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Biomedical subjects

A Bollen

Publications and source records attributed to A Bollen.

At least 109 records · Page 6Linked to original sources
PubMed1994

Virucidal effect of myeloperoxidase on human immunodeficiency virus type 1-infected T cells.

Myeloperoxidase is virucidal to human immunodeficiency virus type 1 (HIV-1) in the persistently infected CEM human T-cell line or in acutely infected human peripheral blood mononuclear cells, as judged by viral infectivity and P24 radioimmunoassay. HIV-1 was specifically inactivated by low doses of the human myeloperoxidase (1.4 to 14.3 mU/ml) and the cells were spared. A higher enzyme concentration (143 mU/m) was cytotoxic, but uninfected CEM cells and normal lymphocytes were resistant to > or = 143 mU of myeloperoxidase per ml. The enzyme was virucidal with the Cl- present in medium and did not require exogenous H2O2. Catalase, an antioxidant enzyme, partially inhibited the virucidal effect of myeloperoxidase. Hence, the H2O2 probably came from the HIV-infected cells themselves. These in vitro findings indicate that the myeloperoxidase system is capable of inactivating HIV-1 of infected cells.

Antiviral Agents↗
PubMed1994

Annexin II marks astrocytic brain tumors of high histologic grade.

Annexin II is highly expressed in glioblastoma multiforme [Reeves, S. A.; Chavez-Kappel, C.; Davis, R.; Rosenblum, M.; Israel, M. A. Developmental regulation of annexin II (lipocortin 2) in human brain and expression in high grade glioma. Cancer Res. 52:6871-6876; 1992] and is a likely second messenger in mitogenic pathways known to be important for the growth of these tumors. We have examined tumor tissue from patients diagnosed with low-, intermediate-, or high-grade astrocytic tumors for expression of annexin II by immunohistochemistry, and found that annexin II levels varied significantly among these three tumors (P < 0.0005). Levels were highest in glioblastoma multiforme, intermediate in anaplastic astrocytomas, and lowest in astrocytomas. In contrast to the usual cytoplasmic localization of annexin II, distinct nuclear staining was found in many of the specimens. Reactive astrocytes found in gliotic brain also stained with anti-annexin II antibody. We examined matched specimens for a correlation between annexin II staining intensity and the bromodeoxyuridine labeling index and found that, while tumors with the most intense annexin II staining had highest bromodeoxyuridine labeling indexes, there was not a strong association between these two parameters. The association between annexin II staining and histologic grade in astrocytic malignancies indicates that annexin II may be an important marker of high-grade glial tumors, and suggests that this marker may be useful for the pathologic classification of glial tumors and the clinical evaluation of brain tumor patients.

Adult↗
PubMed1994

Properties and regulation of the coupling to adenylate cyclase of secretin receptors stably transfected in Chinese hamster ovary cells.

The binding properties, coupling to adenylate cyclase, and desensitization of secretin receptors stably expressed in transfected Chinese hamster ovary (CHO) cells were compared in two clones expressing high (CHO-SnR-c5, 450 +/- 80 fmol/mg of protein) and low (CHO-SnR-c1, 40 +/- 25 fmol/mg of protein) receptor densities. The Kd values for receptor occupancy by secretin, selected analogues, and fragments were identical in CHO-SnR-c1 and -c5 cells and identical to those described for native receptors from NG 108-15 cells. The Kact values for adenylate cyclase stimulation were identical to the Kd values in CHO-SnR-c1 cells but 5-10-fold higher than those in CHO-SnR-c5 cells. The Kact values in both CHO-SnR-c1 and -c5 cell lines were reduced in the presence of the nonhydrolyzable GTP derivative guanosine-5'-(beta, gamma-imido)triphosphate and after pretreatment of the cells with cholera toxin. Preincubation of both CHO-SnR-c1 and -c5 cell lines with secretin for 24 hr reduced their binding capacity and reduced secretin efficacy in CHO-SnR-c1 cells and secretin potency in CHO-SnR-c5 cells. These results suggest efficient coupling of the secretin receptor to the adenylate cyclase machinery and the existence of spare receptors in the clone expressing higher receptor density. Pretreatment of the two cell lines with the reducing agent dithiothreitol reduced the binding capacity and induced the appearance of a low affinity binding component. In both cell lines, dithiothreitol pretreatment decreased secretin potency but not secretin efficacy, suggesting the necessity of integrity of the disulfide bridges for optimal receptor recognition.

