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At least 919 records · Page 51Linked to original sources
PubMed2003

Identification and characterization of the conjugal transfer region of the pCg1 plasmid from naphthalene-degrading Pseudomonas putida Cg1.

Hybridization and restriction fragment length polymorphism data (K. G. Stuart-Keil, A. M. Hohnstock, K. P. Drees, J. B. Herrick, and E. L. Madsen, Appl. Environ. Microbiol. 64:3633-3640, 1998) have shown that pCg1, a naphthalene catabolic plasmid carried by Pseudomonas putida Cg1, is homologous to the archetypal naphthalene catabolic plasmid, pDTG1, in P. putida NCIB 9816-4. Sequencing of the latter plasmid allowed PCR primers to be designed for amplifying and sequencing the conjugal transfer region in pCg1. The mating pair formation (mpf) gene, mpfA encoding the putative precursor of the conjugative pilin subunit from pCg1, was identified along with other trb-like mpf genes. Sequence comparison revealed that the 10 mpf genes in pCg1 and pDTG1 are closely related (61 to 84% identity) in sequence and operon structure to the putative mpf genes of catabolic plasmid pWW0 (TOL plasmid of P. putida) and pM3 (antibiotic resistance plasmid of Pseudomonas. spp). A polar mutation caused by insertional inactivation in mpfA of pCg1 and reverse transcriptase PCR analysis of mRNA showed that this mpf region was involved in conjugation and was transcribed from a promoter located upstream of an open reading frame adjacent to mpfA. lacZ transcriptional fusions revealed that mpf genes of pCg1 were expressed constitutively both in liquid and on solid media. This expression did not respond to host exposure to naphthalene. Conjugation frequency on semisolid media was consistently 10- to 100-fold higher than that in liquid media. Thus, conjugation of pCg1 in P. putida Cg1 was enhanced by expression of genes in the mpf region and by surfaces where conditions fostering stable, high-density cell-to-cell contact are manifest.

Bacterial Proteins↗
PubMed1999

Homotypic and heterotypic antibody responses to a 56-kilodalton protein of Orientia tsutsugamushi.

We analyzed homotypic and heterotypic antibody responses to a type-specific antigen (Tsa), a 56-kDa protein of Orientia tsutsugamushi, by using sera from mice immunized with strains Gilliam, Karp, Kato, and Boryong. We generated a series of deletion constructs of the tsa gene and expressed them as MalE fusion proteins. Variable domain I (VD I) showed strong responses to homotypic antibodies. Antigenic domain II (AD II) from Boryong and Karp showed cross-reactivities to each other. VD III showed no responses to any of the antibodies. Sera from Kato-immunized mice showed only homotypic responses to AD III. On the other hand, sera of the mice immunized with Gilliam, Karp, or Boryong showed homotypic as well as heterotypic responses to this region. VD IV showed the strongest heterotypic antibody responses among the fragments tested. These data suggest that VD I is important in homotypic antibody responses and that AD II, AD III, and VD IV are important in heterotypic antibody responses of the mice to Tsa.

Animals↗
PubMed1984

Structure of the terminal reducing heptasaccharide of polysaccharide 1 isolated from the Bordetella pertussis endotoxin.

The tetrasaccharide beta-D-glucopyranosyl-(1,3)-beta-D-glucopyranuronyl-(1, 2)-L-glycero-alpha-D-manno-heptopyranosyl-(1,5)-3-deoxy-D-manno-2- octulosonic acid was isolated after treatment of polysaccharide 1 of Bordetella pertussis endotoxin with nitrous acid. Taking into account previously identified di- and trisaccharide fragments and analytical data obtained for the intact polysaccharide 1, we present the structure of a heptasaccharide that is thought to represent the region immediately adjacent to the hydrophobic (lipid A) moiety of lipopolysaccharide 1 of the B. pertussis endotoxin. This heptasaccharide represents 50 to 60% of the complete polysaccharide structure.

Bordetella pertussis↗
PubMed2001

Nested deletions of the SRL pathogenicity island of Shigella flexneri 2a.

