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PubMed1967

Chemistry and structure of nucleic acids of bacteriophages. Many forms of nucleic acids of bacteriophages show the ways that information is stored and reproduced.

The nucleic acids of bacteriophages are characterized by a surprising multiformity. RNA and DNA may occur, the latter in single- or double-stranded form, circular or linear, with or without breaks or single-strand ends. Terminal redundancy may exist and the populations of linear phages may be uniform or randomly permuted. A double-stranded circular DNA does not occur in extracellular bacteriophage, but is often if not always formed after infection of the bacterial host. Phage DNA may be glucosylated or methylated to a certain extent, and the glucose and methyl residues may influence the stability of the DNA inside the host.

Bacteriophages↗
PubMed1992

Phosphoglycerate mutase from Streptomyces coelicolor A3(2): purification and characterization of the enzyme and cloning and sequence analysis of the gene.

The enzyme 3-phosphoglycerate mutase was purified 192-fold from Streptomyces coelicolor, and its N-terminal sequence was determined. The enzyme is tetrameric with a subunit Mr of 29,000. It is 2,3-bisphosphoglycerate dependent and inhibited by vanadate. The gene encoding the enzyme was cloned by using a synthetic oligonucleotide probe designed from the N-terminal peptide sequence, and the complete coding sequence was determined. The deduced amino acid sequence is 64% identical to that of the phosphoglycerate mutase of Saccharomyces cerevisiae and has substantial identity to those of other phosphoglycerate mutases.

2,3-Diphosphoglycerate↗
PubMed2006

Efficient in vitro refolding and characterization of a new peptide from the scorpion Buthotus saulcyi venom produced in Escherichia coli.

The selective toxicity of depressant scorpion neurotoxins to insects is useful in studying the insect sodium channel gating, as well as being relevant to several other applications. In order to carry out structure/activity studies, the functional expression of such polypeptides is required. In the work reported here, the cDNA of a new peptide from the venom of the scorpion Buthotus saulcyi was cloned and sequenced. It codes for a 64 residues peptide (BsaulI) with 8 highly-conserved cysteines. This peptide shares high sequence similarity with depressant insect toxins of other scorpion species. Large amounts of insoluble BsaulI protein were expressed in Escherichia coli. Purification of this peptide was carried out under denaturing conditions. Renaturation was performed by pulsed dilution of the denatured BsaulI in the refolding buffer. Production of refolded Bsaul1, however, is approximately an order of magnitude higher than that obtained with similar scorpion depressant toxins. Intrinsic fluorescence, far-UV circular dichroism spectra and biological activity assays indicate that the peptide adopts a folded structure.

Amino Acid Sequence↗
PubMed2007

Mitochondrial genome of Savalia savaglia (Cnidaria, Hexacorallia) and early metazoan phylogeny.

Mitochondrial genomes have recently become widely used in animal phylogeny, mainly to infer the relationships between vertebrates and other bilaterians. However, only 11 of 723 complete mitochondrial genomes available in the public databases are of early metazoans, including cnidarians (Anthozoa, mainly Scleractinia) and sponges. Although some cnidarians (Medusozoa) are known to possess atypical linear mitochondrial DNA, the anthozoan mitochondrial genome is circular and its organization is similar to that of other metazoans. Because the phylogenetic relationships among Anthozoa as well as their relation to other early metazoans still need to be clarified, we tested whether sequencing the complete mitochondrial genome of Savalia savaglia, an anthozoan belonging to the order Zoantharia (=Zoanthidea), could be useful to infer such relationships. Compared to other anthozoans, S. savaglia's genome is unusually long (20,766 bp) due to the presence of several noncoding intergenic regions (3691 bp). The genome contains all 13 protein coding genes commonly found in metazoans, but like other Anthozoa it lacks most of the tRNAs. Phylogenetic analyses of S. savaglia mitochondrial sequences show Zoantharia branching closely to other Hexacorallia, either as a sister group to Actiniaria or as a sister group to Actiniaria and Scleractinia. The close relationships suggested between Zoantharia and Actiniaria are reinforced by strong similarities in their gene order and the presence of similar introns in the COI and ND5 genes. Our study suggests that mitochondrial genomes can be a source of potentially valuable information on the phylogeny of Hexacorallia and may provide new insights into the evolution of early metazoans.

