Biomedical subjects

A Bollen

Publications and source records attributed to A Bollen.

At least 37 records · Page 2Linked to original sources

PubMed2000

A human milk factor susceptible to cathepsin D inhibitors enhances human immunodeficiency virus type 1 infectivity and allows virus entry into a mammary epithelial cell line.

Human immunodeficiency virus type 1 (HIV-1) growth in lymphocyte cultures was increased when the virus inoculum was incubated in breast milk. The enhancing effect of milk was abolished by anti-cathepsin D antibody or by pepstatin A, a cathepsin D inhibitor. The cathepsin D-producing CD4-negative MCF7 mammary cells supported the growth of some HIV-1 isolates. An MCF7 line chronically producing HIV-1 IIIb was obtained. Cathepsin D may induce conformational modification of viral gp120, allowing direct interaction with a coreceptor. We demonstrated the presence of CXCR4 mRNA in MCF7 cells.

Animals↗

PubMed1999

Cloning, expression and purification of recombinant cotton rat interferon-gamma.

The hispid cotton rat (Sigmodon hispidus) has proven to be an excellent small animal model; however, immunological studies have been limited due to a lack of available reagents. We report cloning of the cotton rat interferon-gamma (IFN-gamma) cDNA from concanavalin A-stimulated spleen cells using a combination of reverse transcription polymerase amplification reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) protocols. The open reading frame of 513 nucleotides encodes a 170 amino acid (aa) protein followed by a stop codon with a predicted molecular mass of 19548Da. Cotton rat IFN-gamma shares 63, 60, 43 and 43% identity with the hamster, gerbil, mouse and rat counterpart, respectively. IFN-gamma nucleotide sequence corresponding to aa 18-153 was expressed in Escherichia coli under tryptophan promoter control, either fused to a single initiating codon or fused to the thioredoxin coding sequence. Both expression products were found exclusively in bacterial inclusion bodies. Two purification schemes have been developed to purify the product fused to a single methionine. One of them is fast and leads to the recovery of a pure product suitable for use in antibody production. The second protocol, which includes chromatographic steps, allows the use of the purified product for in vitro demonstration of biological activity in a viral cytopathic reduction assay on cotton rat cells.

Amino Acid Sequence↗

PubMed1999

The sulfonium ion linkage in myeloperoxidase. Direct spectroscopic detection by isotopic labeling and effect of mutation.

The heme group of myeloperoxidase is covalently linked via two ester bonds to the protein and a unique sulfonium ion linkage involving Met(243). Mutation of Met(243) into Thr, Gln, and Val, which are the corresponding residues of eosinophil peroxidase, lactoperoxidase, and thyroid peroxidase, respectively, and into Cys was performed. The Soret band in the optical absorbance spectrum in the oxidized mutants is now found at approximately 411 nm. Both the pyridine hemochrome spectra and resonance Raman spectra of the mutants are affected by the mutation. In the Met(243) mutants the affinity for chloride has decreased 100-fold. All mutants have lost their chlorination activity, except for the M243T mutant, which still has 15% activity left. By Fourier transform infared difference spectroscopy it was possible to specifically detect the (13)CD(3)-labeled methionyl sulfonium ion linkage. We conclude that the sulfonium ion linkage serves two roles. First, it serves as an electron-withdrawing substituent via its positive charge, and, second, together with its neighboring residue Glu(242), it appears to be responsible for the lower symmetry of the heme group and distortion from the planar conformation normally seen in heme-containing proteins.

Amino Acid Substitution↗

PubMed1999

Protein kinase activity in Helicobacter pylori.

Based on the predictive analysis of the cellular protein content from the complete genome sequence of Helicobacter pylori, discrepant results were previously reported concerning the occurrence of a protein kinase in this bacterium. To solve this ambiguity, we have directly assayed cellular extracts for their capacity of phosphorylating endogenous proteins. At least eight different proteins, ranging from 24 to 200 kDa, were found to be phosphorylated to a varying extent. Individual measurement of their phosphoamino acid composition showed that they all were modified at serine residues. These data indicate that H. pylori does contain a protein-serine kinase activity.