In this study, we determined the boundaries of a 99-kb deletable element of Shigella flexneri 2a strain YSH6000. The element, designated the multiple-antibiotic resistance deletable element (MRDE), had recently been found to contain a 66-kb pathogenicity island (PAI)-like element (designated the SRL PAI) which carries the Shigella resistance locus (SRL), encoding resistance determinants to streptomycin, ampicillin, chloramphenicol, and tetracycline. The YSH6000 MRDE was found to be flanked by two identical IS91 elements present at the S. flexneri homologs of the Escherichia coli genes putA and mdoA on NotI fragment D. Sequence data from two YSH6000-derived MRDE deletants, YSH6000T and S2430, revealed that deletion of the MRDE occurred between the two flanking IS91 elements, resulting in a single IS91 element spanning the two original IS91 loci. Selection for the loss of tetracycline resistance confirmed that the MRDE deletion occurred reproducibly from the same chromosomal site and also showed that the SRL PAI and the SRL itself were capable of independent deletion from the chromosome, thus revealing a unique set of nested deletions. The excision frequency of the SRL PAI was estimated to be 10(-5) per cell in the wild type, and mutation of a P4-like integrase gene (int) at the left end of the SRL PAI revealed that int mediates precise deletion of the PAI.

Anti-Bacterial Agents↗
PubMed1999

Isolation of Lyme disease Borrelia from puffins (Fratercula arctica) and seabird ticks (Ixodes uriae) on the Faeroe Islands.

This is the first report on the isolation of Lyme disease Borrelia from seabirds on the Faeroe Islands and the characteristics of its enzootic cycle. The major components of the Borrelia cycle include the puffin (Fratercula arctica) as the reservoir and Ixodes uriae as the vector. The importance of this cycle and its impact on the spread of human Lyme borreliosis have not yet been established. Borrelia spirochetes isolated from 2 of 102 sampled puffins were compared to the borreliae previously obtained from seabird ticks, I. uriae. The rrf-rrl intergenic spacer and the rrs and the ospC genes were sequenced and a series of phylogenetic trees were constructed. Sequence data and restriction fragment length polymorphism analysis grouped the strains together with Borrelia garinii. In a seroepidemiological survey performed with residents involved in puffin hunting on the Faeroe Islands, 3 of 81 serum samples were found to be positive by two commonly used clinical tests: a flagellin-based enzyme-linked immunosorbent assay (ELISA) and Western blotting. These three positive serum samples also had high optical density values in a whole-cell ELISA. The finding of seropositive Faeroe Islanders who are regularly exposed to I. uriae indicate that there may be a transfer of B. garinii by this tick species to humans.

Animals↗
PubMed2001

Genotyping Neisseria gonorrhoeae using fluorescent amplified fragment length polymorphism analysis.

An evaluation of fluorescent amplified fragment length polymorphism (FAFLP) for typing Neisseria gonorrhoeae using 52 isolates revealed that its discriminatory power approached that of Opa-typing in identifying epidemiologically linked isolates. Automated, accurate sizing of FAFLP amplified fragments permits objective data analysis and storage, making it an attractive method for large surveillance projects.

Bacterial Typing Techniques↗
PubMed1985

Transformation by Epstein-Barr virus requires DNA sequences in the region of BamHI fragments Y and H.

Eight independent recombinant Epstein-Barr virus genomes, each of which was a transforming strain, were made by superinfecting cell lines containing Epstein-Barr virus DNA (Raji or B95-8 strain) with a nontransforming virus (P3HR1 strain). A knowledge of the constitution of each transforming recombinant allowed the localization of the defect in the genome of the nontransforming parent to a 12-megadalton sequence within the EcoRI A fragment. Within this region, the nontransforming virus has a deletion of the BamHI Y fragment and about half of the sequences in the adjacent BamHI H fragment. The present data suggest that this deletion is responsible for the nontransforming phenotype. Furthermore, mapping a deletion in one of the recombinant genomes allowed the conclusion that a sequence (comprising about 20% of the Epstein-Barr virus genome) from the center of BamHI-D to BamHI-I' is not necessary for the maintenance of transformation by Epstein-Barr virus.

Base Sequence↗
PubMed2004

Human immunodeficiency virus type 1-derived lentivirus vectors pseudotyped with envelope glycoproteins derived from Ross River virus and Semliki Forest virus.