Animals↗
PubMed1992

Expression and characterization of human mitochondrial ferredoxin reductase in Escherichia coli.

Ferredoxin reductase (Fd-reductase) supplies reducing equivalents obtained from NADPH to mitochondrial cytochrome P450 enzymes via the small iron-sulfur protein ferredoxin. Two cDNAs (differing by the presence or absence of an 18-bp insert in the coding region) for the human Fd-reductase were subcloned into a newly constructed general purpose expression vector, p delta blue; protein expression under control of the bacteriophage lambda pL promoter was then induced in Escherichia coli. Western blot analysis of subcellular fractions indicated that Fd-reductase protein expressed from both plasmids was present in both inclusion bodies and soluble fractions. However, only the form lacking the insert exhibited Fd-reductase activity. The active material was purified and was found to have electrophoretic, chromatographic, optical, and circular dichroism properties comparable to the bovine homologue. The apparent Km of the expressed protein for NADPH was determined to be 0.7 +/- 0.1 microM and the apparent Km for human ferredoxin was found to be 106 +/- 8 nM. While yields of active enzyme were relatively low (approximately 0.1 mg/liter of culture), the production of Fd-reductase in E. coli will allow structural and mechanistic studies of the enzyme and its interactions with ferredoxin.

Base Sequence↗
PubMed1989

Structural analysis of human skeletal beta-tropomyosin produced in E. coli.

We have cloned the cDNA coding the beta-tropomyosin of human muscle in an expression vector whose expression depends upon a promotor that can be induced by isopropyl-beta-thiogalactopyranoside. We show that a new protein was synthesized by bacteria containing the engineered plasmid. This protein was heat stable and reacted with antibodies against tropomyosin. We have purified this protein and further identified it by determining its amino acid composition and sequencing the NH2 terminal. Unlike the native muscle tropomyosin, the NH2 terminal is not acetylated and contains a methionine. The circular dichroism spectrum is compatible with 100% alpha-helices. These results show that the protein synthesized in E. coli possesses a native structure.

Amino Acid Sequence↗
PubMed1997

Expression, purification and characterization of the homeodomain of rat ISL-1 protein.

Isl-1 is a member of a family of Homeodomains containing proteins that possess an N-terminal pair of zinc binding LIM domains. The Isl-1 gene in rat codes for a protein that binds to the insulin gene enhancer and is also involved in regulation of amylin and proglucagon genes. A DNA sequence coding for 66 amino acid residues containing the C-terminal homeodomain fragment of Isl-1 was expressed as a soluble protein in Escherichia coli. Here, we describe a procedure which allows the rapid native purification of recombinant homeodomain protein fused to an N-terminal tag of six histidines. The purified homeodomain showed DNA-binding activity to its cognate DNA sequence. An enhanced binding activity is observed in the presence of a reducing agent in electrophoretic mobility shift assays. The DNA binding was further characterized by circular dichroism spectroscopy. Addition of DNA to the homeodomain did not change the overall secondary structure content, but the thermal and chemical denaturing profiles were altered. A stabilization of the secondary structure was observed upon DNA binding. The free energy of unfolding at 23 degrees C was 7 kJ mol(-1) in absence of DNA and 29 kJ mol(-1) in the presence of DNA.

Animals↗
PubMed1990

Molecular characterization of recombinant human acidic fibroblast growth factor produced in E. coli: comparative studies with human basic fibroblast growth factor.