Adenosine Triphosphate↗

PubMed1999

HIV-1 infectivity and host range modification by cathepsin D present in human vaginal secretions.

OBJECTIVE: To investigate HIV-1 infectivity in the natural environment of vaginal secretions. DESIGN: Vaginal wash samples collected from 14 healthy women were incubated in vitro with various HIV-1 strains for 10 min at 37 degrees C and then assayed for infectivity on primary lymphocyte cultures, or on CEM cells, or on CD4- ME180 cells derived from vaginal epithelium. METHODS: HIV-1 infectivity was measured by early virus growth in the various host cells tested using a quantitative p24 assay and by the Karber procedure. RESULTS: Preincubation of HIV-1(IIIB) with vaginal wash samples or 2 microg/ml cathepsin D increased the ability of the virus to grow in lymphocyte cultures. The vaginal wash effect was abolished by 5 microg/ml pepstatin A, an inhibitor of aspartyl proteases. Presence of precursor and mature forms of cathepsin D in vaginal wash was demonstrated after passage through a pepstatin A-agarose column. Median tissue culture infective doses of HIV-1(IIIB) and HIV-1(JRFL) strains were increased 14.4-fold and 18-fold, respectively, after preincubation in vaginal wash sample, and were increased by pretreatment with 2 microg/ml cathepsin D. When CD4 receptors of CEMss cells were blocked by OKT4a monoclonal antibody, the cells lost susceptibility to HIV-1 (IIIB), but supported the growth of virus pretreated with vaginal wash sample or cathepsin D. These treated viruses were able to initiate infection of CD4-ME180 epithelial cells, which were not receptive to untreated virus. ME180 cells were shown to possess the messenger of CXC-chemokine receptor-4. CONCLUSIONS: Vaginal secretions may help HIV-1 transmission to women by increasing infectivity for CD4+ cells and allowing entrance into some CD4-epithelial cells.

CD4-Positive T-Lymphocytes↗

PubMed1999

Characterization of the 70 kDa polypeptide of the Na/Ca exchanger.

The Na/Ca exchanger is associated with 160, 120 and 70 kDa polypeptides whose nature is poorly understood. We have purified and characterized the Na/Ca exchanger from bovine cardiac sarcolemmal vesicles (SLVs) by using ion-exchange and affinity chromatographies. The Na/Ca exchanger-enriched fraction was reconstituted into asolectin liposomes [lipid to protein ratio 10:1 (w/w)] that showed Na/Ca exchange activity. Under non-reducing conditions, SDS/PAGE showed a single 70 kDa polypeptide, which was further characterized by immunoblots with different antibodies: SWant, raised against the purified exchanger protein; NH2-terminus, residues 1-21; NCX1, residues 393-406; and Exon F, residues 622-644. Immunoblots under reducing conditions with SWant, NH2-terminus and NCX1 showed three bands migrating at 160, 120 and 70 kDa for SLV preparations, whereas Exon F reacted only with the 160 and 120 kDa bands. Under non-reducing conditions, immunoblots with purified reconstituted Na/Ca exchanger showed a single band at 70 kDa reacting with SWant, NH2-terminus and NCX1 but not with Exon F. We conclude that the 70 kDa protein is associated with Na/Ca exchange activity, has the same N-terminal sequence as the cloned bovine cardiac exchanger, and has its length decreased by at least 35% from its C-terminal portion as compared with that of the wild-type exchanger.

Animals↗

PubMed1999

Neutrophilic myeloperoxidase-macrophage interactions perpetuate chronic inflammation associated with experimental arthritis.

