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At least 667 records · Page 37Linked to original sources
PubMed1997

Flow in a simple model skeletal muscle ventricle: comparison between numerical and physical simulations.

Flow patterns generated during ventricular filling have been investigated for three different combinations of flow rate and injection volume. The numerical solutions from a commercially available computational fluid dynamics package were compared with observations made under identical flow conditions in a physical model for the purpose of code validation. Particle pathlines were generated from the numerical velocity data and compared with corresponding flow-visualization pictures. A vortex formed at the inlet to the ventricle in both cases: During the filling phase, the vortex expanded and traveled toward the apex of the ventricle until, at the end of filling, the vortex occupied the full radial extent of the ventricle; the vortex continued to travel once the filling process had ended. The vortices in vitro were more circular in shape and occupied a smaller volume than those generated by the numerical model. Nevertheless, comparison of the trajectories of the vortex centres showed that there was good agreement for the three conditions studied. Postprocessing of velocity data from the numerical solution yielded wall shear-stress measurements and particle pathlines that clearly illustrate the mass-transport qualities of the traveling vortex structure. For the cases considered here, the vortex transit produced a time-dependent shear stress distribution that had a peak value of 20 dynes cm-2, with substantially lower levels of shear stress in those regions not reached by the traveling vortex. We suggest that vortex formation and travel could reduce the residence time of fluid within a skeletal muscle ventricle, provided that the vortex travels the complete length of the ventricle before fluid is ejected at the start of the next cycle.

Blood Flow Velocity↗
PubMed1995

Hyperexpressed EcoRII renatured from inclusion bodies and native enzyme both exhibit essential cooperativity with two DNA sites.

EcoRII was the first restriction endonuclease (ENase) reported needing the cooperative interaction with at least two DNA sites for activity. We constructed an EcoRII-overproducing strain of Escherichia coli by placing the coding sequence under control of the T7 gene 10 regulatory elements. The yield of EcoRII expression could be increased to about 10% of total soluble cellular protein. Inclusion bodies are formed that mainly consist of insoluble EcoRII molecules. After solubilization by 6 M guanidine hydrochloride refolding of the enzyme was achieved by dilution into appropriate buffer. The endonuclease was purified to homogeneity from both the soluble protein fraction and the protein renatured from inclusion bodies. Their identity was proven by circular dichroism and analysis of enzyme activity with respect to the special substrate requirements of EcoRII. It is shown that EcoRII cleavage of oligodeoxyribonucleotide duplexes (oligo duplexes) with only one recognition site follows a sigmoidal concentration dependence, i.e., they cannot be cleaved below a distinct low DNA concentration where simultaneous interaction with two substrate molecules is no longer possible. We demonstrate that the restriction of oligo duplexes containing two recognition sites does not show this concentration dependence, confirming an intramolecular site cooperativity.

Base Sequence↗
PubMed2005

Complete mitochondrial genomes of the three brown algae (Heterokonta: Phaeophyceae) Dictyota dichotoma, Fucus vesiculosus and Desmarestia viridis.

We report the complete mitochondrial sequences of three brown algae (Dictyota dichotoma, Fucus vesiculosus and Desmarestia viridis) belonging to three phaeophycean lineages. They have circular mapping organization and contain almost the same set of mitochondrial genes, despite their size differences (31,617, 36,392 and 39,049 bp, respectively). These include the genes for three rRNAs (23S, 16S and 5S), 25-26 tRNAs, 35 known mitochondrial proteins and 3-4 ORFs. This gene set complements two previously studied brown algal mtDNAs, Pylaiella littoralis and Laminaria digitata. Exceptions to the very similar overall organization include the displacement of orfs, tRNA genes and four protein-coding genes found at different locations in the D. dichotoma mitochondrial genome. We present a phylogenetic analysis based on ten concatenated genes (7,479 nucleotides) and 29 taxa. Stramenopiles were always monophyletic with heterotrophic species at the base. Results support both multiple primary and multiple secondary acquisitions of plastids.