Synthetic cDNA coding for human acidic fibroblast growth factor (haFGF) was expressed in E. coli under the control of the T7 promoter. The haFGF produced was purified extensively using heparin-Sepharose and phenyl-Sepharose columns. The mitogenic activity of haFGF on 3T3 and endothelial cells was significantly potentiated in the presence of heparin (10-50 micrograms/ml), while angiogenic activity was observed on chick embryo chorioallantoic membrane without exogenously added heparin. This significant potentiation of mitogenic activity was observed specifically with haFGF, not human basic fibroblast growth factor (hbFGF). Circular dichroism spectra of haFGF was not affected by the presence of heparin. The affinity of haFGF for heparin was examined using heparin affinity HPLC and was precisely confirmed to be relatively lower than that of hbFGF. These results implied that haFGF was potentiated by heparin and that this potentiation did not involve a significant change in the conformation of the haFGF molecule. The affinity of haFGF for copper was also confirmed to be higher than that of hbFGF using a copper affinity HPLC column. In addition, under acidic conditions, haFGF appeared more stable than hbFGF and was further stabilized in the presence of heparin.

Cell Division↗
PubMed1988

N-ras-like sequences on chromosomes 9, 6 and 22 with a polymorphism at the chromosome 9 locus.

Two clones, pCN1 and pCN2, which together form full-length cDNA for N-ras, were used to search for restriction fragment length polymorphisms. pCN2, which entirely consists of 3' non-translated sequences, revealed more bands on DNA transfer hybridizations than could be accounted for using the known restriction map of N-ras. None of the extra cross hybridizing sequences is located on chromosome 1. One of these sequences showed a high-frequency two-allele polymorphism with the restriction enzyme TaqI and maps to the short arm of chromosome 9. Of the remaining two sequences, one maps to chromosome 22 and the other maps to the short arm of chromosome 6. pCN1A, which contains the 5' untranslated regions and all the coding exons of N-ras only hybridized to the chromosome 1 site. No polymorphisms have been found for pCN1 with TaqI, MspI, BclI, BglI, EcoRI, BstXI, XbaI, BamHI, BglII or HindIII.

Chromosomes, Human↗
PubMed1976

Clo DF13 plasmid deoxyribonucleic acid-directed in vitro synthesis of biologically active cloacin DF13 and clo DF13 immunity protein.

Clo DF13 plasmid deoxyribonucleic acid (DNA) was used as a template to direct transcription and translation in a DNA-dependent cell-free system prepared from Escherichia coli. Analysis of the invitro products on sodium dodecylsulfate-polyacrylamide gels revealed that Clo DF13 DNA directs the synthesis of at least 10 polypeptides ranging in molecular weight from approximately 7,000 to 70,000. Two of these polypeptides could be identified, with respect to their physiochemical and biological characteristics, as the products of the Clo DF13 genes coding for cloacin DF13 and Clo DF13 immunity protein. These results confirm previous findings, obtained which Clo DF13-harbouring minicells of E. coli, that the structural tenes for the latter proteins residue on the Clo DF13 genome.

Bacterial Proteins↗
PubMed1999

Molecular characterization and interstrain variability of pHPS1, a plasmid isolated from the Sydney strain (SS1) of Helicobacter pylori.

The 5846-bp circular plasmid pHPS1 of Helicobacter pylori Sydney strain, SS1, was cloned, sequenced, and structurally characterized. The SS1 strain is widely used in animal studies of H. pylori infection. The sequence of pHPS1 revealed three open reading frames (ORFs), all of which are transcribed. Two ORFs encode putative plasmid replication proteins, RepA and RepB, similar to replicases resident on theta plasmids. In contrast, the function of ORF2 remains cryptic due to the absence of sequence similarity with any known protein in sequence databases. In addition, species specificity of these three coding regions was shown using DNA dot blot hybridization in 57 diverse clinical H. pylori isolates and 32 Helicobacter and Campylobacter strains. RepA appears to be the predominant plasmid replication protein of H. pylori and the deduced amino acid sequence was highly conserved (76-96%) in 8 H. pylori isolates, including SS1. RepB was detected in 3 H. pylori isolates examined in this study, 2 of which possess only the repB gene. Analysis of the protein sequences of these two replicases, together with previously characterized H. pylori plasmid replication proteins, supports the formation of a distinct class of H. pylori plasmid proteins. Moreover, comprehensive analysis of the whole genome sequence of H. pylori strain 26695, pHPS1, and other H. pylori plasmid sequences that are available revealed interesting insights as to the occurrence of plasmid-mediated recombination within H. pylori. Common regions between plasmids and chromosome sequences of H. pylori were identified in this study which could only have arisen by genetic recombination, thus providing the first line of evidence, albeit indirectly, of the contribution of H. pylori plasmids in generating an extensive genetic heterogeneity characteristic of this important gastroduodenal pathogen.