Rheumatoid arthritis is a systemic disease of unknown etiology. The purpose of this study was to elucidate an unrecognized interaction between neutrophilic myeloperoxidase (MPO) and macrophages (Mphi) which could perpetuate the inflammatory response associated with arthritis. A monoarticular arthritis was induced by intra-articular injection of group A streptococcus cell wall fragments (PG-APS) into the ankle joint of female Lewis rats. After swelling/erythema subsided, joints were reinjected with either recombinant MPO or enzymatically inactive MPO (iMPO). Joint measurements were made daily and arthritis was confirmed by histology. Neither iMPO nor MPO could initiate "clinical" arthritis; however, either form of the enzyme injected after PG-APS induced a dose-dependent increase in erythema and swelling. Mannans, which block the binding of MPO to Mo, ablated clinical symptoms. Also, the presence of tumor necrosis factor alpha was observed only in diseased joints using immunocytochemistry.

Animals↗

PubMed1999

Expression of a recombinant Toxoplasma gondii ROP2 fragment as a fusion protein in bacteria circumvents insolubility and proteolytic degradation.

A 268-amino-acid-residue carboxy-terminal antigenic fragment of the Toxoplasma gondii rhoptry protein ROP2 (recROP2(t), residues 196-464) was expressed in Escherichia coli. This recombinant fragment was produced at low concentration and in a highly insoluble form. By contrast, the level of recROP2(t) production was drastically greater when the same coding sequence was fused to the C-terminus of thioredoxin (TRX) or to the maltose-binding protein (MBP) gene. While both fusion proteins were found to be mainly insoluble, solubilization could be achieved without significant degradation. MBP was more efficient than TRX in increasing the recovery of soluble protein with more than 10% of total MBP-recROP2(t) being readily expressed in a soluble form. Moreover, the insoluble form of MBP-recROP2(t) could be correctly refolded with a recovery of more than 80%. Both forms of MBP-recROP2(t) were purified to homogeneity by amylose chromatography. In contrast, the refolding of TRX-recROP2(t) promoted aggregation of the protein, which was prevented by the use of zwitterionic detergent during the one-step purification by gel filtration. Subsequent proteolytic cleavages of purified TRX-recROP2(t) and of MBP-recROP2(t) led respectively to the complete degradation or to the truncation of the recROP2(t) moiety. However, recROP2(t), despite the presence of the fusion partners, adopted a suitable conformation recognized by human serum-derived antibodies from T. gondii-seropositive individuals. Finally, both fusion proteins were able to induce specific humoral and cell-mediated immune response to the ROP2 fragment. Such fusions could represent an alternative to study the immunogenicity of T. gondii proteins which are difficult to produce because of insolubility and degradation.

ATP-Binding Cassette Transporters↗

PubMed1999

The Met243 sulfonium ion linkage is responsible for the anomalous magnetic circular dichroism and optical spectral properties of myeloperoxidase.

The heme group of myeloperoxidase shows anomalous optical properties, and the enzyme possesses the unique ability to catalyze the oxidation of chloride. However, the nature of the covalently bound heme macrocycle has been difficult to identify. In this work, the electronic and magnetic properties of the heme groups in oxidized and reduced forms of wild-type and Met243Thr mutant myeloperoxidase and wild-type lactoperoxidase have been investigated using variable-temperature (1.6-273 K) magnetic circular dichroism (MCD) spectroscopy along with parallel optical absorption and electron paramagnetic resonance studies. The results provide assessment of the spin state mixtures of the oxidized and reduced samples at ambient and liquid helium temperatures and show that the anomalous MCD properties of myeloperoxidase, e.g. red-shifted and inverted signs for bands in the high-spin ferric and low-spin ferrous forms compared to other heme peroxidases and heme proteins in general, are a direct consequence of a novel electron-withdrawing sulfonium ion heme linkage involving Met243.

Circular Dichroism↗

PubMed1999

A PCR based DNA hybridisation capture system for the detection of human cytomegalovirus. A comparative study with other identification methods.