Adenylyl Cyclases↗
PubMed1993

Intracellular retention and degradation of human mutant variant of a alpha 1-antitrypsin in stably transfected Chinese hamster ovary cell lines.

Normal (PiM) and mutant (PiZ) variants of human alpha 1-antitrypsin (alpha 1-AT) cDNA, cloned into the pTnd eucaryotic expression vector, were used to derive recombinant Chinese hamster ovary cell lines permanently expressing the corresponding proteins. Secretion, accumulation and glycosylation of PiM and PiZ alpha 1-AT proteins were studied in the presence of various transport-impairing drugs. Pulse-chase, followed by immunoprecipitation as well as immunofluorescence experiments showed that the PiZ alpha 1-AT undergoes continuous degradation that was prevented by Brefeldin A but not by incubation of cells at 16 degrees C. Moreover, monensin partially impaired the glycosylation of both PiM and PiZ alpha 1-AT but not their secretion nor the degradation of PiZ alpha 1-AT. Those results suggest that PiZ alpha 1-AT degradation occurs in the cis-Golgi network, a compartment located between the endoplasmic reticulum and the Golgi stack. The process did not apparently involve lysosomes since it was insensitive to chloroquine. In addition, inhibition of PiM and PiZ alpha 1-AT glycosylation and secretion by tunicamycin did not result in the accumulation of the protein, but instead in its rapid lag-free degradation. Treatment of cells with the A23187 ionophore, for a short (60 min) but not a long (24 h) period, improved the secretion of PiZ alpha 1-AT in a similar way as it affects retention of naturally endoplasmic-reticulum-resident proteins, suggesting that the small proportion of PiZ alpha 1-AT which is not degraded or secreted, but accumulates in the endoplasmic reticulum, is back transported as a partially glycosylated species from the post endoplasmic reticulum compartment in which degradation takes place.

Animals↗
PubMed1993

Characterization of rabies glycoprotein expressed in yeast.

The rabies virus surface glycoprotein was synthesized in Saccharomyces cerevisiae using an expression vector which contains an inducible promoter from the copper metallothionein gene. The rabies G protein was also expressed constitutively in yeast when cloned under control of the triose dehydrogenase promoter. Polypeptides of 65-68 kDa, which migrated at the same molecular weight as authentic viral rabies G protein species, were synthesized by yeast transformants as detected by immunoblotting with rabies specific antiserum. In addition, these polypeptides were immunoprecipitated with several rabies G-specific monoclonal antibodies which neutralize virus infectivity. The recombinant rabies G proteins were glycosylated and associated with membranes in yeast. When injected into guinea pigs, yeast extracts containing the rabies G protein protected animals from lethal rabies virus challenge when administered intramuscularly. However, the same material did not protect mice from a lethal rabies intracerebral challenge.

Animals↗
PubMed1993

Regulation of macrophage function by human recombinant myeloperoxidase.

Myeloperoxidase is an enzyme which is found in the azurophilic granules of neutrophils and is associated with bactericidal, fungicidal, and tumoricidal activity. The present studies show that human recombinant myeloperoxidase (rec-MyPo) can regulate a number of macrophage (M phi) capacities and functions. Macrophages from mice exposed to rec-MyPo in vitro released reactive oxygen intermediates, tumor necrosis factor alpha (TNF alpha), and interferon alpha/beta (IFN alpha/beta). Enhanced target cell killing was also demonstrated with TNF alpha sensitive but not TNF alpha insensitive cells. Intravenous injection of rec-MyPo induced high titers of systemic TNF alpha and IFN alpha/beta. These results indicate that MyPo can function as an immunomodulator both in vitro and in vivo. Because of these actions, it is apparent that MyPo represents a previously unrecognized endogenous immunomodulator.