Chromosome Mapping↗
PubMed1996

Sequence-specific resonance assignments of the 1H-NMR spectra and structural characterization in solution of the HIV-1 transframe protein p6.

The frameshift protein p6* encoded directly upstream of the protease in the human immunodeficiency virus type 1 (HIV-1) pol reading frame is thought to be a natural inhibitor of protease activation and to play a role in the polyprotein processing of Gag and Gag-Pol precursors. To allow structural characterization of the p6* transframe protein, the p6* coding region was cloned into the vector pGEX-KG and expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST) under the control of the tac promoter. Thrombin cleavage of the construct resulted in a 70-amino-acid polypeptide which is extended by two additional residues at the N-terminus compared to the natural p6* sequence. The native purification procedure including an affinity and a size-exclusion chromatography step yielded sufficient amounts of highly pure protein suitable for NMR spectroscopy. Fluorescence, circular dichroism and 1H-NMR spectroscopy were applied to characterize the structure of protein. Two-dimensional NMR spectra provided essentially complete sequence-specific resonance assignments at pH 5.9. Although there is evidence for a helix-forming tendency in the N-terminus of the protein, the experiments indicate that p6* has no overall stable secondary or tertiary structure with the single tryptophan exposed in aqueous solution. However, the results reported herein open the way to characterize further the interaction of p6* with the HIV-1 protease in structural and functional in vitro studies.

Amino Acid Sequence↗
PubMed2002

Laser-assisted optoporation of single cells.

The plasma membrane of Chinese hamster ovary cells was made permeable using the focused beam of an argon ion laser (488 nm) and phenol red as a light absorbing dye. Small circular dark spots on the cell surface appeared immediately after laser irradiation and disappeared within about 5 min. They were related to transient changes in membrane properties, which could be visualized using the fluorescent marker laurdan, and were probably due to a local increase in temperature. According to a colony forming assay, cell viability was maintained by using light doses up to 2.5 MJ/cm(2) applied for 1 s. In addition to measurements of the efflux of the cytoplasmic marker calcein, cell transfection using a green fluorescent protein (GFP) coding plasmid was studied: brightly fluorescent GFP with an emission maximum around 510 nm was observed within part of the cells after 24 h. The transfection rates after laser irradiation were around 30% for younger subcultures and less than 10% for aging cells. This may be due to age dependent changes in the phase transition of membrane lipids from gel phase to liquid crystalline phase. High transfection rates, visual control and universality towards various cell lines are possibly the main advantages of laser-assisted optoporation in comparison with presently existing methods of cell transfection.

Animals↗
PubMed1992

Action of thrombin receptor polypeptide in gastric smooth muscle: identification of a core pentapeptide retaining full thrombin-mimetic intrinsic activity.