A simple, sensitive and specific colourimetric hybridisation method for the detection of HCMV DNA in clinical specimens is described. This method combines a PCR assay with a sensitive sandwich hybridisation assay. It relies on the use of a specific capture probe linked covalently to polystyrene microplates and a specific polybiotinylated detection probe. Amplified DNA fragments, sandwiched between these two probes, are detected by an enzymatic colour reaction. This PCR-based colourimetric hybridisation method was compared with other known HCMV detection methods. Clinical specimens (n = 145, corresponding to 106 patients) were tested by both a nested PCR assay and this colourimetric hybridisation method; and by either the culture method or the pp65 antigenaemia test depending on the type of sample used. The results showed that the PCR-based hybridisation method has a specificity similar to tissue culture, known as the conventional gold standard method, and could be used for the examination of the clinical specimens.

Adolescent↗

PubMed1999

Role of charged amino acids conserved in the vasoactive intestinal polypeptide/secretin family of receptors on the secretin receptor functionality.

The secretin receptor is a member of a large family of G-protein-coupled receptors that recognize polypeptide hormone and/or neuropeptides. Charged, conserved residues might play a key role in their function, either by interacting with the ligand or by stabilizing the receptor structure. Of the four charged amino acids that are conserved in the whole secretin receptor family, D49 and R83 (in the N-terminal domain) were probably important for the secretin receptor structure: replacement of D49 by H or R and of R83 by D severely reduced both the maximal response to secretin and its potency. No functional secretin receptor could be detected after replacement of R83 by L. Mutation of D49 to E, A, or N had no effect or reduced 5-fold the potency of secretin. The highly conserved positive charges found at the extracellular ends of TM III (K194) and IV (R255) were important for the secretin receptor function, as K194 mutation to A or Q and R255 mutation to Q or D decreased the secretin's affinity 15- to 1000-fold, respectively. Six extracellular charged residues are conserved in closely related receptors but not in the whole family. K121 (TM I) and R277 (TM V) were not important for functional secretin receptor expression. D174 (TM II) was necessary to stabilize the active receptor structure: the D174N mutant receptors were unable to stimulate normally the adenylate cyclase in response to secretin, and functional D174A receptors could not be found. Mutation of R255, E259 (second extracellular loop), and E351 (third extracellular loop) to uncharged residues reduced only 10- to 100-fold the secretin potency without changing its efficacy: these residues either stabilized the active receptor conformation or formed hydrogen rather than ionic bonds with secretin. Mutation of K121 (TM I) to Q or L and of R277 (TM V) to E or Q did not affect the receptor functional properties.

Adenylyl Cyclases↗

PubMed1999

Characterization of the Asp94 and Glu242 mutants in myeloperoxidase, the residues linking the heme group via ester bonds.

The heme group of all mammalian peroxidases is covalently linked to the protein matrix via two esterbonds, as we have recently shown by Fourier transform infrared (FTIR) difference spectroscopy [Kooter, I. M., Pierik, A.J., Merkx, M., Averill, B.A., Moguilevsky, N., Bollen, A. & Wever, R. (1997) J. Am. Chem. Soc. 119, 11542-11543]. We have examined the effects of mutation of Asp94 and Glu242, responsible for those ester bonds in myeloperoxidase, on the spectroscopic properties and catalytic activity of this enzyme. Mutation of Asp94 in myeloperoxidase results in two species. The first species has spectroscopic characteristics similar to that of wild-type myeloperoxidase. The second species has spectroscopic characteristics similar to that of Met243-->Gln mutant, and it is therefore concluded that, besides loss of the ester bond involving Asp94, this species also has lost the sulfonium ion linkage that is also characteristic of myeloperoxidase. The Asp94-->Asn mutant still has about 30% residual peroxidase activity while for the Asp94-->Val mutant only a few percentage activity is left. When Glu242 is mutated the sulfonium ion linkage is not affected, but this residue together with its neighbouring residue Met243, according to resonance Raman spectra, is responsible for the low symmetry of the heme group. Mutation of either of these residues results in loss of the bowed distortion from the planar conformation, and in a heme group with higher symmetry. For the Glu242-->Gln mutant 8% residual peroxidase activity is found.

Aspartic Acid↗

PubMed1999

Ribozyme-mediated decrease in mumps virus nucleocapsid mRNA level and progeny in infected vero cells.

