Search PubMed⌕ Search

Biomedical subjects

A Bollen

Publications and source records attributed to A Bollen.

At least 91 records · Page 5Linked to original sources
PubMed1995

Purification and characterization of recombinant varicella-zoster virus glycoprotein gpII, secreted by Chinese hamster ovary cells.

Chinese hamster ovary cells have been engineered to secrete an anchorless form of the varicella-zoster virus gpII protein. Purification of the recombinant product was achieved by a combination of hydrophobic and gel filtration chromatography giving rise to a protein more than 85% pure. Recombinant gpII was composed of several polypeptides which, on the basis of amino-terminal sequence analysis, corresponded to a 93-kDa precursor and to the N- and C-terminal subunits of the molecule (64 and 39/36 kDa, respectively). All polypeptides carried N-linked high-mannose and complex glycosylations, whereas O-glycosylations were carried by the precursor and the N-terminal subunits only. Surprisingly, purified recombinant gpII spontaneously formed large oligomeric structures of variable size. These complexes contained noncovalently linked lipids. Mice inoculated with the recombinant gpII absorbed onto the weak adjuvant, aluminium hydroxide, produced virus neutralizing antibodies. The recombinant gpII may thus constitute a good candidate for the development of a subunit vaccine against varicella-zoster virus.

Amino Acid Sequence↗
PubMed1995

Neutrophil-macrophage interaction: a paradigm for chronic inflammation.

Autoimmune diseases, such as rheumatoid arthritis and inflammatory bowel disease are characterized by chronic inflammatory responses resulting in tissue damage. These diseases have a number of common denominators including: abnormal cytokine expression, aberrant antigen-antibody complexes, T cell anomalies, and increased numbers of neutrophils and macrophages. We propose that the interaction between neutrophils and macrophages induces a state of chronic inflammation which contributes to the disease state. One of the central players in this scenario is myeloperoxidase (MyPo). This enzyme functions in the 'cytotoxic triad' and is involved in cell killing. Studies done by the present investigators have known that MyPo, which is released from neutrophils, induces macrophages to secrete interleukin-1, interferon alpha beta and tumor necrosis factor alpha. Furthermore, our studies have suggested a major immunoregulatory role of this enzyme. We propose that the release of MyPo from neutrophils and subsequent binding to macrophages initiates a cascade of events which enhance the production of reactive oxygen intermediates and cytokine expression resulting in the chronic inflammatory state associated with autoimmune diseases.

Animals↗
PubMed1995

Rapid identification of Mycobacterium xenopi from bacterial colonies or "Bactec" culture by the polymerase chain reaction and a luminescent sandwich hybridization assay.

Oligonucleotide primers were used in the polymerase chain reaction (PCR) to amplify a specific 584-bp DNA fragment, located in the 16S RNA gene of Mycobacterium xenopi. This set of primers, X222 and X224, was able to discriminate between the pathogen and other mycobacterial species as well as non-mycobacterial strains; it detected down to 3 fg of M. xenopi DNA, i.e. about one genome equivalent. These oligonucleotide primers proved suitable for the routine identification of M. xenopi cultures, starting from one single colony on solid medium or from a liquid culture in Middelbrook 12B "Bactec" medium. In addition, a luminescent hybridization assay was designed for use on PCR-amplified DNA. This system, which, for capture, relied on a matrix-bound oligonucleotide (M30) specific for the genus Mycobacterium and, for detection, on a biotinylated xenopi-specific X221 probe, proved fully specific, highly sensitive and rapid for the evaluation of M. xenopi Bactec cultures at low growth index.

DNA, Bacterial↗
PubMed1995

Protection of hamsters against experimental mumps virus (MuV) infection by antibodies raised against the MuV surface glycoproteins expressed from recombinant vaccinia virus vectors.

The fusion (F) and haemagglutinin-neuraminidase (HN) glycoproteins of mumps virus (MuV) have been produced in CV1 cells via vaccinia virus recombinants. Recombinant proteins accumulated in infected cells and were glycosylated. Upon reduction, the F protein product was completely converted into its subunits. Hamsters infected with vaccinia recombinants expressing either the F or HN proteins produced antibodies recognizing MuV antigens and neutralizing MuV infectivity in vitro. These antibodies provided protection against MuV-induced encephalitis in newborn hamsters.

