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Biomedical subjects

A Bollen

Publications and source records attributed to A Bollen.

At least 19 recordsLinked to original sources

Mitigation of azinphos-methyl in a vegetated stream: comparison of runoff- and spray-drift.

The effectiveness of aquatic macrophytes in reducing runoff- and spray-drift-induced azinphos-methyl (AZP) input was compared in a vegetated stream. Water, sediment and plant samples were taken at increasing distances from a point of input during a spray-drift event and two runoff (10 and 22 mm/day) events. Peak concentrations of AZP decreased significantly (R2=0.99; p<0.0001; n=5) from 0.24 microg/l to 0.11 microg/l during the 10mm runoff event. No reduction took place during the 22 mm event. AZP concentrations were reduced by 90% following spray-drift input, with peak concentrations decreasing significantly (R2=0.93; p=0.0084; n=5) from 4.3 microg/l to 1.7 microg/l with increasing distance from the point of input. Plant samples taken after the spray-drift event showed increased AZP concentrations in comparison to before the event indicating sorption of the pesticide to the macrophytes. Although peak concentrations of AZP were as effectively mitigated during the 10mm runoff event as during the spray-drift event, predictive modelling revealed that maximum concentrations expected during a worst-case scenario 10mm runoff event (0 days after application) are an order of magnitude lower than what can be expected for a worst-case spray-drift and 22 mm runoff event, suggesting that spray-drift-derived pesticide concentrations are more effectively mitigated than those of runoff.

Azinphosmethyl↗

Complexity of the major surface protease (msp) gene organization in Leishmania (Viannia) braziliensis: evolutionary and functional implications.

The major surface protease (msp or gp63) of Leishmania plays a major role in the host-parasite interaction. We analysed here the structure of the msp gene locus in Leishmania (Viannia) braziliensis and compared it to results obtained in other species. Physical mapping of cosmid contigs revealed a minimum of 37 genes per haploid genome and at least 8 different msp gene families. Within the same organism, these genes showed a nucleotide sequence varying in certain stretches from 3 to 34%, and a mosaic structure. From an evolutionary point of view, major differences were observed between subgenera Viannia and Leishmania, both in terms of msp gene number and sequence. Within subgenus Viannia, phenetic analysis revealed three clusters in which sequence variants of L. (Viannia) braziliensis and L. (Viannia) guyanensis were interspersed. Functional implications of our results were explored from predicted L. (Viannia) braziliensis protein sequences: regions encoding the msp catalytic site showed a conserved sequence, while regions encoding surface domains possibly involved in the host-parasite interaction (macrophage adhesion sites and immunodominant B-cell and T-cell epitopes) were variable. We speculate that this would be an adaptive strategy of the parasite.

Animals↗

Aquaporin-1 expression in human glial tumors suggests a potential novel therapeutic target for tumor-associated edema.

Aquaporins are membrane proteins involved in water transport in many fluid-transporting tissues. The objective of this study was to investigate the expression of aquaporins in malignant glial tumors associated with cerebral edema. Eighteen human glial tumors were obtained from the UCSF Neurosurgery Tissue Bank. Aquaporin-1 (AQP1) expression was evaluated via Western blot and immunohistochemistry. Intense upregulation of AQP1 expression was found in all glioblastomas. The robust expression of aquaporins in glioblastomas suggests a pathologic role for these membrane water channels, and raises the possibility that selective AQP inhibition might offer a new therapeutic option for tumor-associated cerebral edema.

Aquaporin 1↗

Absence of immunoglobulin E synthesis and airway eosinophilia by vaccination with plasmid DNA encoding ProDer p 1.