The effects of endogenously expressed ribozymes directed to the mumps virus nucleocapsid (NP) mRNA were studied during viral infection. To this end, eukaryotic expression vectors encoding ribozymes or controls of passive hybridization effects were constructed and used to transfect mumps permissive Vero cells. Transcripts spanning trans-acting ribozymes of the hammerhead and hairpin types were designed to hydrolyze the first 5'GUC-3' sequence downstream from the initiation site and to hybridize to a 16 base sequence containing the putative cleavage site. Control vectors encoded mutated and catalytically inactive forms of the ribozymes or a 16 base antisense version of the target sequence. When stably expressed in cells, both ribozymes and passive control RNAs reduced viral yields. A ribozyme-mediated effect on viral growth was, however, observed, as both ribozyme types reduced viral titers by approximately 80%, well above the highest inhibition level of approximately 35% found when noncatalytic RNAs were expressed. In addition, levels of NP mRNA were generally lower in cells expressing catalytic RNAs, supporting the observed inhibition of viral growth. Although cleavage in vitro of a synthetic analog of the NP mRNA was demonstrated using RNAs isolated from ribozyme-expressing cells, in vivo cleavage products were not detectable despite the use of sensitive methods, possibly because of degradation phenomena. We also suggest here that additional controls should be conducted when semicompetitive RT-PCR methods are used to evaluate intracellular cleavage by ribozymes, as the results may depend on the initial target RNA concentration.

Animals↗

PubMed1999

Differential neutralizing antibody responses to varicella-zoster virus glycoproteins B and E following naked DNA immunization.

The only available vaccine against varicella-zoster virus (VZV) consists of the VZV-Oka attenuated but persistent virus strain. Development of a safer, subunit vaccine is therefore desirable. In this prospect, nucleic acid vaccines, expressing truncated forms of VZV glycoproteins B (recgB) and E (recgE) from which the anchor and the cytoplasmic domains were deleted, were used to immunize mice. Vaccination with recgB encoding plasmid elicited a strong and specific humoral immune response. Total IgG and neutralizing titres were comparable to those previously obtained by vaccination with purified and adjuvanted native recgB. In contrast, mice immunization with recgE encoding plasmid only induced a very weak immune response whereas we previously showed that vaccination with adjuvanted native or denatured recgE protein led to high neutralizing titres. The weakness of the immune response induced by recgE-encoding plasmid depended neither on the deletion of the anchor domain in the gE gene nor on the animal model. Analysis of antibody isotypes produced by plasmid immunizations revealed a response slightly dominated by IgG2a. Taken together, the data indicate that a VZV subunit vaccine based on adjuvanted recombinant glycoprotein E is more promising than a nucleic acid-based vaccine strategy. As regards recgB, both vaccination approaches might be appropriate.

Animals↗

PubMed1999

Chromosomal abnormalities in glioblastoma multiforme by comparative genomic hybridization: correlation with radiation treatment outcome.

Glioblastoma multiforme (GM) is the most common and most malignant astrocytoma in adults. After surgery, radiation therapy extends patient survival; however, in vivo response to radiation therapy is variable. The purpose of this investigation was to determine whether the cytogenetic abnormalities of GM differ according to patient response to radiation therapy. Radiation response was defined by either progression [radiation-resistant (RR)] or resolution [radiation-sensitive (RS)] of tumor at the first postradiation radiographic imaging evaluation. Twenty RR and 10 RS frozen tissue specimens were subjected to cytogenetic analysis by comparative genomic hybridization. RS and RR specimens had different cytogenetic aberrations that mapped predominantly to chromosomes 7, 9, 10, 13, and 19. Relative gain of 7 occurred in 70% of the RR and 30% of the RS cases and was the most significant difference involving a single change between the two groups (P = 0.06). RR and RS specimens also differed in their patterns of simultaneous cytogenetic aberrations. A simultaneous gain of chromosomes 7 and 19 was found in 30% of the RR cases but was absent in the RS group. Concurrent loss of 9p23-24 and 13q14 regions was absent in the RS cohort but occurred in 30% of the RR series. This latter cytogenetic pattern was also associated with older age. Amplifications were more common in the RR series, but the difference did not reach statistical significance. The data suggest that GM with different in vivo responses to radiation therapy also differ cytogenetically.