Adjuvants, Immunologic↗
PubMed1993

Stable integration and expression of the Plasmodium falciparum circumsporozoite protein coding sequence in mycobacteria.

The DNA coding for the circumsporozoite protein of Plasmodium falciparum (CSP; aa 1-412) has been placed under the control of the mycobacterial promoter derived from the gene encoding the 64-kDa antigen of Mycobacterium bovis-BCG. This expression cassette was cloned into pJRD184, an Escherichia coli multicloning site vector, together with the kanamycin resistance gene from Tn903 and the attachment site and integrase gene from the temperate mycobacteriophage FRAT1. One of the resulting plasmids, pNIV2173, introduced by electroporation into both Mycobacterium smegmatis and M. bovis-BCG, integrated at a specific site in the genome of each recipient. Recombinants expressed immunoreactive polypeptides, ranging in size from 62 to 43 kDa, at a level of about 1% of total soluble proteins. Part of this material was present in the culture medium indicating that mycobacterial recombinants were able to secrete the CSP. The M. smegmatis and M. bovis-BCG recombinants, transformed with pNIV2173 and grown in absence of antibiotic, were followed for more than 400 and 50 generations respectively. Over this time span, neither DNA rearrangement nor loss of expression was observed. Inoculation of the recombinant BCG to mice did not induce humoral response to CSP nor proliferative response to CSP Th2R CD4+ T lymphocyte epitope.

Animals↗
PubMed1993

Production of authentic human proapolipoprotein A-I in Escherichia coli: strategies for the removal of the amino-terminal methionine.

Several methods were compared with respect to the production of authentic, N-terminal methionine-free proapolipoprotein A-I in engineered Escherichia coli bacteria. A first approach consisted of treating the purified methionylated recombinant protein with an amino-peptidase, purified from Aeromonas proteolytica. A second series of strategies was based on the construction of proapo A-I encoding cassettes carrying built-in recognition sites suitable for specific in vitro cleavage of the products with kallikrein and enterokinase, respectively. Along the same line, a fusion between ubiquitin and proapo A-I was produced in E. coli with the prospect to achieve post-purification cleavage with yeast ubiquitin hydrolase. Finally, proapo A-I was fused to the signal peptide of the bacterial outer membrane protein, OmpA, aiming at an in situ conversion to authentic proapo A-I during secretion to the bacterial periplasm. The data showed that, out of these five systems, the OmpA signal peptide system and, to a lesser extent, the one involving the fusion to ubiquitin were the most efficient in yielding authentic proapo A-I from engineered Escherichia coli.

Amino Acid Sequence↗
PubMed1993

Induction of neutralizing antibodies by varicella-zoster virus gpII glycoprotein expressed from recombinant vaccinia virus.

The gpII glycoprotein of varicella-zoster virus (VZV) was produced in CV1 cells via vaccinia virus recombinants. Two different DNA constructs were expressed: the first one encodes the complete gpII protein (gpII s+a+) and the second a truncated species lacking the membrane anchorage domain (gpII s+a-). To achieve expression both coding sequences had to be engineered at the 5' end by substituting the unusually short (24 bp) natural signal sequence by a more conventional one encoding 29 amino acids. Recombinant gpII proteins were detected in vaccinia virus-infected cells by ELISA and immunoprecipitation. Both forms of recombinant gpII proteins were produced as glycosylated single-chain molecules of respectively 110K and 90K. Upon reduction these were only partially converted into subunits. A rabbit infected with the vaccinia virus recombinant expressing the complete gpII produced antibodies which recognized VZV antigens and neutralized VZV infectivity in vitro, independent of complement.