BACKGROUND: Various studies have shown that immunization with naked DNA encoding allergens induces T helper 1(Th1)-biased non-allergic responses. OBJECTIVE: To evaluate the polarization of the immune responses induced by vaccinations with plasmid DNA encoding the major mite allergen precursor ProDer p 1. METHODS: A DNA vaccine was constructed on the basis of a synthetic cDNA encoding ProDer p 1 with optimized codon usage. The immunogenicity of ProDer p 1 DNA in CBA/J mice was compared with that of purified natural Der p 1 or recombinant ProDer p 1 adjuvanted with alum. Vaccinated mice were subsequently exposed to aerosolized house dust mite extracts to provoke airway inflammation. The presence of inflammatory cells was examined in bronchoalveolar lavage (BAL) fluids and allergen-specific T cell reactivity was measured. RESULTS: Naive mice immunized with ProDer p 1 DNA developed Th1 immune responses characterized by high titres of specific IgG2a antibodies, low titres of specific IgG1 and, remarkably, the absence of anti-ProDer p 1 IgE. No specific responses were observed in animals vaccinated with the blank DNA vector. By contrast, natural Der p 1 or recombinant ProDer p 1 adsorbed to alum induced pronounced Th2 allergic responses with strong specific IgG1 and IgE titres. Spleen cells from DNA ProDer p 1-vaccinated mice secreted high levels of IFN-gamma and low production of IL-5. Conversely, both adjuvanted allergens stimulated typical Th2-type cytokine profile characterized by high and low levels of IL-5 and IFN-gamma, respectively. Whereas BAL eosinophilia was clearly observed in Der p 1-immunized animals, ProDer p 1 DNA as well as ProDer p 1 vaccinations prevented airway eosinophil infiltrations. CONCLUSIONS: These results suggest that vaccination with DNA encoding ProDer p 1 effectively fails to induce the allergen-induced IgE synthesis and airway cell infiltration. Plasmid DNA encoding ProDer p 1 may provide a novel approach for the treatment of house dust mite allergy.

Animals↗

High-level expression of recombinant house dust mite allergen Der p 1 in Pichia pastoris.

BACKGROUND: The major house dust mite Der p 1 allergen is associated with allergic disease. Heterologous over-expression of biologically active Der p 1 was previously attempted but with limited success. OBJECTIVE: The aim of this study was to establish an efficient system for the production of recombinant Der p 1. METHODS: The proform of Der p 1 was expressed in Pichia pastoris as a fusion with the alpha mating factor signal sequence. The recombinant product was purified from culture medium and compared to Der p 1 isolated from mite culture, in terms of enzymatic activity as well as IgE binding capacity. RESULTS: ProDer p 1 was efficiently secreted into culture medium as a hyperglycosylated protein of 40-60 kDa. Postpurification dialysis in acidic buffer was required for the autocatalytic processing of Der p 1. During this treatment, the prosequence was efficiently removed to give highly glycosylated recombinant mature Der p 1. Competition ELISA experiments as well as cysteine proteinase activity assays indicated that recombinant processed Der p 1 was similar to natural Der p 1 isolated from mite cultures in terms of IgE binding and enzymatic activities. However, the histamine releasing activity of recombinant Der p 1 was slightly weaker than that of natural Der p 1. CONCLUSION: This efficient system for recombinant Der p 1 expression leads the way for the design of new diagnostics for house dust mite allergy, epitope mapping, allergen engineering, structural and immunological studies and new immunotherapeutic treatments.

Antigens, Dermatophagoides↗

DNA immunisation. New histochemical and morphometric data.

Splenic germinal center reactions were measured during primary response to a plasmidic DNA intramuscular injection. Cardiotoxin-pretreated Balb/c mice were immunized with DNA plasmids encodmg or not the SAG1 protein, a membrane antigen of Toxoplasma gondii. Specific anti-SAG1 antibodies were detected on days 16 and 36 after injection of coding plasmids. The results of ELISAs showed that the SAG1-specific antibodies are of the IgG2a class. Morphometric analyses were done on serial immunostained cryosections of spleen and draining or non-draining lymph nodes. This new approach made it possible to evaluate the chronological changes induced by DNA immunisation in the germinal centres (in number and in size). Significant increases in the number of germinal centres were measured in the spleen and only in draining lymph nodes after plasmid injection, the measured changes of the germinal centers appeared to result from the adjuvant stimulatory effect of the plasmidic DNA since both the coding and the noncoding plasmid DNA induced them. No measurable changes were recorded in the T-dependent zone of lymph organs.

Animals↗

Human recombinant myeloperoxidase antiviral activity on cytomegalovirus.