Adult↗

PubMed1999

Mutations of aromatic residues in the first transmembrane helix impair signalling by the secretin receptor.

The secretin amino terminal residues are essential for high affinity binding to the cognate receptor and for the subsequent activation of adenylate cyclase. It has been already established that two basic residues of the receptor TM 2 are involved in the interaction with aspartate 3 of the ligand. The present work investigated the hypothesis that two conserved tyrosine residues of the TM 1 (Tyrosines 124 and 128) could also participate to the positioning of the amino terminus of the ligand. Tyrosines 124 and 128 were mutated into alanine and histidine residues, and the properties of the mutant receptors, expressed in CHO cells, were compared with those of the wild-type receptor. Mutation of tyrosine 124 to Ala or His decreased the affinity of the receptor for secretin, [Glu3]secretin, [Asn3]secretin and the secretin fragment 2-27, and reduced the intrinsic activity of [Asn3]secretin. Mutation of tyrosine 128 to Ala, but not to His reduced 50-fold secretin and [Asn3]secretin affinity but only 3-fold that of [Glu3]secretin. Secretin and [Glu3]Sn were equipotent in that mutant receptor. These results suggested that tyrosine 128 of the secretin receptor interacted directly with the [Asp3] residue of secretin and thus that the amino terminal domain of secretin interacts with amino acids buried in both the TM 1 and TM 2 helices.

Adenylyl Cyclases↗

PubMed1999

Properties of a recombinant human secretin receptor: a comparison with the rat and rabbit receptors.

A secretin receptor was cloned from a commercial human pancreatic complementary DNA (cDNA) bank. The amino acid sequence deduced from the nucleotide sequence differed slightly from the three different sequences previously published, suggesting a genetic polymorphism of the human receptor. The binding properties of the receptor were evaluated by testing natural secretin, related peptides, and synthetic analogs or fragments on membranes of Chinese hamster ovary (CHO) cells expressing the receptor after transfection. The second-messenger coupling was evaluated by adenylate cyclase measurement. The human secretin receptor was compared with the rat and the rabbit receptors. In the three animals species, rat and human secretin were equipotent; rabbit secretin was equipotent on human and rabbit secretin receptors and less potent on the rat receptor. Similar data were obtained for the [Arg16]-secretin analog. Deletion of histidine 1 and replacement of aspartate 3 reduced the affinity of the peptides for the three receptors; however, the reduction was more pronounced on rat than on human and rabbit secretin receptors. Finally, the low affinity of the rat and human receptors for vasoactive intestinal peptide (VIP) was identical; the rabbit receptor, however, had a 20-fold higher affinity. Thus the human secretin receptor shows properties of both rat and rabbit receptors. Evaluation of the properties of chimeric receptors will be useful to fit the ligand on the receptors.

Adenylyl Cyclases↗

PubMed1998

Recombinant bovine lactoperoxidase as a tool to study the heme environment in mammalian peroxidases.

The cDNA encoding bovine lactoperoxidase (LPO) has been expressed in CHO cells. The recombinant LPO was secreted as an enzymatically active single chain molecule presenting two immunoreactive forms of 88 kDa and 82 kDa, differing by their glycosylation. rLPO exhibited the characteristic absorbance spectrum with a Soret peak at 413 nm. Engineering of rLPO into a myeloperoxidase (MPO)-like molecule was attempted by substituting Gln-376 by Met, a residue known to achieve covalent binding with the heme in MPO. However, the resulting bovine LPO mutant failed to acquire the peculiar absorbance spectrum and the chlorinating activity of MPO, underlining the complex nature of interactions in the heme vicinity.

Amino Acid Sequence↗