Ross River virus (RRV) and Semliki Forest virus (SFV) are two alphaviruses that have a high degree of amino acid homology, as well as a very broad host range. We show here that envelope glycoproteins derived from both viruses can pseudotype human immunodeficiency virus type 1 (HIV-1)-derived lentivirus vectors. Both RRV and SFV glycoproteins considerably expand the host range of the lentivirus vector, and vectors can be efficiently concentrated by ultracentrifugation. A systematic analysis comparing the alphaviral glycoproteins to the vesicular stomatitis virus glycoprotein (VSV-G) revealed that lentivirus vectors incorporate RRV glycoproteins with an efficiency comparable to that of VSV-G. Both pseudotypes have comparable physical titers, but infectious titers with the RRV pseudotype are lower than with VSV-G. Incorporation of SFV glycoproteins into lentivirus vector is less efficient, leading to decreased physical and infectious titers. The transduction rates with VSV-G-, RRV-, and SFV-pseudotyped lentivirus vectors into adherent cell lines can be significantly increased by using a combination of Polybrene and plates coated with CH-296 recombinant fibronectin fragments. Together, our data suggest that RRV and SFV glycoproteins might be suitable as alternatives to VSV-G for pseudotyping lentivirus vectors.

Animals↗
PubMed2003

Molecular cytogenetic analysis of wheat-barley hybrids using genomic in situ hybridization and barley microsatellite markers.

In the present investigation, genomic in situ hybridization (GISH) and barley microsatellite markers were used to analyse the genome constitution of wheat-barley hybrids from two backcross generations (BC1 and BC2). Two BC1 plants carried 3 and 6 barley chromosomes, respectively, according to GISH data. Additional chromosomal fragments were detected using microsatellites. Five BC2 plants possessed complete barley chromosomes or chromosome segments and six BC2 plants did not preserve barley genetic material. Molecular markers revealed segments of the barley genome with the size of one marker only, which probably resulted from recombination between wheat and barley chromosomes. The screening of backcrossed populations from intergeneric hybrids could be effectively conducted using both genomic in situ hybridization and molecular microsatellite markers. GISH images presented a general overview of the genome constitution of the hybrid plants, while microsatellite analysis revealed the genetic identity of the alien chromosomes and chromosomal segments introgressed. These methods were complementary and provided comprehensive information about the genomic constitution of the plants produced.

Chimera↗
PubMed1993

Induction of normal human eosinophil migration in vitro by substance P.

To investigate the role of tachykinins in allergic inflammation, the effect of substance P (SP) on normal human eosinophil migration was examined in Boyden chamber type assays. SP stimulated eosinophil migration in vitro with an EC50 of about 1-10 pM and maximal effects were seen at 100 pM. A carboxy-terminal fragment of SP possessed appreciable activity whereas an amino-terminal fragment was inactive. Data represented in this paper and previous studies suggest that eosinophils are attracted by SP and activated to enhanced mediator release and cytotoxic activity.

Chemotaxis, Leukocyte↗
PubMed1997

Overexpression of CuZn-superoxide dismutase reduces hippocampal injury after global ischemia in transgenic mice.

BACKGROUND AND PURPOSE: The role of copper, zinc-superoxide dismutase (CuZn-SOD) in hippocampal injury after transient global ischemia was studied using transgenic (Tg) mice and wild-type littermates. METHODS: Global ischemia was induced by bilateral common carotid artery occlusion. The hemisphere with the hypoplastic posterior communicating artery was determined and then the hippocampus in this hemisphere was evaluated qualitatively using a score of 0 to 4 and quantitatively using an image analyzer. RESULTS: Hippocampal injury was reduced in Tg mice after both 5 and 10 minutes of ischemia. In the 5-minute ischemia group, the mean score of the injury was significantly lower in Tg than nontransgenic (nTg) mice at 3 days. In the 10-minute group, the hippocampal injury was reduced more in Tg than nTg mice at 1 day. Quantitative evaluation by an image analyzer confirmed the qualitative data. Neurons with fragmented DNA were also studied in the hippocampal injury. In the 5-minute group, despite the reduction of the injury in Tg mice, their neurons with fragmented DNA were relatively increased at 1 day. In the 10-minute group, this ratio was almost the same in both nTg and Tg mice. CONCLUSIONS: CuZn-SOD plays a protective role in the pathogenesis of selective hippocampal injury after brief ischemia, whether the insult is relatively mild or intense. Furthermore, CuZn-SOD may reduce both necrotic and DNA fragmented neuronal death after global ischemia.

Animals↗
PubMed1992

Plasticity of early endosomes.

