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Biomedical subjects

L Bernier

Publications and source records attributed to L Bernier.

At least 19 recordsLinked to original sources
PubMed2001

Comparative study of a novel nucleoside analogue (Troxatyl, troxacitabine, BCH-4556) and AraC against leukemic human tumor xenografts expressing high or low cytidine deaminase activity.

PURPOSE: Troxacitabine (beta-L-dioxolane cytidine, BCH-4556; Troxatyl, BioChem Pharma Inc.) is a novel nucleoside analogue, which in experiments demonstrated potent antitumor activity against both leukemias and solid tumors. Since troxacitabine is a cytidine nucleoside analogue like AraC (1-beta-D-arabinofuranosylcytosine), which is currently used in the treatment of acute myelogenous leukemia, we compared the in vivo antileukemic activity of troxacitabine with that of AraC in human leukemia xenograft models. METHODS: The antiproliferative activity of troxacitabine and AraC was analyzed on hemapoietic cell lines by use of a thymidine incorporation assay. For in vivo studies, we compared troxacitabine with AraC by using equitotoxic schedules of the two nucleosides optimized for therapeutic activity. The antileukemic activity of both drugs was evaluated by measurement of their effect on the percent increased lifespan. RESULTS: AraC had good in vitro antiproliferative activity (IC50 = 14 nM) but was ineffective in vivo against the HL60 promyelocyte leukemia cell line (treated vs control, T/C = 105%). Troxacitabine, which in contrast to AraC is not a substrate for cytidine deaminase, showed potent in vitro and in vivo activity in the same model (IC50 = 53 nM and T/C = 272% to 422%). The poor in vivo activity of AraC against HL60 leukemia cells could be due to the high cytidine deaminase (CDA; EC 3.5.4.5) activity in this cell line. This hypothesis was tested with CCRF-CEM T-lymphoblastoid leukemia cells which have undetectable levels of CDA activity. Short-term exposure of these leukemia cell lines to both drugs indicated that AraC was indeed significantly more effective in the CCRF-CEM cell line than in HL60. In contrast, the antiproliferative activity of troxacitabine was similar for both cell lines. These observations were extended to in vivo studies. Mice bearing CCRF-CEM tumor xenografts were treated with AraC and troxacitabine. In this model, T/C values were comparable for both drugs and ranged from 138% to 157%. CONCLUSIONS: Our findings indicate that troxacitabine is likely to be effective not only against solid tumors with high CDA activity but also in leukemias which have developed resistance to AraC due to increased CDA levels; this suggests that troxacitabine is a promising agent for the treatment of cancer. Indeed, significant antileukemic activity has been observed with troxacitabine in a phase I clinical trial in patients with primary refractory or relapsed acute myeloid leukemias (AML).

Animals↗
PubMed2001

Establishment of a gene transfer system for Pseudozyma flocculosa, an antagonistic fungus of powdery mildew fungi.

A reliable DNA-mediated transformation system has been developed for Pseudozyma flocculosa, a fungus that is antagonistic to powdery-mildew fungi. Plasmids harboring various selectable markers under the control of different promoters were tested. Molecular analyses demonstrated that successful transformation could be achieved using a plasmid that confers resistance to hygromycin B under the control of the Ustilago maydis hsp70 promoter and terminator sequences. On average, 1-40 (mean = 20) transformants were obtained per 10 microg of linearized DNA per 10(8) protoplasts. Southern analysis of the transformants revealed that, in each case, the vector had integrated in multiple tandem copies into the genome of P. flocculosa, and that integration events were random. Pulsed-field gel electrophoresis was employed to separate the genome of P. flocculosa into at least 11 chromosomes with sizes ranging from 0.55 Mb to 2.9 Mb. Hybridization with the plasmid indicated that integration of vector DNA had occurred in one to several chromosomes depending on the transformant examined.

Base Sequence↗
PubMed2001

Identification of a gamma-interferon-responsive element in the promoter of the human macrophage scavenger receptor A gene.