Amino Acid Sequence↗
PubMed1993

Candidicidal activity of the human recombinant myeloperoxidase system.

The effect of the human recombinant myeloperoxidase (recMPO) system on the viability of Candida albicans blastospores was determined. Cells were incubated in the presence of the enzyme, H2O2, and various substrates. In optimal conditions, 100% mortality was reached with only 41 mU of recMPO per ml, confirming its powerful candidicidal activity.

Candida albicans↗
PubMed1993

[Cancer of the cervix, papillomavirus, contraception and tobacco].

This survey started in May 1989 and deals with 2,436 patients treated by 10 gynecologists from the Brussels region. In addition to routine cytological analysis in cervical smears, an additional test for Human Papilloma Virus (HPV 16, 18 and 33) was advised for these women. Gynecologists filled in a questionnaire about these patients. In cases with benign cytology, virus prevalence was 2 times greater in woman who took contraceptive pills, over at least 6 months, than in each of the other groups using either diaphragm or male condoms or no contraception, or at menopausal age. It was strange to find that viral prevalence varied according to the pill trade mark, but not according to the pill hormonal content. No confounding factor in population recruitment could be detected. In contrast to viral infection, prevalence of cancer lesions was not increased in the oral contraception group. Squamous intraepithelial lesions of Low Grade and High Grade (SIL LG and HG) were on the contrary 2 times less frequent in women taking the pill, with or without HPV infection. During follow-up of 223 women, the same HPV genotype persisted in SIL lesions but it disappeared within 4 to 8 weeks in 68% of normal cervical smears. However, beyond this period, the proportion of women with persisting virus remained nearly constant, indicating that a subgroup may be less able to mount a defence against the infection. Risk factor of cigarette smoking for SIL LG or HG was 1.6 in women with HPV and 4.0 in those with no virus. This factor was 3.3 in women with oral contraception and 4.5 in the other group.

Adolescent↗
PubMed1992

Lethal oxidative damage to human immunodeficiency virus by human recombinant myeloperoxidase.

Human recombinant myeloperoxidase was evaluated in a cell-free system for its inactivation properties on the replication of human immunodeficiency virus, HTLV-IIIB. In the presence of a hydrogen peroxide generating system (glucose and glucose oxidase) and sodium thiocyanate, the recombinant enzyme inhibited virus-induced syncytium formation and viral replication without causing any cytopathic effects on SupT1 reporter cells. In addition, U937 monocytoid cells, chronically infected with HIV1, were exposed to recombinant myeloperoxidase (10 U/ml) and monitored during 48 h for the accumulation of intracellular p24 viral antigen. Under these conditions, the recombinant enzyme significantly reduced intracellular viral replication without affecting cell viability.

Cell Line↗
PubMed1992

Site-directed mutants of human myeloperoxidase. A topological approach to the heme-binding site.

Two site-directed mutants of human promyeloperoxidase, MPO(His416----Ala) and MPO(His502----Ala), have been expressed in Chinese hamster ovary cells and purified. Overall purification yields and apparent molecular masses of the mutant proteins were similar to those of the wild-type enzyme. Both mutant species were analyzed spectroscopically to check the presence of the hemic iron in the proteins and were assayed for peroxidase activity. The data show that substitution of His502 leads to the loss, or to an inappropriate configuration, of the heme together with the loss of activity, suggesting that this residue could be the proximal His involved in the binding to the iron centers. On the other hand, substitution of His416 by alanine had no effect on either of the studied parameters.

Amino Acid Sequence↗
PubMed1992

Purification, primary structure and molecular cloning of a rat ribosomal protein showing homology with yeast ribosomal protein YL34.