We have used a guinea pig gastric longitudinal (LM) smooth muscle bioassay system to evaluate the contractile activities of a previously described thrombin receptor-derived polypeptide, S42FLLRNPNDKYEPF55 (one-letter amino acid code) (TRP42-55) and of a series of peptides derived from this sequence. The contractile activities of the polypeptides were compared with the actions of thrombin. Shortened peptides of the sequences S42FLLRNPND50, S42FLLRN47, and S42FLLR46 (TRP42-46) all exhibited contractile activities that were equivalent to or greater than those of the parent polypeptide, TRP42-55. Both TRP42-55 and TRP42-46 mimicked the action of thrombin, in terms of two different signal transduction pathways that were activated either in the LM preparation or in the related but distinct gastric circular muscle assay. In the LM preparation, the peptide FSLLR also exhibited appreciable, but much reduced, activity. Minimal activity was exhibited in the LM by the sequence SFLLA, but the lysine-containing analogue S42FLLK46 was about one fifth as potent as TRP42-46. In contrast, the receptor-derived sequences S42FLL45, S42FL44-NH2, F43LLR46, and S42ALLR46, as well as arginine-containing polypeptides beginning with the SF motif, SFRG and SFRGHITR, were inactive in the LM bioassay system, at concentrations of greater than or equal to 200 microM, as either agonists or antagonists against TRP42-55. In addition to its actions in the LM and circular muscle preparations, the active pentapeptide, TRP42-46, also exhibited thrombin-mimetic intrinsic activity in a rat aortic arterial ring relaxation bioassay, whereas the pentapeptide S42FLLA46 and the tetrapeptide S42FLL45 were inactive. We conclude that the intrinsic biological activity of the thrombin receptor-derived peptide resides in the pentapeptide TRP42-46 and that the phenylalanine and arginine residues at positions 43 and 46 play key roles in the activity of this pentapeptide in smooth muscle systems.

Amino Acid Sequence↗
PubMed1995

Dose homogeneity in boron neutron capture therapy using an epithermal neutron beam.

Simulation models based on the neutron and photon Monte Carlo code MCNP were used to study the therapeutic possibilities of the HB11 epithermal neutron beam at the High Flux Reactor in Petten. Irradiations were simulated in two types of phantoms filled with water or tissue-equivalent material for benchmark treatment planning calculations. In a cuboid phantom the influence of different field sizes on the thermal-neutron-induced dose distribution was investigated. Various shapes of collimators were studied to test their efficacy in optimizing the thermal-neutron distribution over a planning target volume and healthy tissues. Using circular collimators of 8, 12 and 15 cm diameter it was shown that with the 15-cm field a relatively larger volume within 85% of the maximum neutron-induced dose was obtained than with the 8- or 12-cm-diameter field. However, even for this large field the maximum diameter of this volume was 7.5 cm. In an ellipsoid head phantom the neutron-induced dose was calculated assuming the skull to contain 10 ppm 10B, the brain 5 ppm 10B and the tumor 30 ppm 10B. It was found that with a single 15-cm-diameter circular beam a very inhomogenous dose distribution in a typical target volume was obtained. Applying two equally weighted opposing 15-cm-diameter fields, however, a dose homogeneity within +/- 10% in this planning target volume was obtained. The dose in the surrounding healthy brain tissue is 30% at maximum of the dose in the center of the target volume. Contrary to the situation for the 8-cm field, combining four fields of 15 cm diameter gave no large improvement of the dose homogeneity over the target volume or a lower maximum dose in the healthy brain. Dose-volume histograms were evaluated for the planning target volume as well as for the healthy brain to compare different irradiation techniques, yielding a graphical confirmation of the above conclusions. Therapy with BNCT on brain tumors must be performed either with an 8-cm four-field irradiation or with two opposing 15- or 12-cm fields to obtain an optimal dose distribution.

Boron Neutron Capture Therapy↗
PubMed2003

Formation of disulfide bonds and homodimers of the major cat allergen Fel d 1 equivalent to the natural allergen by expression in Escherichia coli.