In the present study, we demonstrate gamma-interferon (gamma-IFN)-inducible scavenger receptor A (SR-A) mRNA expression during the early stages of THP-1 and blood monocyte differentiation. Predominant induction of SR-A type II mRNA parallels the increased accumulation of cholesteryl esters under these conditions. A potential signal transducer and activator of transcription (STAT1) binding site (gamma-interferon activation site) in the SR-A promoter demonstrates gamma-IFN-inducible DNA binding activity and is most likely responsible for the gamma-IFN-dependent expression of an SR-A promoter-luciferase fusion construct. In contrast, gamma-IFN inhibits SR-A expression in mature macrophages as well as after prolonged gamma-IFN incubation of THP-1 monocytes. Taken together, these results demonstrate opposite effects of gamma-IFN on SR-A expression and activity during the early versus late stages of monocyte maturation. gamma-IFN-induced STAT1 activation, leading to increased SR-A expression, could therefore play an important role in the initial steps of foam cell formation and xanthomatosis.

Adult↗
PubMed2000

The double-stranded RNA-binding protein Staufen is incorporated in human immunodeficiency virus type 1: evidence for a role in genomic RNA encapsidation.

Human Staufen (hStau), a double-stranded RNA (dsRNA)-binding protein that is involved in mRNA transport, is incorporated in human immunodeficiency virus type 1 (HIV-1) and in other retroviruses, including HIV-2 and Moloney murine leukemia virus. Sucrose and Optiprep gradient analyses reveal cosedimentation of hStau with purified HIV-1, while subtilisin assays demonstrate that it is internalized. hStau incorporation in HIV-1 is selective, is dependent on an intact functional dsRNA-binding domain, and quantitatively correlates with levels of encapsidated HIV-1 genomic RNA. By coimmunoprecipitation and reverse transcription-PCR analyses, we demonstrate that hStau is associated with HIV-1 genomic RNA in HIV-1-expressing cells and purified virus. Overexpression of hStau enhances virion incorporation levels, and a corresponding, threefold increase in HIV-1 genomic RNA encapsidation levels. This coordinated increase in hStau and genomic RNA packaging had a significant negative effect on viral infectivity. This study is the first to describe hStau within HIV-1 particles and provides evidence that hStau binds HIV-1 genomic RNA, indicating that it may be implicated in retroviral genome selection and packaging into assembling virions.

Binding Sites↗
PubMed2000

Involvement of oxysterols and lysophosphatidylcholine in the oxidized LDL-induced impairment of serum albumin synthesis by HEPG2 cells.

Oxidized low density lipoproteins (Ox-LDLs) are increasingly thought to be a key element in atherogenesis. We have previously reported that serum albumin has important antioxidant properties and that a reduced synthesis of albumin may represent a crucial point in the overall antioxidant defense. In the present work, we aimed at determining whether Ox-LDL could modulate albumin synthesis in cultured human hepatocytes (HepG2 cells). With the use of enzyme immunoassay and radiolabeled leucine incorporation followed by specific immunoprecipitation, Ox-LDL was found to lead to a dose-dependent decrease in albumin secretion. Moreover, the protein synthesis and mRNA levels were decreased in the presence of Ox-LDL, as assessed by Northern blot analysis. Because oxysterols and lysophospholipids are key components of Ox-LDL, we tested the effects of oxysterols (7-ketocholesterol and 25-hydroxycholesterol) and lysophosphatidylcholine on albumin secretion and expression. In our experimental conditions, we found that incubations with oxysterols or lysophosphatidylcholine at pathophysiological concentrations similar to those measured in Ox-LDLs reproduced the above-mentioned inhibitory effects on albumin synthesis. On the basis of our in vitro data, we propose that this newly described biological effect of Ox-LDL might partly explain the findings of epidemiological studies indicating that reduced levels of serum albumin are associated with increased mortality.

Antioxidants↗
PubMed1999

A glycosyl hydrolase activity of mammalian reovirus sigma1 protein can contribute to viral infection through a mucus layer.

