. A head orientation (tilt) related position signal can be obtained from population vector coding of tonic otolith afferent signals during static or dynamic head tilts, which in turn could produce compensatory eye position signals in the LVOR. On the other hand, eye angular velocity signals may be extracted, as proposed in this study, from the population response of tilt-velocity sensitive otolith afferents. Such afferents are shown to encode instantaneous head orientation relative to gravity at onset of a head movement and, as the movement continues, the projection of head angular velocity onto the earth-horizontal plane, indicating the instantaneous direction of movement relative to gravity. Angular velocity components along the earth-vertical direction which are not directly encoded by otolith afferents can be detected by central signal processing. Central reconstruction of 3D head angular velocity allows to obtain information about absolute head orientation in space even in the absence of semi-circular canal related information. Such information is important for generating compensatory eye movements as well as for dynamic control of posture.

Afferent Pathways↗
PubMed2007

Reductive evolution and niche adaptation inferred from the genome of Mycobacterium ulcerans, the causative agent of Buruli ulcer.

Mycobacterium ulcerans is found in aquatic ecosystems and causes Buruli ulcer in humans, a neglected but devastating necrotic disease of subcutaneous tissue that is rampant throughout West and Central Africa. Here, we report the complete 5.8-Mb genome sequence of M. ulcerans and show that it comprises two circular replicons, a chromosome of 5632 kb and a virulence plasmid of 174 kb. The plasmid is required for production of the polyketide toxin mycolactone, which provokes necrosis. Comparisons with the recently completed 6.6-Mb genome of Mycobacterium marinum revealed >98% nucleotide sequence identity and genome-wide synteny. However, as well as the plasmid, M. ulcerans has accumulated 213 copies of the insertion sequence IS2404, 91 copies of IS2606, 771 pseudogenes, two bacteriophages, and multiple DNA deletions and rearrangements. These data indicate that M. ulcerans has recently evolved via lateral gene transfer and reductive evolution from the generalist, more rapid-growing environmental species M. marinum to become a niche-adapted specialist. Predictions based on genome inspection for the production of modified mycobacterial virulence factors, such as the highly abundant phthiodiolone lipids, were confirmed by structural analyses. Similarly, 11 protein-coding sequences identified as M. ulcerans-specific by comparative genomics were verified as such by PCR screening a diverse collection of 33 strains of M. ulcerans and M. marinum. This work offers significant insight into the biology and evolution of mycobacterial pathogens and is an important component of international efforts to counter Buruli ulcer.

Adaptation, Physiological↗
PubMed2004

Modeling three-dimensional elastic wave propagation in circular cylindrical structures using a finite-difference approach.

Wave propagation along circular cylindrical structures is important for nondestructive-testing applications and shocks in tubes. To simulate elastic wave propagation phenomena in such structures the governing equations in cylindrical coordinates are solved numerically. To reduce the required amount of computer memory and the computational time, the stress components are eliminated in the equilibrium equations. In the resulting coupled partial differential equations, in which only the three displacement components are involved, the derivatives with respect to spatial coordinates and time are approximated using second order central differences. This leads to the present new approach, which is both accurate and efficient. In order to obtain a stable scheme the displacements must be allocated on a staggered grid. The von Neumann stability analysis is performed and the result is compared with an existing empirical criterion. Mechanical energies are observed in order to validate the finite-difference code. Since no material damping or energy dissipation is taken into account in the equations of motion, the total energy must remain constant over time. Only negligible variations are observed during long-term simulations. Dispersion relations are used to check the physical behavior of the waves calculated with the proposed finite-difference method: Theoretically calculated curves are compared with values obtained by a spectrum estimation method, applied to the results of a simulation.

Journal Article↗
PubMed1997

Subpopulations of gastric myenteric neurons are differentially activated via distinct serotonin receptors: projection, neurochemical coding, and functional implications.

The enteric nervous system coordinates various gut functions. Functional studies suggested that neurotransmitters and neuromodulators, one of the most prominent among them being 5-HT, may act through a specific modulation of ascending and descending enteric pathways. However, it is still mostly unknown how particular components of enteric reflex circuits are controlled. This report describes experiments aimed at identifying a differential activation of enteric pathways by 5-HT. Electrophysiological and immunohistochemical methods were combined to investigate the projection pattern and the transmitter phenotype of 5-HT-sensitive gastric myenteric neurons. Of 294 intracellularly labeled neurons, 60.5% showed responses mediated via 5-HT3 receptors, 11.3% were 5-HT1P-responsive, 3.7% exhibited both 5-HT3 and 5-HT1P receptor-mediated depolarization, and 24.5% were not responding to 5-HT. The 5-HT3-responsive cells were mainly cholinergic (79%) and had ascending projections, whereas the 5-HT1P-responsive cells had primarily descending projections and were nitrergic (67%). Substance P-positive neurons were cholinergic; most of the cells (75%) exhibited 5-HT3 mediated responses and had ascending projections. Muscle strip recordings supported the functional significance of the differential location of 5-HT receptor subtypes. Thus, contractile responses of gastric circular muscle strips were dose-dependently increased by a 5-HT3 and decreased by a 5-HT1P agonist. Results indicated that excitatory ascending enteric pathways consisting of cholinergic, substance Pergic neurons were activated by 5-HT3 receptors, whereas 5-HT1P receptors were involved in activation of inhibitory descending pathways using nitrergic neurons. This suggested that different effects of 5-HT on gastric functions are related to specific activation of receptors located on different subsets of enteric neurons.