In vitro incubation of human cytomegalovirus (Towne strain) with 8 U/ml human recombinant myeloperoxidase plus sodium chloride and glucose nearly abolished viral infectivity. To assay the effect on intracellular infection, cell toxicity of the enzymes was first studied. Even the high dose of 16 U/ml of recombinant myeloperoxidase plus 10 mU/ml glucose oxidase did not decrease MRC5 cell growth. By contrast, this dose reduced proliferation of activated THP1 cells. Even half of the myeloperoxidase dose proved slightly toxic to these cells. Non-cytotoxic concentrations of the reagents were used to monitor their effect on cytomegalovirus infection. In MRC5 cells, even the low dose of 4 U/ml myeloperoxidase plus glucose oxidase inhibited synthesis of cytomegalovirus early antigens, as tested by immunofluorescence. Viral release in the supernatant was decreased by 4 logs. In THP1 cells, which produce endogenously hydrogen peroxide, myeloperoxidase alone (8 U/ml) decreased the formation of early and late antigens by 53 and 44%, respectively.

Antiviral Agents↗

Glu375Gln and Asp225Val mutants: about the nature of the covalent linkages between heme group and apo-Protein in bovine lactoperoxidase.

In analogy with studies previously reported for myeloperoxidase (Kooter, I. M.; Moguilevsky, N.; Bollen, A.; Van der Veen, L. A.; Otto, C.; Dekker, H. L.; Wever, R. J. Biol. Chem. 1999, 274, 26794), we examined for bovine lactoperoxidase the effect of mutation of Asp225 and Glu375, the residues thought to be responsible for the covalent binding of the heme group to the apoprotein. Starting from the plasmid encoding rbLPO (Watanabe, S.; Varsalona, F.; Yoo, Y.; Guillaume, J. P.; Bollen, A.; Shimazaki, K.; Moguilevsky, N. FEBS Letters 1998, 441, 476), which was engineered to carry mutations in correspondence of those residues, the mutants Asp225Val and Glu375Gln were expressed in CHO cells and their products purified and characterized. Unequivocal evidence about the existence of ester linkages as well as their relative contribution to the specific spectroscopic and catalytic properties of bLPO is here discussed.

Amino Acid Substitution↗

Genetic subgroups of anaplastic astrocytomas correlate with patient age and survival.

Astrocytomas are brain tumors with variable responses to radiation and chemotherapy. Tumor grade and patient age are important prognostic factors but do not account for the variability in clinical outcome. We hypothesized that genetic subgroups play a role in the outcome of grade III astrocytomas and studied 80 grade III astrocytomas by comparative genomic hybridization. Some chromosomal aberrations (+7p/q, -9p, -10q, -13q, +19q) were related to aberrations that are frequent in grade IV astrocytoma, whereas others (+10p, -11q, +11p, -Xq) were more frequent in grade III astrocytoma. +7p, +19 and -4q were more frequent in tumors from older patients while -11p was more frequent in tumors from younger patients. Finally, gains of 7p and 7q were associated with shorter patient survival, independent of age. Our results indicate that genetic events underlie the well-known effects of age on survival in grade III astrocytoma and demonstrate the importance of molecular classification in astrocytic tumors.

Adolescent↗

Chromosomal abnormalities subdivide ependymal tumors into clinically relevant groups.

Ependymoma occurs most frequently within the central nervous system of children and young adults. We determined relative chromosomal copy-number aberrations in 44 ependymomas using comparative genomic hybridization. The study included 24 intracranial and 20 spinal cord tumors from pediatric and adult patients. Frequent chromosomal aberrations in intracranial tumors were gain of 1q and losses on 6q, 9, and 13. Gain of 1q and loss on 9 were preferentially associated with histological grade 3 tumors. On the other hand, gain on chromosome 7 was recognized almost exclusively in spinal cord tumors, and was associated with various other chromosomal aberrations including frequent loss of 22q. We conclude that cytogenetic analysis of ependymomas may help to classify these tumors and provide leads concerning their initiation and progression. The relationship of these aberrations to patient outcome needs to be addressed.

Adolescent↗

The surface antigen SAG3 mediates the attachment of Toxoplasma gondii to cell-surface proteoglycans.