The mammalian reovirus sigma1 protein is responsible for viral attachment to host cells and hemagglutination properties of the virus. In the present study, sequence similarity between sigma1 and chicken-type lysozymes prompted us to investigate additional functions of the sigma1 protein. Expression in Pichia pastoris yeast cells showed that sigma1 can actually cleave lysozyme substrates, including complex sugars found in bacterial cell walls. Replacement by site-directed mutagenesis of acidic amino acid residues in sigma1 by their respective isosteric, uncharged, amino acid residues has allowed us to identify Glu36 and Asp54 as the catalytic pair involved in sigma1-mediated glycosidase activity. The enzyme appears inactive in virions but its activity is unmasked upon generation of infectious subviral particles (ISVPs) by partial proteolytic removal of the outer capsid proteins. Purified sigma1 protein and ISVPs can also hydrolyze mucins, heavily glycosylated glycoproteins that are a major component of the mucus layer overlaying the intestinal epithelium. Furthermore, reovirus infection of epithelial Madin Darby canine kidney cells was inhibited tenfold in cells expressing mucin at their apical surface, while this inhibition was overcome by ISVPs. Unmasking of sigma1 mucinolytic activity in the intestine, consecutive to proteolytic cleavage of virions to ISVPs, thus likely contributes to the known increase in infectivity of reovirus ISVPs compared to complete virions. This work presents the first evidence that some mammalian viruses have evolved mechanisms to facilitate their penetration through the protective barrier of the mucus layer in the intestinal tract.

Amino Acid Sequence↗
PubMed1999

Apolipoprotein E and atherosclerosis: insight from animal and human studies.

Major advances have been made in our understanding of the role of apolipoprotein E (apoE) in the onset and development of atherosclerosis. Increasing evidence from both animal and human studies suggests that apoE is able to protect against atherosclerosis by: a) promoting efficient uptake of triglyceride-rich lipoproteins from the circulation; b) maintaining normal macrophage lipid homeostasis; c) playing a role in cellular cholesterol efflux and reverse cholesterol transport; d) acting as an antioxidant; e) inhibiting platelet aggregation; and f) modulating immune function. In humans, apoE is polymorphic, and this genetic variation has a strong effect on its antiatherogenic characteristics. Thus, compared to the epsilon3 allele, the epsilon4 allele promotes atherosclerosis, whereas the epsilon2 allele is either pro- or anti-atherogenic, depending on the influence of both environmental and genetic factors. ApoE and its gene are prime targets for therapeutic intervention aimed at preventing or treating atherosclerotic vascular disease.

Animals↗
PubMed1999

Mitochondrial iron sequestration in dopamine-challenged astroglia: role of heme oxygenase-1 and the permeability transition pore.

Little is currently known concerning the mechanisms responsible for the excessive deposition of redox-active iron in the substantia nigra of subjects with Parkinson's disease (PD). In the present study, we demonstrate that dopamine promotes the selective sequestration of non-transferrin-derived iron by the mitochondrial compartment of cultured rat astroglia and that the mechanism underlying this novel dopamine effect is oxidative in nature. We also provide evidence that up-regulation of the stress protein heme oxygenase-1 (HO-1) is both necessary and sufficient for mitochondrial iron trapping in dopamine-challenged astroglia. Finally, we show that opening of the mitochondrial transition pore (MTP) mediates the influx of non-transferrin-derived iron into mitochondria of dopamine-stimulated and HO-1-transfected astroglia. Our findings provide an explanation for the pathological iron sequestration, mitochondrial insufficiency, and amplification of oxidative injury reported in the brains of PD subjects. Pharmacological blockade of transition metal trapping by "stressed" astroglial mitochondria (e.g., using HO-1 inhibitors or modulators of the MTP) may afford effective neuroprotection in patients with PD and other neurological afflictions.

Animals↗
PubMed1999

Rubidium marking of Anopheles stephensi (Diptera: Culicidae).