Base Sequence↗
PubMed1989

Overexpression of native human beta 2-microglobulin in Escherichia coli and its purification.

beta 2-Microglobulin (beta 2M), the small subunit of human leukocyte antigen (HLA) class-I proteins, has been synthesized in Escherichia coli and purified in mg amounts. A beta 2m cDNA clone was fused in-frame behind DNA encoding the signal sequence for the outer membrane protein, OmpA. Three different constructions were made, whose products differed by the insertion of either an extra Ala residue, the hexapeptide AEFLEA [single-letter amino acid (aa) code], or no aa between the OmpA signal sequence and beta 2M-coding sequence. All three protein products were correctly processed by bacterial signal peptidase, as determined by N-terminal sequencing, and all three were secreted as soluble proteins into the periplasmic space. However, the signal sequence of the preprotein with the inserted hexapeptide, AEFLEA, was cleaved to a much greater degree than the other two preproteins. When there was no insertion, the mature protein was identical to human beta 2M, as analyzed by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis, circular dichroism, and native isoelectric focusing. This 'bacterial beta 2M', radiolabeled with Bolton-Hunter reagent, was able to exchange into papain-solubilized HLA-B7, as determined by Sephadex G-75 chromatography and immune precipitation, indicating that bacterial beta 2M could complex with the heavy chain of HLA-B7.

Blotting, Western↗
PubMed2003

Complete nucleotide sequence of the chloroplast genome from a leptosporangiate fern, Adiantum capillus-veneris L.

We determined the complete nucleotide sequence of the chloroplast genome of the leptosporangiate fern, Adiantum capillus-veneris L. (Pteridaceae). The circular genome is 150,568 bp, with a large single-copy region (LSC) of 82,282 bp, a small-single copy region (SSC) of 21,392 bp and inverted repeats (IR) of 23,447 bp each. We compared the sequence to other published chloroplast genomes to infer the location of putative genes. When the IR is considered only once, we assigned 118 genes, of which 85 encode proteins, 29 encode tRNAs and 4 encode rRNAs. Four protein-coding genes, all four rRNA genes and six tRNA genes occur in the IR. Most (57) putative protein-coding genes appear to start with an ATG codon, but we also detected five other possible start codons, some of which suggest tRNA editing. We also found 26 apparent stop codons in 18 putative genes, also suggestive of RNA editing. We found all but one of the tRNA genes necessary to encode the complete repertoire required for translation. The missing trnK gene appears to have been disrupted by a large inversion, relative to other published chloroplast genomes. We detected several structural rearrangements that may provide useful information for phylogenetic studies.

Base Sequence↗
PubMed2001

Construction of a chimeric shuttle plasmid via a heterodimer system: secretion of an scFv protein from Bacillus brevis cells capable of inhibiting hemagglutination.

Passive immunization is an attractive therapy for preventing oral diseases including dental caries and periodontal disease. For this purpose, we attempted to produce a single chain variable fragment, scFv, which inhibited hemagglutination using the Bacillus brevis protein-producing system. To accomplish this, a novel strategy, a heterodimer system, was used for the construction of a chimeric shuttle plasmid. Initially, a set of new plasmids, kanamycin-resistant donor and erythromycin-resistant general cloning plasmids, were constructed. p15A ori was a common replication origin in these plasmids, while the pUB110 rep and minus origin (MO) were cloned into the donor plasmid. Next, the secretion domain of the B. subtilis alpha-amylase gene and the G2-4 gene, coding for the scFv protein, were cloned into the general cloning plasmid and fused by PCR. Both the donor plasmid and the general cloning plasmid containing the fused gene were digested with NotI and them ligated, a dimeric plasmid being constructed. The key restriction sites, AscI, are arranged such that the pUB110 rep-MO moiety was switched from the donor to the general cloning plasmid following AscI digestion. The chimeric shuttle plasmid was readily constructed by simple re-circularization and a B. brevis transformant producing the scFv protein in the culture fluid was isolated.

Amino Acid Sequence↗
PubMed1985

The FLP protein of the 2-micron plasmid of yeast. Purification of the protein from Escherichia coli cells expressing the cloned FLP gene.

Most laboratory strains of the yeast Saccharomyces cerevisiae contain many copies of an autonomously replicating plasmid called 2-micron circle DNA. This plasmid codes for a site-specific recombinase, the FLP protein which promotes recombination across two 599-base pair inverted repeats of the plasmid DNA. We have cloned the FLP gene under the control of a strong Escherichia coli promoter and have hyperproduced the protein in that organism. Cell-free extracts from this source promote highly efficient site-specific recombination in vitro and we have used this activity to purify the FLP protein substantially. The enzyme acts efficiently on circular and linear substrates and requires only monovalent or divalent cations for activity.

Amino Acid Sequence↗
PubMed1990

Detection of a protein encoded by a class II mitochondrial intron of Podospora anserina.