The attachment of Toxoplasma gondii to target cells is mediated by recognition of cellular heparan sulfate proteoglycans (HSPGs). The present study was performed to determine whether SAG1 and SAG3, two of the parasite surface antigens anchored to the membrane via glycosylphosphatidylinositol groups (GPIs), are involved in the tachyzoite binding to proteoglycans. The use of recombinant soluble forms of these proteins allowed us to demonstrate that SAG3, but not SAG1, interacts specifically with cellular HSPGs. Indeed, soluble recombinant SAG3 protein (recSAG3) was found to bind to immobilized heparin, whereas recSAG1 did not interact with this glycoaminoglycan. The specific adherence of recSAG3 to CHO cells was inhibited by soluble glycoconjugates, of which heparin, fucoidan and dextran sulfate were the most effective. Moreover, binding of recSAG3 to two HSPGs-deficient cell mutants was reduced by up to 80%. Proteoglycan sulfation was critical for SAG3 adherence to HSPGs as incubation of cells in the presence of sodium chlorate drastically reduced the recSAG3 binding. Finally, preincubation of CHO cells with recSAG3 blocked the adsorption of radiolabelled Toxoplasma tachyzoites. Taken together, these results indicate that SAG3 is a first glycoaminoglycan-binding protein associated with Toxoplasma, and SAG3-HSPGs interactions are involved in the parasite attachment to target cells.

Animals↗

Three-dimensional magnetic resonance spectroscopic imaging of histologically confirmed brain tumors.

The goal of this study was to determine whether presurgical metabolite levels measured by 3D MR Spectroscopic Imaging (MRSI) can accurately detect viable cancer within human brain tumor masses. A total of 31 patients (33 exams, 39 pathology correlations) with brain tumors were studied prior to surgical biopsy and/or resection. The 3D MRSI was obtained with a spatial resolution of 0.2 to 1 cc throughout the majority of the mass and adjacent brain tissue using PRESS-CSI localization. Levels of choline, creatine and NAA were estimated from the locations of the resected tissue and normalized to normal appearing brain tissue. The data were correlated with subsequent histologic analysis of the biopsy tissue samples. Although there were large variations in the metabolite ratios, all regions of confirmed cancer demonstrated significant choline levels and a mean choline/NAA ratio of 5.84 + 2.58 with the lowest value being 1.3. This lowest value is greater than 4 standard deviations above the mean (0.52 +/- 0.13) found in 8 normal volunteers. The choline signal intensities in confirmed cancers were significantly elevated compared to normal appearing brain tissue with a mean ratio of 1.71 +/- 0.69. Spectra with no significant metabolite levels were observed in the non-enhancing necrotic core of the tumor masses. The results of this study indicate that 3D MRSI of brain tumors can detect abnormal metabolite levels in regions of viable cancer and grades and can differentiate cancer from necrosis and/or normal brain tissue.

Biomarkers, Tumor↗

Characterization of the type III secretion locus of Bordetella pertussis.

Multiple sequence comparisons of proteins of the LcrD/FlbF family allowed the design of primers that specifically amplify sequences coding for type III secretion components. Amplification of Bordetella pertussis DNA with these primers yielded a fragment that was further used as a probe for screening a genomic library. The nucleotide sequence of a positive clone revealed a 2100-bp gene, called bcrD, which specifies a 75-kDa polypeptide homologous to the Yersinia LcrD protein. Chromosome walking allowed the characterization of a 35-kb DNA segment that contains the entire locus and flanking housekeeping genes. The B. pertussis type III secretion locus consists of more than 30 open reading frames (ORFs), most of which are identical to annotated genes of Bordetella spp and share similarities with known type III secretion genes of related bacteria. In order to assess the function of this locus, we engineered a bcrD null mutant. However, none of the tested phenotypes, such as protein secretion, cellular invasion, cytotoxicity or mouse lung colonization, differentiated the mutant from its parental strain. Studies of bcrD and bscN expressions indicated that, under our experimental conditions, these genes are not expressed in vitro. Restriction analyses on pulsed-field gel electrophoresis allowed the type III locus mapping at coordinate position 1,590 kb on the Tohama I strain chromosome.

Animals↗

High-level expression in mammalian cells of recombinant house dust mite allergen ProDer p 1 with optimized codon usage.