Animals↗
PubMed1995

Neutrophil and recombinant myeloperoxidase as antigens in ANCA positive systemic vasculitis.

Myeloperoxidase (MPO) is one of the major autoantigens recognized by anti-neutrophil cytoplasm antibodies. The association of this antigen with specific disease entities requires that there is a source of pure antigen present in large quantities. Further delineation of the molecular mechanisms involved in the antigen-antibody interaction requires the ability to manipulate the molecule. The expression of recombinant MPO in Chinese hamster ovary cells has produced a source of pure protein, suitable for molecular studies. We have shown that this protein is an antigen recognized by 95% of anti-MPO antibodies from patients with systemic vasculitis. This recombinant molecule will be of use in providing an additional specific solid-phase assay for these antibodies and further forms of this protein which mirror the antigenicity of native MPO more exactly may replace chemically purified antigen. It will also be of great value in studies examining the epitopes recognized by anti-MPO antibodies and in studies of immunoregulation and T cell activation.

Animals↗
PubMed1995

Exogenous myeloperoxidase enhances bacterial phagocytosis and intracellular killing by macrophages.

It is well documented that myeloperoxidase (MyPo) contributes to the bacterial activities of neutrophils and monocytes. Since mature macrophages (M phi) are devoid of this enzyme, its participation in M phi-mediated phagocytes and bacterial killing has not been completely defined. The present study demonstrates the exogenously added MyPo, at physiological levels, enhances both phagocytosis and killing of Escherichia coli. Murine peritoneal M phi were exposed to various concentrations of MyPo for different time intervals. Viable opsonized E. coli was added either prior to or after addition of MyPo. Thioglycolate-induced but not resident M pho exhibited an increase in the number of phagocytizing cells. Both resident and thioglycolate-induced M phi demonstrated increased bactericidal activity. Physiological levels of soluble MyPo also induced a significant increase in chemiluminescence. Since luminol-dependent chemiluminescence measures reactive oxygen intermediate production, studies were done to determine whether superoxide anion or H2O2 was involved in MyPo-induced M pho killing. Both superoxide dismutase and catalase ablated MyPo-induced bactericidal activity. The above data suggest that soluble MyPo, released from neutrophils at a site of infection or inflammation, can enhance both phagocytosis and killing of microorganisms.

Animals↗
PubMed1995

Diagnosis of Helicobacter pylori infection by PCR: comparison with other invasive techniques and detection of cagA gene in gastric biopsy specimens.

A PCR assay for the detection of Helicobacter pylori in gastric biopsy specimens with specific primers for ureC gene amplification (herein referred to as ureC PCR) was compared with other routine invasive methods (culture, the rapid-urease test, and Giemsa staining of histological sections) with samples from a group of 104 consecutive dyspeptic patients. Bacteria were found in 40 (38.5%), 38 (36.5%), 36 (34.6%), and 35 (33.7%) of the patients by ureC PCR, culture, the rapid-urease test, and Giemsa stain, respectively. Sixty-three patients had negative cultures, negative histological examinations, and negative rapid-urease test results, and 61 of these patients were also negative by ureC PCR. ureC PCR detected H. pylori in two culture-negative patients. In parallel, a PCR-based assay to detect the H. pylori cytotoxin-associated antigen (cagA) gene, a putative virulence gene, was also developed. To assess the likelihood of detection of H. pylori genes directly from gastric biopsy samples and from the corresponding H. pylori isolates, specimens from 31 patients were subjected to PCR with ureC- and cagA-targeting primers. All 31 biopsy specimens and the corresponding H. pylori isolates were positive in the ureC PCR. H. pylori strains that were cagA positive also gave positive cagA PCR fragments with biopsy specimens from the same patients. All ureC PCR-positive patients were examined; biopsy specimens from 10 of 11 (91.7%) duodenal ulcer patients harbored H. pylori cagA-positive strains, whereas 19 of26 (73%) of those from patients with chronic gastritis only were found to be cagA positive. These findings indicate first that ureC PCR is at least as sensitive as culture for diagnosing H. pylori infection and second that the presence of the H. pylori cagA gene can also be detected directly in biopsy specimens by PCR amplification.