In the filamentous fungus Podospora anserina, the amplification as circular DNA molecules of the first intron (intron alpha) of the CO1 mitochondrial gene, encoding the cytochrome oxidase subunit 1, is known to be strongly associated with aging of strains. In this study we have attempted to detect the protein potentially encoded by the open reading frame (ORF) contained in this intron. This was done by the Western blot technique using specific antisera raised against three polypeptides encoded by three non-overlapping fragments of this ORF adapted to the universal code and overexpressed in Escherichia coli. We examined about thirty independent subclones of Podospora derived from two different geographic races (A, s), using wild-type and mutant strains, young and senescent cultures. A 100 kDa polypeptide, encoded by the class II intron alpha, was detected in five senescent subclones which all showed strong amplification of the intronic alpha sequence (Sen DNA alpha).

Cloning, Molecular↗
PubMed2006

Detection of a phage genome carrying a zonula occludens like toxin gene (zot) in clinical isolates of Stenotrophomonas maltophilia.

During a study of the genetic diversity of Stenotrophomonas strains, we found an autonomous replicating DNA molecule in chromosomal DNA preparations of the clinical Stenotrophomonas maltophilia strain c5. The entire sequence of 6,907 bp of the isolated DNA molecule was determined, which was called phiSMA9. Seven ORFs, which code for proteins with considerable similarity to proteins in databases, were identified in the DNA sequence. The largest ORF shows high sequence similarities to the pI protein of the filamentous phage phiLf, which was later shown to be identical to toxin Zot of Vibrio cholerae. Beside the Zot-like protein, six other proteins with similarities to known phage proteins such as a phage replication protein RstA and phage absorption or coat protein are encoded on phiSMA9, which indicate that this circular DNA molecule represents the replicative form of a linear phage genome. A PCR-based screening showed that only five from the totally investigated 47 Stenotrophomonas strains of clinical and environmental origin harbor these genes. Altogether, we describe the first genome of a phage for the nosocomial pathogen Stenotrophomonas, which contains a Zot toxin like gene and might be regarded as the first Stenotrophomonas virulence factor.

Amino Acid Sequence↗
PubMed2000

DNA sequence analysis of the complete mitochondrial genome of the green alga Scenedesmus obliquus: evidence for UAG being a leucine and UCA being a non-sense codon.

The complete DNA sequence of the mitochondrial genome of the chlorophyceen alga Scenedesmus obliquus was determined. The circular genome of 42781bp contains a basic set of 13 mitochondrial genes, which are conserved among plant or algal chondriomes. In addition, two scrambled rRNA and 27 tRNA genes are present, together with four intronic sequences (group I and II) and five open reading frames (ORFs), which show no significant homology to other ORFs from organellar genomes. The comparison with deduced amino acid sequences from 13 conserved mitochondrial genes gives rise to the conclusion that two deviations from the standard genetic code must be present in S. obliquus mitochondria: (i) UAG codes for leucine as was already found in some other algal mitochondria; (ii) UCA is a stop codon, which seems unique for mitochondrial genomes. This was supported by our finding that a tRNA-Leu gene possesses a UCA anticodon and by a missing tRNA-serine, able to decode the UCA codon. Consistent with these data is the absence of any UCA codon from conserved mitochondrial ORFs. This codon occurs only close to the end of all ORFs, while UAA or UGA codons are found at some distance from any conserved ORF. Codon changes by RNA editing can be excluded, since RT-PCR analysis does not reveal any evidence for post-transcriptional RNA modifications of the primary transcript.

Algal Proteins↗
PubMed1997

Identification of the epitope for a monoclonal antibody that blocks platelet aggregation induced by type III collagen.

A library of eight conformation-dependent monoclonal antibodies that react with distinct epitopes on native human type III collagen has been examined for the ability of these antibodies to inhibit platelet aggregation induced by this collagen. Six of these antibodies had no effects; one, 1E7-D7/Col3, delayed the onset and slowed the rate of platelet aggregation, while another, 2G8-B1/Col3, completely inhibited aggregation. In order to identify the epitope recognized by this inhibitory antibody, a series of peptides that could fold to form triple-helical fragments was examined. Each peptide included six Gly-Xaa-Yaa triplets from the human type III collagen sequence, where Xaa and Yaa represent the particular amino acids in the sequence, and a C-terminal (Gly-Pro-Hyp)4 sequence to enhance triple-helical stability. Using these peptides we have identified the epitope as a nine-amino-acid sequence, GLAGAOGLR (where O is the one-letter code for 4-hydroxyproline), starting at position 520 in the human type III collagen helical domain. This sequence is proximal to the site proposed for the interaction of type III collagen with alpha2beta1-integrin of platelets.