BACKGROUND: The major house dust mite allergen Der p 1 is associated with allergic diseases such as asthma. Production of recombinant Der p 1 was previously attempted, but with limited success. The present study describes the expression of recombinant (rec) ProDer p 1, a recombinant precursor form of Der p 1, in CHO cells. METHODS: As optimization of the codon usage may allow successful overexpression of protein in mammalian cells, a synthetic gene encoding ProDer p 1 was designed on the basis of the codon usage frequently found in highly expressed human genes. Gene synthesis was accomplished from a set of 14 mutually priming overlapping oligonucleotides and after two runs of polymerase chain reaction. RESULTS: COS cells transiently transfected with the synthetic ProDer p 1 gene produced up to 5--10 times as much ProDer p 1 compared with the expression level obtained after transfection with the authentic gene. To stably express the recombinant allergen, CHO-K1 cells were transfected with the ProDer p 1 synthetic gene, and one amplified recombinant clone produced up to 30 mg of recProDer p 1 per liter in the culture medium before purification. recProDer p 1 was secreted as an enzymatically inactive single-chain molecule presenting three glycosylated immunoreactive forms of 41, 38 and 36 kD. When examined with respect to direct binding, recProDer p 1 and natural Der p 1 displayed very similar IgE reactivities. However, IgE inhibition and histamine release assays showed a much higher reactivity to natural Der p 1 compared to recProDer p 1. CONCLUSIONS: These data indicated that codon optimization represents an attractive strategy for high-level production of allergen in mammalian cells.

Amino Acid Sequence↗

NCX1 Na/Ca exchanger splice variants in pancreatic islet cells.

In the rat pancreatic beta-cell, Na/Ca exchange displays a quite high capacity. The cell is equipped with two alternatively spliced Na/Ca exchanger-1 (NCX1) isoforms, namely NCX1.3 and NCX1.7. To examine the existence of a possible functional difference between these splice variants, they were cloned, together with the heart variant NCX1.1, and expressed in human embryonic kidney-293 (HEK293) and Chinese hamster ovary (CHO) cells. In these two systems, the three splice variants showed a comparable level of intracellular Na+ (Na+(i))-dependent extracellular Ca2+(Ca2+(o)) uptake. Different levels of NCX1.3 and NCX1.7 transcripts were found in four rodent species, together with a marked interspecies difference in Na/Ca exchange activity. Three additional splice variants were found (NCX1.2, NCX1.9 and NCX1.13) in guinea-pigs, hamsters and mice, again in different proportions. Our data provide evidence for the activity of the three NCX1 splice variants. They also show the existence of a differential and species-specific transcription pattern of NCX1 gene products in pancreatic islet cells.

Alternative Splicing↗

Cloning, expression and purification of recombinant cotton rat interleukin-5.

The coding sequence of the hispid cotton rat (Sigmodon hispidus) interleukin-5 (IL-5) was isolated by a combination of reverse transcription (RT)-PCR and RACE protocols from concanavalin A stimulated spleen cells. The open reading frame of 399 bp encodes a polypeptide of 132 amino acids. Comparison with the rat, mouse, gerbil and human counterparts revealed 88, 88, 87 and 75% identity at the nucleotide level and 88, 90, 89 and 70% at the amino acid level, respectively. The entire coding sequence, minus the putative signal peptide sequence, was inserted into an inducible Escherichia coli expression vector. The recombinant protein possessed an expected molecular mass of 14kDa and was located in bacterial inclusion bodies. A purification scheme under reducing and denaturing conditions followed by subsequent successive dialysis steps led to the recovery of a recombinant dimeric cotton rat IL-5. The biological activity of the recombinant protein was demonstrated in a murine cell line proliferation assay. This activity was specifically inhibited by rat monoclonal antibodies directed against mouse IL-5. Together with specific antibodies that can be generated easily, cotton rat IL-5 constitutes a useful tool for extending the use of the cotton rat animal model in the study of various human pathogens.

Amino Acid Sequence↗

Bovine lactoperoxidase and its recombinant: comparison of structure and some biochemical properties.

Biochemical properties of bovine lactoperoxidase isolated from milk and recombinant bovine lactoperoxidase expressed by Chinese hamster ovary cells were compared. The natural and recombinant lactoperoxidases showed the same conformational features as determined by circular dichroism (CD) measurements. The alpha-helix, beta-structure, and unordered structure contents were found to be 17. 8, 54.2, and 28.0% for the natural lactoperoxidase and 18.6, 50.1, and 31.3% for the recombinant lactoperoxidase, respectively. The microenvironments of aromatic amino acid residues in both lactoperoxidases seemed to be the same, although the CD spectral band due to the Soret band differed slightly. A difference in the pH-dependent spectral changes of absorbance at 413 nm was observed. From a pepsin hydrolysate of lactoperoxidase, a heme-binding peptide was isolated by reverse-phase HPLC and its amino acid sequence was examined.

Animals↗