Adult↗
PubMed1995

Simultaneous presence of different Borrelia burgdorferi genospecies in biological fluids of Lyme disease patients.

Oligonucleotide primers based on Borrelia burgdorferi sensu lato ospA gene sequences have been designed for use in the PCR to type all (SL primers) or each (GI to GIII primers) of the B. burgdorferi sensu lato genospecies involved in Lyme disease. These genospecies-specific primers were then used in the PCR on 24 biological fluids collected from 18 neuroborreliosis patients. Among the samples tested, 20 contained DNA from Borrelia garinii, 11 contained DNA from B. burgdorferi sensu stricto, and 10 contained DNA from Borrelia afzelii. In toto, 10 patients appeared to have been infected by a single genospecies and 8 were infected by more than one Lyme disease-associated genospecies. Serum specimens from six patients were absorbed with heterologous antigens and tested by Western blotting (immunoblotting). In four cases, residual immunodetection revealed specific epitopes of genospecies also detected by PCR; in two of them, the concordant results indicated pluri-infection of the patients. In the other two cases, Western blotting showed specific antibodies for two genospecies of Borrelia, while PCR detected DNA from only one. In summary, the data underscored the relatively high prevalence of pluri-infections in Lyme disease and confirmed the association of B. garinii with neuroborreliosis.

Animals↗
PubMed1995

Assignment of Borrelia burgdorferi strains G25 and VS461 to the Borrelia garinii and Borrelia afzelii genospecies, respectively: a comparison of OspA protein sequences.

The nucleotide sequence encoding the Outer Surface Protein A (OspA) from two Borrelia burgdorferi sensu lato isolates, G25 and VS461, has been determined. On the basis of a phylogenetic analysis, strains G25 and VS461 were respectively assigned to the B. garinii and B. afzelii genospecies. Comparative analysis of OspA proteins from 26 different B. burgdorferi sensu lato strains involved in Lyme disease indicated a higher heterogeneity in the B. garinii genospecies than in the two other genospecies, B. burgdorferi sensu stricto and B. afzelii.

Amino Acid Sequence↗
PubMed1995

Correlation of prenatal events with the development of polymicrogyria.

We report two cases of polymicrogyria in which maternal insults were well documented during the first half of the second trimester of pregnancy. The clinical histories, MR studies, and, in one patient, autopsy results were carefully reviewed, and the timing and nature of the injuries correlated with known developmental events at the time of the injury. Our results support the theory that polymicrogyria results from injury to, or disruption of normal cellular interactions at, the external limiting membrane (the "glial-pial barrier").

Abnormalities, Drug-Induced↗
PubMed1995

Automatic synthesis of multifork-like structures for multiple labeling of oligonucleotides: application to diagnosis of Lyme disease.

Multifork-like structures have been designed for multiple labelling of oligonucleotides. They rely on the synthesis of new non nucleosidic phosphoramidites which can be used in automatic DNA synthesis. The new compounds allowed tagging of any oligonucleotide and provided reactive primary amino groups for non isotopic labelling. Polybiotinylated oligonucleotides have been tested in hybridization assays using a corresponding complementary sequence covalently linked to polystyrene tubes. Detection limits ranged from 3.10(5) to 3.10(6) molecules, depending on the number of biotins. Multilabelled oligonucleotides have been included into a capture format to detect DNA of Borrelia burgdorferi, the causal agent of Lyme disease. In short, 5'-phosphate oligonucleotides were chemically condensed onto aminated microwells and served to capture PCR-amplified target DNA which, in turn, hybridized with polybiotinylated complementary oligonucleotides. Detection of the hybrids was achieved through streptavidin-conjugated peroxidase and a colorimetric substrate. The solid phase sandwich DNA hybridization assay proved highly specific, very sensitive, rapid and user friendly for the identification of spirochetal DNA in human biological specimens.

Borrelia burgdorferi Group↗
PubMed1994

Properties of the VIP-PACAP type II receptor stably expressed in CHO cells.