Dander from the domestic cat (Felis domesticus) is one of the most common causes of IgE-mediated allergy. Attempts to produce tetrameric folded major allergen Fel d 1 by recombinant methods with structural features similar to the natural allergen have been only partially successful. In this study, a recombinant folded Fel d 1 with molecular and biological properties similar to the natural counterpart was produced. A synthetic gene coding for direct fusion of the Fel d 1 chain 2 N-terminally to chain 1 was constructed by overlapping oligonucleotides in PCR. Escherichia coli expression resulted in a non-covalently associated homodimer with an apparent molecular mass of 30 kDa defined by size exclusion chromatography. Furthermore, each 19,177-Da subunit displayed a disulfide pattern identical to that found in the natural Fel d 1, i.e. Cys3(1) Cys73(2), Cys44(1)-Cys48(2), Cys70(1)-Cys7(2), as determined by electrospray mass spectrometry after tryptic digestion. Circular dichroism analysis showed identical folds of natural and recombinant Fel d 1. Furthermore, recombinant Fel d l reacted specifically with serum IgE, inducing expression of CD203c on basophils and lymphoproliferative responses in cat-allergic patients. The results show that the overall fold and immunological properties of the recombinant Fel d 1 are very similar to those of natural Fel d 1. Moreover, the recombinant Fel d 1 construct provides a tool for defining the three-dimensional structure of Fel d 1 and represents a reagent for diagnosis and allergen-specific immunotherapy of cat allergy.

Allergens↗
PubMed1974

Electron microscopy and immunoelectronmicroscopy of cytoplasmic hepatitis B antigen in hepatocytes.

The localization of hepatitis B antigen (HB Ag) and the nature of the virus-like particles in hepatocytes of patients with HB antigenemia are controversial. In many reports, numerous virus-like particles have been demonstrated in hepatocytic nuclei; the few reported in the cytoplasm are insufficient in number to explain the intense cytoplasmic fluorescence after staining with fluoresceinated antibody to HB Ag (HB Ab). We found numerous tubular and circular structures, measuring 20 to 30 nm in diameter, in the cisternae of the excess smooth endoplasmic reticulum (ER) of varying numbers of hepatocytes in 13 of 16 HB Ag carriers and in 4 of 9 patients with HB Ag-positive chronic hepatitis corresponding to cytoplasmic HB Ag-specific fluorescence. Direct immunoelectronmicroscopy using peroxidase-labeled HB Ab revealed that the intracisternal bodies and the surrounding membranes contain HB antigenic determinants. These bodies are an ultrastructural correlate of cytoplasmic HB Ag. It is suggested that they are virally coded coat material rather than the mature hepatitis B virus or its core.

Antigens, Viral↗
PubMed2004

A carboxypeptidase inhibitor from the tick Rhipicephalus bursa: isolation, cDNA cloning, recombinant expression, and characterization.

A novel proteinaceous metallo-carboxypeptidase inhibitor, named tick carboxypeptidase inhibitor (TCI), was isolated from the ixodid tick Rhipicephalus bursa and N-terminally sequenced. The complete cDNA encoding this protein was cloned from tick mRNA by reverse transcription-PCR and rapid amplification of cDNA ends techniques. The full-length TCI cDNA contains an open reading frame coding for a precursor protein of 97 amino acid residues that consists of a predicted signal peptide of 22 residues and of mature TCI, a 75-residue cysteine-rich protein (12 Cys). The deduced amino acid sequence shows no homology to other known proteins; the C terminus, however, resembles those of other protein metallo-carboxypeptidase inhibitors, suggesting a common mechanism of inhibition. Recombinant TCI expressed in Escherichia coli is fully functional and inhibits carboxypeptidases of the A/B subfamily with equilibrium dissociation constants in the nanomolar range. Structural analyses by circular dichroism and nuclear magnetic resonance indicate that TCI is a protein strongly constrained by disulfide bonds, unusually stable over a wide pH range and highly resistant to denaturing conditions. As a tight binding inhibitor of plasma carboxypeptidase B, also known as thrombin-activatable fibrinolysis inhibitor, recombinant TCI stimulates fibrinolysis in vitro and thus may have potential for applications to prevent or treat thrombotic disorders.

Amino Acid Sequence↗