Amino Acid Sequence↗
PubMed1997

The Borrelia burgdorferi circular plasmid cp26: conservation of plasmid structure and targeted inactivation of the ospC gene.

The 26 to 28kb circular plasmid of B. burgdorferi sensu lato (cp26) is ubiquitous among bacteria of this group and contains loci implicated in the mouse-tick transmission cycle. Restriction mapping and Southern hybridization indicated that the structure of cp26 is conserved among isolates from different origins and culture passage histories. The cp26 ospC gene encodes an outer surface protein whose synthesis within infected ticks increases when the ticks feed, and whose synthesis in culture increases after a temperature upshift. Previous studies of ospC coding sequences showed them to have stretches of sequence apparently derived from the ospC genes of distantly related isolates by homologous recombination after DNA transfer. We found conservation of the promoter regions of the ospC and guaA genes, which are divergently transcribed. We also demonstrated that the increase in OspC protein after a temperature upshift parallels increases in mRNA levels, as expected if regulatory regions adjoin the conserved sequences in the promoter regions. Finally, we used directed insertion to inactivate the ospC gene of a non-infectious isolate. This first example of directed gene inactivation in B. burgdorferi shows that the OspC protein is not required for stable maintenance of cp26 or growth in culture.

Antigens, Bacterial↗
PubMed1987

Transformation of vegetative cells of Bacillus thuringiensis by plasmid DNA.

Plasmid DNA-mediated transformation of vegetative cells of Bacillus thuringiensis was studied with the following two plasmids: pBC16 coding for tetracycline resistance and pC194 expressing chloramphenicol resistance. A key step was the induction of competence by treatment of the bacteria with 50 mM Tris hydrochloride buffer (pH 8.9) containing 30% sucrose. Transformation frequency was strongly influenced by culture density during the uptake of DNA and required the presence of polyethylene glycol. Growth in a minimal medium supplemented with Casamino Acids gave 35 times more transformants than growth in a rich medium. The highest frequencies were obtained with covalently closed circular DNA. With all parameters optimized, the frequency was 10(-3) transformants per viable cell or 10(4) transformants per microgram of DNA. Cells previously frozen were also used as recipients in transformation experiments; such cells gave frequencies similar to those obtained with freshly grown cells. The procedure was optimized for B. thuringiensis subsp. gelechiae, but B. thuringiensis subsp. kurstaki, B. thuringiensis subsp. galleriae, B. thuringiensis subsp. thuringiensis, and B. thuringiensis subsp. israelensis were also transformed. Compared with protoplast transformation, our method is much faster and 3 orders of magnitude more efficient per microgram of added DNA.

Bacillus thuringiensis↗
PubMed1994

The Myb oncogene product induces DNA-bending.

The nuclear oncogene v-myb and its cellular counterpart c-myb code for proteins that bind to DNA in a sequence specific manner and act as regulators of transcription. The Myb protein contains DNA binding and trans-regulatory domains which are important for its function. The DNA binding domain of Myb protein has been shown to contain three imperfectly conserved repeats of 50-52 amino acids that constitute the amino terminal end. In this communication, we show that Myb protein induces conformational change in DNA after protein-DNA complex formation. Circular permutation assays indicate that Myb protein induces DNA bending at the site of binding. Phasing analysis confirm the DNA bending and allowed the detection of relative orientation of bend. Myb proteins which comprise only DNA-binding domains either with three repeats or two repeats also bend DNA in the same orientation as the larger proteins with both DNA-binding and transactivating domains. However, the transactivating region seems to influence the magnitude of bend angle. We used molecular modeling to analyse the structure of Myb-DNA complex formation resulting in the bending of DNA. Data presented here show that Myb protein, like other transcriptional regulators, bends DNA upon binding allowing the interaction of regulatory elements.

Base Sequence↗
PubMed2000

Minicircular plastid DNA in the dinoflagellate Amphidinium operculatum.

Plastid DNA was purified from the dinoflagellate Amphidinium operculatum. The genes atpB, petD, psaA, psbA and psbB have been shown to reside on single-gene minicircles of a uniform size of 2.3-2.4 kb. The psaA and psbB genes lack conventional initiation codons in the expected positions, and may use GTA for translation initiation. There are marked biases in codon preference. The predicted PsbA protein lacks the C-terminal extension which is present in all other photosynthetic organisms except Euglena gracilis, and there are other anomalies elsewhere in the predicted amino acid sequences. The non-coding regions of the minicircles contain a "core" region which includes a number of stretches that are highly conserved across all minicircles and modular regions that are conserved within subsets of the minicircles.