We observed that the structural organization of early endosomes was significantly modified after cell surface biotinylation followed by incubation in the presence of low concentrations of avidin. Under these conditions early endosomes increased in size to form structures which extended over several micrometers and which had an intra-luminal content with a characteristic electron-dense appearance. The modified early endosomes were not formed when either avidin or biotinylation was omitted, suggesting that they resulted from the cross-linking of internalized biotinylated proteins by avidin. Accumulation of a fluid-phase tracer was increased after the avidin-biotin treatment (145% after 45 min). Both recycling and transport to the late endosomes still occurred, albeit to a somewhat lower extent than in control cells. Quantitative electron microscopy showed that the volume of the endosomal compartment was increased approximately 1.5-fold but that the surface area of the compartment decreased relative to its volume after avidin-biotin treatment. Finally, overexpression of a rab5 mutant, which is known to inhibit early endosome fusion in vitro, prevented the formation of these structures in vivo and caused early endosome fragmentation. Altogether, our data suggest that early endosomes exhibit a high plasticity in vivo. Cross-linking appears to interfere with this dynamic process but does not arrest membrane traffic to/from early endosomes.

Animals↗
PubMed1982

Chemical studies on the structure of human serum high-density lipoprotein (HDL). Photochemical crosslinking of azido-labelled lipids in HDL.

The lipid classes of native human serum high-density lipoprotein (HDL) were exchanged against phosphatidylcholine, sphingomyelin and cholesteryl ester species substituted with photosensitive fatty acyl residues and against 25-azido-27-norcholesterol. The photosensitive fatty acyl residues were 5-and 16-azidopalmitic, 12-azidooleic and 18-azidolinoleic acid, all labelled with high tritium radioactivity. The lipid exchange method previously described was used. After UV irradiation and delipidation the apoproteins AI and AII, photocrosslinked with the radioactive lipids, were separated. The yield of covalently crosslinked lipid molecules amounted to 30% of the photosensitive lipid molecules incorporated into the HDL particle. ApoAI and apoAII were labelled by each of the photosensitive lipid classes, intergrated in the HDL particle although in very different stoichiometry. The regiospecific photochemical labelling of the lipopolypeptides AI and AII was established by cyanogen bromide cleavage and the separation of the four apoAI and two apoAII CNBr fragments. The analytical data prove the close steric relation and interaction of defined protein regions with hydrophobic regions of the lipid molecules by chemical means. These results are discussed with respect of the HDL structure.

Azides↗
PubMed2000

Crystal structure of an antibody bound to an immunodominant peptide epitope: novel features in peptide-antibody recognition.

The crystal structure of Fab of an Ab PC283 complexed with its corresponding peptide Ag, PS1 (HQLDPAFGANSTNPD), derived from the hepatitis B virus surface Ag was determined. The PS1 stretch Gln2P to Phe7P is present in the Ag binding site of the Ab, while the next three residues of the peptide are raised above the binding groove. The residues Ser11P, Thr12P, and Asn13P then loop back onto the Ag-binding site of the Ab. The last two residues, Pro14P and Asp15P, extend outside the binding site without forming any contacts with the Ab. The PC283-PS1 complex is among the few examples where the light chain complementarity-determining regions show more interactions than the heavy chain complementarity-determining regions, and a distal framework residue is involved in Ag binding. As seen from the crystal structure, most of the contacts between peptide and Ab are through the five residues, Leu3-Asp4-Pro5-Ala6-Phe7, of PS1. The paratope is predominantly hydrophobic with aromatic residues lining the binding pocket, although a salt bridge also contributes to stabilizing the Ag-Ab interaction. The molecular surface area buried upon PS1 binding is 756 A(2) for the peptide and 625 A(2) for the Fab, which is higher than what has been seen to date for Ab-peptide complexes. A comparison between PC283 structure and a homology model of its germline ancestor suggests that paratope optimization for PS1 occurs by improving both charge and shape complementarity.

Amino Acid Sequence↗
PubMed1999

Neurotrophic factors regulate cathepsin S in macrophages and microglia: A role in the degradation of myelin basic protein and amyloid beta peptide.