The VIP receptor cloned from rat lung (VIP1 receptor from the group of the PACAP-VIP type II receptors) was inserted into a mammalian expression vector and stably transfected into Chinese hamster ovary cells (CHO). Two clones were selected, expressing respectively a high (850 +/- 50 fmol/mg protein, for clone 3) and a low (100 +/- 30 fmol/mg protein for clone 16) number of receptors. Both clones had the same apparent Kd value of binding for VIP and related peptides. The receptor expressed had the same binding properties as the natural VIP receptor, judged from the relative potency of VIP and PACAP analogues and fragments. The EC50 value of adenylate cyclase activation were 3 to 10 fold lower in clone 3 than in 16. The values observed in clone 16 were closer to the binding Kd values. The differences between the two clones were explained by the existence of spare receptors in clone 3, since: (a) the relative efficacy of some fragments were lower in clone 16 than in clone 3; (b) pretreatment of the cells with VIP reduced the number of receptors in both clones and increased the EC50 value for VIP in clone 3 but decreased peptide efficacy in clone 16 without significant change of the EC50 value.

Adenylyl Cyclases↗
PubMed1994

Stable expression of human H1-histamine-receptor cDNA in Chinese hamster ovary cells. Pharmacological characterisation of the protein, tissue distribution of messenger RNA and chromosomal localisation of the gene.

A cDNA clone for the histamine H1 receptor was isolated from a human lung cDNA library; it encoded a protein of 487 amino acids which showed characteristic features of G-protein-coupled receptors. The percentages of identity of the deduced amino acid sequence with bovine, rat and guinea pig H1 histamine receptors were 82.6%, 79.4% and 73.3%, respectively, whereas these percentages decreased to 74.6%, 66% and 56.7% for the amino acid sequence of the third intracellular loop. The human H1-receptor cDNA was transfected into Chinese hamster ovary cells (CHO) via an eukaryotic expression vector; the receptor protein present on cell membranes specifically bound [3H]mepyramine with a Kd of 3.7 nM. The binding was displaced by H1-histamine-receptor antagonists and histamine. Northern blot analysis indicated the presence of two histamine H1 receptor mRNAs of 3.5 kb and 4.1 kb in various human tissues and an additional mRNA of 4.8 kb restricted to the human brain. Finally, by means of somatic cell hybrids segregating either human or rat chromosomes, the gene for histamine H1 receptor was found to reside on human chromosome 3 and rat chromosome 4.

Amino Acid Sequence↗
PubMed1994

Site-directed mutagenesis of human myeloperoxidase: further identification of residues involved in catalytic activity and heme interaction.

Evidence for the involvement of four spatially clustered residues, Asp260(94), His261(95), Glu408(242) and Met409(243), in catalytic and spectral properties of human myeloperoxidase was provided by the analysis of site-directed mutants wherein these amino acids have been substituted by asparagine, alanine, glutamine and glutamine respectively. Although none of the mutations prevented folding, heme incorporation or secretion of the enzyme from transfected Chinese Hamster Ovary cell lines, the Glu408(242) to Gln and the Met409(243) to Gln substitutions led to a full blue-shift of the Soret peak, whereas the Asp260(94) to Asn modification led to a partial blue-shift. On the other hand, His261(95)->Ala and Met409(243)->Gln mutants totally lost the typical peroxidasic activity of the enzyme, whereas the Asp260(94)->Asn mutant was only partially active. These results confirm that His261(95) is the distal histidine essential for the catalytic activity of the enzyme while Asp260(94), Met409(243) and Glu408(242) are necessary for maintaining the correct conformation of the active site and all four residues that interact closely with the periphery of the heme contribute to the unique spectral properties of the heme in MPO.

Amino Acid Sequence↗
PubMed1994

Correct in vivo processing of a chimeric ubiquitin-proapolipoprotein A-I fusion protein in baculovirus-infected insect cells.

The cDNA coding for human proapolipoprotein A-I was expressed as a ubiquitin fusion under the control of the polyhedrin promoter in baculovirus-infected Sf9 Spodoptera frugiperda insect cells. The fusion protein was expressed at high level and was quantitatively cleaved in vivo. The cleaved product was purified and its N-terminal amino acid sequence was established. The data showed that authentic proapolipoprotein A-I has been produced, and thus demonstrated the existence in Spodoptera frugiperda insect cells of a specific ubiquitin hydrolase activity.

Amino Acid Sequence↗