Immature Anopheles stephensi Liston were reared in untreated water and water containing eight 2-fold dilutions of rubidium (Rb) from 1,000 to 7.8 ppm to determine the concentration that allowed reliable detection and produced the least toxic effects as measured by adult emergence, weight, and survival. The amount of Rb detected in mosquitoes increased positively with increasing concentrations in the rearing water. Concentrations > or = 31.2 ppm Rb in the rearing water provided high and consistent detection levels of > or = 3,500 ppm Rb/mg of adult mosquito. There were no adverse effects of Rb on the weights of mosquitoes. However, increased Rb concentrations in the rearing water were associated with decreased emergence and survival. After 8 d, percentage emergence from Rb concentrations of 0-31.2 ppm was > or = 50%. At day 21, Rb concentrations of 0-31.2 ppm showed < or = 29% reduction of female survival compared with controls. The recommended concentration for reliable Rb detection with minimal toxic effects in An. stephensi was 32 ppm.

Animals↗
PubMed1997

Cerato-ulmin, a hydrophobin secreted by the causal agents of Dutch elm disease, is a parasitic fitness factor.

Dutch elm disease is caused by the aggressive Ophiostoma novo-ulmi and the nonaggressive O. ulmi. Both secrete the protein cerato-ulmin (CU). To determine what role CU plays in the pathology of Dutch elm disease, we constructed a CU overexpression mutant of the nonaggressive O. ulmi H5. Stable integration of a single copy of the cu gene from the aggressive O. novo-ulmi into the genome of the nonaggressive isolate resulted in increased secretion of CU protein. Trials with American elm, Ulmus americana, suggested no alteration of virulence of this overexpressing transformant. Using aggressive and nonaggressive wild types, the cu overexpressing mutant, and our cu- mutant (Bowden et al., 1996), we have demonstrated that CU production is correlated with an altered phenotype and more hydrophobic and adherent yeast-like cells. Our results also demonstrate that CU has a role in protecting infectious propagules from desiccation. These biological roles for CU would affect transmission of Dutch elm disease, and we therefore propose that this hydrophobin acts as a parasitic fitness factor.

Cell Adhesion↗
PubMed1997

A meiotically reproducible chromosome length polymorphism in the ascomycete fungus Ophiostoma ulmi (sensu lato).

We have followed the transmission of Ophiostoma ulmi s.l. chromosome length polymorphisms (CLPs) into the F2 generation to determine the reproducibility of a genome rearrangement culminating in the conversion of a 1.0 Mb chromosome into a 800 kb chromosome. The 1.0 Mb chromosome in strain CESS16K is thus far unique among O ulmi s.l. wild-type strains, as no other wild-type strains have been observed with chromosomes smaller than 2.3 Mb. It has been previously shown that the 1.0 Mb chromosome is mitotically stable, carries at least one normally expressed gene, and is transmitted through meiosis. In this study, a series of crosses were performed to further elucidate the pattern of inheritance of the 1.0 Mb chromosome and the process of conversion of the 1.0 Mb species to 800 kb. In crosses where the 1.0 Mb chromosome was allowed to pair with itself or with the 800 kb chromosome, all progeny inherited a copy of the 1.0 Mb or 800 kb form, further demonstrating the A-type nature of these small chromosomes. When a cross was repeated between the strains CESS16K (1.0 Mb chromosome) and FG245Br-O (no 1.0 Mb or 800 kb chromosome), the occurrence of a 800 kb chromosome was observed in 9% of the progeny. A reciprocal cross between an 800 kb strain and a strain with no 800 kb or 1.0 Mb chromosome was conducted, and a progeny strain containing a 1.0 Mb chromosome was recovered. The reproducibility and reciprocality of the 1.0 Mb to 800 kb chromosome conversion demonstrates that meiotic processes are responsible for this CLP, and that O. ulmi s.l. strains with various divergent genome architectures can remain sexually compatible.

Ascomycota↗
PubMed1996

Biological and clinicopathological features associated with hepatitis C virus type 5 infections.