Amino Acid Sequence↗
PubMed2005

Dose perturbation of a novel cobalt chromium coronary stent on 32P intravascular brachytherapy: a monte carlo study.

Intravascular brachytherapy has been adopted for the indication of in-stent restenosis on the basis of results of clinical trials using mainly stainless steel stents. Recently, a new stent made of cobalt-chromium L-605 alloy (CoCr, p=9.22 g/cm3) (MULTI-LINK VISION) was introduced as an alternative to the 316L stainless steel stent design (SS, p=7.87 g/cm3) (MULTI-LINK PENTA). In this work, we used the Monte Carlo code MCNPX to compare the dose distribution for the 32P GALILEO source in CoCr and SS 8 mm stent models. The dose perturbation factor (DPF), defined as the ratio of the dose in water with the presence of a stent to the dose without a stent, was used to compare results. Both stent designs were virtually expanded to diameters of 2.0, 3.0, and 4.0 mm using finite element models. The complicated strut shapes of both the CoCr and SS stents were simplified using circular rings with an effective width to yield a metal-to-tissue ratio identical to that of the actual stents. The mean DPF at a 1 mm tissue depth, over the entire stented length of 8 mm, was 0.935 for the CoCr stent and 0.911 for the SS stent. The mean DPF at the intima (0.05 mm radial distance from the strut outer surface), over the entire stented length of 8 mm, was 0.950 for CoCr, and 0.926 for SS. The maximum DPFs directly behind the CoCr and SS struts were 0.689 and 0.644, respectively. All DPF estimates have a standard deviation of +/-0.6%(k=2), approximating the 95% confidence interval. Although the CoCr stent has a higher effective atomic number and greater density than the SS stent, the DPFs for the two stents are similar, probably because the metal-to-tissue ratio and strut thickness of the CoCr stent are lower than those of the SS stent.

Brachytherapy↗
PubMed2005

Complete sequence and organization of the cucumber (Cucumis sativus L. cv. Baekmibaekdadagi) chloroplast genome.

The nucleotide sequence of the cucumber (Cucumis sativus L. cv. Baekmibaekdadagi) chloroplast genome was completed. The circular double-stranded DNA, consisting of 155,527 bp, contained a pair of inverted repeat regions (IRa and IRb) of 25,187 bp each, which were separated by small and large single copy regions of 86,879 and 18,274 bp, respectively. The presence and relative positions of 113 genes (76 peptide-encoding genes, 30 tRNA genes, four rRNA genes, and three conserved open reading frames) were identified. The major portion (55.76%) of the C. sativus chloroplast genome consisted of gene-coding regions (49.13% protein coding and 6.63% RNA regions; 27.81% LSC, 9.46% SSC and 18.49% IR regions), while intergenic spacers (including 20 introns) made up 44.24%. The overall G-C content of C. sativus chloroplast genome was 36.95%. Sixteen genes contained one intron, while two genes had two introns. The expansion/contraction manner of IR at IRb/LSC and IR/SSC border in Cucumis was similar to that of Lotus and Arabidopsis, and the manner at IRa/LSC was similar to Lotus and Nicotiana. In total, 56 simple sequence repeats (more than 10 bases) were identified in the C. sativus chloroplast genome.

Chloroplasts↗
PubMed1994

Homologous recombination and gene replacement at the dihydrofolate reductase-thymidylate synthase locus in Toxoplasma gondii.

To investigate the feasibility of genomic transgene expression and gene targeting in Toxoplasma gondii, parasites have been transfected with constructs differing in the length of contiguous genomic sequence spanning the dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene. We have previously reported that vectors derived from a DHFR-TS cDNA 'minigene' containing mutations in the DHFR coding sequence confer pyrimethamine resistance to transfected parasites (Donald and Roos, 1993). Stably resistant parasite clones arise at high frequency, generally by virtue of transgene integration into parasite chromosomes at locations scattered throughout the genome. In contrast, using a vector which contains 8 kb of contiguous genomic sequence (vs. < 2 kb for the cDNA-derived vectors), approximately half of the integration events occur by homologous recombination. Homologous recombination appears to occur at even higher frequency when a 16 kb genomic clone is used. Circular plasmids were more efficient than linearized molecules at producing homologous recombination in this system, integrating by reciprocal crossing-over to produce a duplication of the DHFR-TS locus. Double crossing-over (or gene conversion) was also observed at low frequency, resulting in complete allelic replacement in this haploid stage of the parasite. The ability to produce either homologous or non-homologous recombinants, by the selection of appropriate transformation constructs, has considerable genetic potential.

Alleles↗