Animals↗
PubMed1997

[New developments in parameter-oriented roentgen densitometry perfusion analysis within the scope of heart catheter studies].

X-ray densitometric evaluation of digital subtraction coronary arteriograms allows a qualitative and quantitative detection of contrast medium propagation through the epicardial coronary arteries, the capillary system and the coronary venous system. So-called "time-density-curves" (TDCs) can be generated following Lambert-Beer's law similar to indicator dilution curves by using contrast medium as the indicator. Several time and density parameters can be derived from these TDCs, which are related to local myocardial perfusion. Different animal validation studies have shown the applicability of this concept for in-vivo evaluation of coronary blood flow and myocardial perfusion. Nevertheless, absolute measurement of volumetric coronary blood flow or myocardial perfusion failed. Therefore, relative changes in coronary blood flow or myocardial perfusion in response to pharmacologically induced maximum hyperemia were measured and coronary or myocardial perfusion reserve was calculated as the ratio of hyperemic flow or perfusion divided by baseline values. Despite theoretical attractions for an application during routine cardiac catheterization, this densitometric approach did not get a wide acceptance. Primary reason for this limited use in specialized centers was the time consuming process of densitometric evaluation of the subtraction coronary arteriograms, which require digital cine angiography and necessitates enormous computer hard ware. This main limitation has been overcome since more powerful computer hard ware (processor speed, hard disk space, digitization boards) has become rapidly available during the last years at more moderate pricing and digital techniques today are state of the art in cardiac catheterization laboratories. In addition, soft ware program packages allowed an automatization of the digitization and densitometric evaluation process. These programs include ECG triggered cine image digitization with improved temporal resolution, semiautomatic definition of regions-of-interest including definition of reference regions-of-interest for the detection of background density changes and quality-controlled densitometric parameter analysis. This progress made an application during routine cardiac catheterization feasible. In animal validation studies this improved X-ray densitometric approach for evaluation of local myocardial perfusion was validated versus colour-coded microsphere techniques. The time parameter "rise time", defined as the time from the start of local contrast medium induced density change to its maximum revealed a close correlation (r2 = 0.965) to the results of the microsphere technique over a wide range of perfusion. We have applied this technique before and after coronary interventions such as balloon angioplasty and stenting. Results documented an improvement of poststenotic myocardial perfusion reserve immediately after coronary balloon angioplasty and an additional improvement after adjunct coronary stenting. Only after stenting but usually not after coronary balloon angioplasty alone poststenotic myocardial perfusion reserve gained the intraindividual reference level, measured in a perfusion bed supplied by an epicardial coronary artery without stenoses. These results documented the functional benefit of coronary stenting on poststenotic myocardial perfusion in addition to the well known morphologic benefit with the creation of a larger and more circular conduit.

Absorptiometry, Photon↗
PubMed1978

Differential expression of the gene for the large subunit of ribulose bisphosphate carboxylase in maize leaf cell types.

Mesophyll cells and bundle sheath cells, the dimorphic photosynthetic cell types in the C4 plant Zea mays, differ in protein composition. In particular, the large subunit of the chloroplast enzyme ribulose-1,5-bisphosphate carbocylase (EC 4.1.1.39) is found entirely or almost exclusively in bundle sheath cells. The DNA sequence coding for this polypeptide is contained in the chloroplast DNA of both mesophyll cells and bundle sheath cells. RNA complementary to this coding sequence has been detected in RNA from bundle sheath cells, but is almost or entirely absent from mesophyll cells. Similarly, translatable mRNA for this polypeptide has been detected in RNA from bundle sheath cells, but not from mesophyll cells. Portions of the 4200 base pair maize plastid DNA sequence Bam 9 outside the large subunit gene region appear to be transcribed in mesophyll cells. Thus differential gene expression at the mRNA level has been directly demonstrated to occur in these two cell types.

Carboxy-Lyases↗