1. A new 17-kDa mammalian ribosomal protein (PR17) was purified to homogeneity from the rat exocrine pancreas. The purification procedure was based on acidic extraction of a heat-denatured homogenate, ammonium-sulfate precipitation, hydrophobic chromatography on phenyl-Sepharose and analytical reverse-phase HPLC on mu Bondapak C18. Fractions of interest were collected using an antiserum directed against the first (1-14) moiety of somatostatin (1-28). 30 micrograms pure RP17 were obtained from 1 g fresh pancreas. 2. A short 111-b cDNA encoding RP17 was amplified from rat pancreatic first-strand cDNA template by using two 64-fold degenerate heptadecamer primers in the DNA-polymerase-chain reaction. From the sequence of amplified cDNA, an unambiguous oligonucleotide probe was designed to screen a rat pancreatic cDNA library. A cDNA clone coding for RP17 was isolated, whose nucleotide sequence, with an open reading frame coding for 155 amino acids (molecular mass of 17,199 Da), confirmed the partial amino acid sequences directly obtained from the purified protein. 3. Northern-blot analysis showed that a similar 0.75-kb transcript was present in rat pancreas, in the rat pancreatic acinar cell line AR 4-2J and in the human neuroblastoma cell-line NB-OK-1, the highest level being in the latter two preparations, despite similar levels of RP17 in all three preparations, as tested with a rabbit antiserum directed against purified RP17. 4. The N-terminal sequence of both RP17 and the ribosomal protein YL43 from Saccharomyces cerevisiae (39 amino acid residues) showed a high degree of identity (77%), indicating that RP17 is a mammalian homolog of yeast ribosomal protein YL43.

Amino Acid Sequence↗
PubMed1992

Molecular cloning, characterization and purification of ornithine carbamoyltransferase from Mycobacterium bovis BCG.

A genomic library of Mycobacterium bovis BCG has been constructed by cloning DNA partially digested with Sau3A into the Escherichia coli expression vector pAS1. The gene coding for ornithine carbamoyl-transferase (EC.2.1.3.3; OTCase), hereafter referred to as argF, was isolated from the library by complementation of a double argF-argI mutant of E. coli and its sequence was determined. The translation initiation codon used, GTG, was identified by comparing the amino acid sequence deduced from the gene with the N-terminal sequence of the corresponding purified protein. On this basis, the M. bovis BCG OTCase monomer consists of 307 amino acid residues and displays about 44% identity with other OTCases, the most closely related homologue being the anabolic enzyme of Pseudomonas aeruginosa. The native enzyme has an estimated molecular mass of 110 kDa, suggesting a trimeric structure as is the case for most of the anabolic OTCases known from various organisms.

Amino Acid Sequence↗
PubMed1991

Spectral and enzymatic properties of human recombinant myeloperoxidase: comparison with the mature enzyme.

Human recombinant myeloperoxidase (recMPO), purified from an engineered Chinese hamster ovary (CHO) cell line, has been characterized and compared to the mature enzyme isolated from polymorphonuclear leukocytes. Both molecules appear essentially similar in physicochemical enzymatic terms according to the following observations. 1. The unprocessed recombinant protein displays the characteristic light absorption spectra of ferric mature MPO and exhibits its typical spectral changes in the presence of dithionite or hydrogen peroxide. 2. The addition of 14C-labeled 5-aminolevulinic acid, a heme precursor, to the culture medium of recombinant CHO cells yields labeled recMPO, indicating the presence of a heme-like structure in the molecule. 3. Like mature MPO, recMPO has a peroxidatic activity and catalyzes the oxidation of chloride ions in the presence of hydrogen peroxide, producing hypochlorous acid as measured by the monochlorodimedon assay. For both enzymes, the chlorinating activity optimally occurs around pH 5.0 at about 100 microM of hydrogen peroxide and is strongly inhibited by methimazole. 4. Diethylpyrocarbonate significantly reduces the enzymatic activity of both molecules, suggesting that histidine residues may be of prime importance in the active site of the enzymes. 5. According to infrared spectroscopy data, both enzymes present a very similar secondary structure organization. In conclusion, the data suggest that the processing of the precursor enzyme (recMPO) into the mature form occurs without major structural and functional consequences.

Animals↗