BACKGROUND: Cathepsin S is a member of the family of cysteine lysosomal proteases preferentially expressed in macrophages and microglia and is active after prolonged incubation in neutral pH. Upon activation of macrophages by a number of inflammatory mediators, there is an increase in secreted cathepsin S activity accompanied by a decrease in cellular cathepsin S activity and protein content, as well as a decrease in cathepsin S mRNA. The decrease in cathepsin S mRNA and protein at the cellular level is in contrast to the response observed in some in vivo scenarios. MATERIALS AND METHODS: We investigated the effect of basic fibroblast growth factor (bFGF) and nerve growth factor (NGF), two growth factors present during cell injury and inflammation but not known to activate macrophages and microglia, on the expression of cathepsin S, cathepsin B, and cathepsin L mRNAs in these cells, and on cathepsin S activity. We then tested the ability of cathepsin S to degrade myelin basic protein, and amyloid beta peptide at both acidic and neutral pH. RESULTS: Basic FGF and NGF treatment of macrophages and microglia significantly increased the levels of cathepsin S, B, and L mRNAs (2- to 5-fold). Basic FGF also increased cathepsin S activity intra- and extracellularly. Recombinant human cathepsin S was able to degrade myelin basic protein and monomeric and dimeric amyloid beta peptide at both acidic and neutral pH, as well as to process human amyloid precursor protein generating amyloidogenic fragments. CONCLUSIONS: These data suggest that bFGF and NGF may be the molecular signals that positively regulate the expression and activity of cysteine lysosomal proteases (cathepsin S in particular) in macrophages and microglia in vivo, and that there is an interplay between these factors and the activators of inflammation. Disruption of the balance between these two categories of signals may underlie the pathological changes that involve cysteine proteases. http://link.springer-ny.com/link/service/journals/00020/bibs /5n5p334. html

Amyloid beta-Peptides↗
PubMed1999

Isolation and identification of Fur binding genes in Escherichia coli.

Fur (ferric uptake regulation) binding fragments were isolated by in vitro binding of purified Fur protein with Sau3AI-digested genomic DNA fragments. The Fur-bound DNA fragments were filtered on nitrocellulose paper, isolated, cloned, and sequenced. The protein binding was confirmed by gel retardation assay for five DNA fragments. The sequence data were used to identify the genes by comparison with the GenBank data. The proposed Fur binding regions lie on or near the putative promoter regions of marAB (multiple antibiotic resistance), pyrC (dihydroorotase), mreB (mecillinam resistance) and an unidentified gene (ecouw93) near argI and in the middle of the treBC (trehalose permease enzyme II) coding region. The proposed Fur binding sites of the known iron regulating operators including the genes of this work are AAT(pyrimidine) and A(purine)TT. The two conserved sequences are 10 bases apart and palindromic to each other, which might suggest the classical pattern of protein binding toward one side of the DNA in contrast to the concept of the Fur protein wrapping around the DNA.

Bacterial Proteins↗
PubMed2000

Mitochondrio-nuclear translocation of AIF in apoptosis and necrosis.

Apoptosis inducing factor (AIF) is a novel apoptotic effector protein that induces chromatin condensation and large-scale ( approximately 50 kbp) DNA fragmentation when added to purified nuclei in vitro. Confocal and electron microscopy reveal that, in normal cells, AIF is strictly confined to mitochondria and thus colocalizes with heat shock protein 60 (hsp60). On induction of apoptosis by staurosporin, c-Myc, etoposide, or ceramide, AIF (but not hsp60) translocates to the nucleus. This suggests that only the outer mitochondrial membrane (which retains AIF in the intermembrane space) but not the inner membrane (which retains hsp60 in the matrix) becomes protein permeable. The mitochondrio-nuclear redistribution of AIF is prevented by a Bcl-2 protein specifically targeted to mitochondrial membranes. The pan-caspase inhibitor Z-VAD. fmk does not prevent the staurosporin-induced translocation of AIF, although it does inhibit oligonucleosomal DNA fragmentation and arrests chromatin condensation at an early stage. ATP depletion is sufficient to cause AIF translocation to the nucleus, and this phenomenon is accelerated by the apoptosis inducer staurosporin. However, in conditions in which both glycolytic and respiratory ATP generation is inhibited, cells fail to manifest any sign of chromatin condensation and advanced DNA fragmentation, thus manifesting a 'necrotic' phenotype. Both in the presence of Z-VAD. fmk and in conditions of ATP depletion, AIF translocation correlates with the appearance of large-scale DNA fragmentation. Altogether, these data are compatible with the hypothesis that AIF is a caspase-independent mitochondrial death effector responsible for partial chromatinolysis.

Adenosine Triphosphate↗
PubMedDate not supplied

Use of a particle beam interface combined with mass spectrometry/negative chemical ionization to determine polar herbicide residues in soil by liquid chromatography.

The use of mass spectrometry/negative chemical ionization (MS/NCI), in combination with selected-ion monitoring, allows sensitive and selective determination of polar and thermally unstable herbicide residues by liquid chromatography coupled to a particle beam interface. The method has been applied to the analysis of soil samples for herbicide residues, using various procedures for their extraction and employing matrix-standard calibration to avoid quantitative errors due to transfer of matter through the interface. Data for the fragment ions found in the MS/NCI procedure and chromatograms from analyses of real samples are also presented.

Chromatography, Liquid↗