BACKGROUND/AIMS: The biological and clinicopathological features of hepatitis C virus infections of type 1 and type 2 have been well documented. However, little is known about the nature of HCV type 5 infections, which have been found to occur in South Africa but rarely elsewhere. METHODS: We investigated the HCV genotypes in 125 viremic blood donors and 125 viremic patients by restriction endonuclease analysis of amplified 5' non-coding region sequences. Donors and patients infected with type 5 were further studied. Serum HCV RNA levels were assessed by a differential-size PCR-aided transcript titration assay. RESULTS: HCV type 5 infections were identified in seven (5.6%) of the blood donors and in five (4.0%) of the patients. Sera from these 12 persons reacted with the core and NS3 antigens in both RIBA-2 and RIBA-3 tests. Six (50%) and 10 (83%) sera reacted with the NS4 antigens in RIBA-2 and RIBA-3 tests, respectively. HCV type 5 was found to replicate to high titers that ranged from 10(6.0) to 10(8.0) molecules/ml. Transfusion was the most frequently observed risk factor (5 of 12) and persons infected with type 5 were generally older than those infected with other types (< 40 years vs. > or = 40 years, p = 0.01). Cirrhosis was found in two of six (33%) donors and three of four (75%) patients. The duration of infection appeared to be an important determinant for the presence of cirrhosis. CONCLUSIONS: In this small group of Canadians infected with HCV type 5, a high proportion developed severe liver disease.

Adult↗
PubMed1996

Identification of numerous hepatitis C virus genotypes in Montreal, Canada.

Hepatitis C virus genotypes were determined for 358 viremic individuals in Montreal, Canada, by restriction endonuclease analysis of PCR products and phylogenetic analysis of core gene sequences. Types 1, 2, and 3 occurred in 62.8, 14.2, and 13.7%, respectively; types 4 and 5 were found in 3.9 and 4.5%, respectively; and genotypes 6a and 7c and a novel genotype each occurred in 0.3%. Types 4, 6, and 7 and the novel genotype were mostly from persons who had immigrated to Canada.

Adult↗
PubMed1995

Inheritance of chromosome-length polymorphisms in Ophiostoma ulmi (sensu lato).

We have investigated the mitotic and meiotic transmission of chromosome-length polymorphisms in Ophiostoma ulmi s.l., the causal agent of Dutch elm disease. The North-American aggressive (NAN) strain CESS16K has an atypical electrophoretic karyotype, carrying two chromosome-sized DNAs (chDNAs) that have not been observed in other members of the NAN biotype. Independent CESS16K chDNA preparations, even after repeated inoculation and recovery from the elm host, and analysis of 16 progeny strains after a cross between the NAN strains FG245Br-O and CESS16K, demonstrated that these unique chDNAs are integral components of the CESS16K genome. Analysis of the progeny, by electrophoretic karyotyping and hybridizations with probes specific to individual chDNAs, presented evidence that genome rearrangements can occur as a consequence of meiosis. Even though novel electrophoretic karyotypes and a novel-sized chromosome were observed in the karyotypes of the progeny strains, the low level of reassortment between the chromosomes carrying length polymorphisms presented evidence that there are constraints to genome plasticity for this fungus.

Ascomycota↗
PubMed1994

Biochemical mapping of neurofibrillary degeneration in a case of progressive supranuclear palsy: evidence for general cortical involvement.

A biochemical study was performed to quantify and map the neurodegenerating process in cortical and subcortical brain areas from a case of progressive supranuclear palsy (PSP). Our approach was based on a Western blot analysis of pathological Tau proteins, which are the basic components of neurofibrillary lesions. We found that: (i) the abnormal Tau proteins can be detected in all cortical areas, sometimes in larger amounts than in some subcortical areas; (ii) these abnormal Tau proteins consist of a doublet called Tau 64 and 69, except for in the entorhinal cortex where we detected, as for Alzheimer brains, the triplet of Tau proteins called Tau 55, 64 and 69; (iii) the amounts of abnormal Tau proteins were higher in some neocortical regions, especially in the frontal lobe, than in the hippocampal formation. Our results show that the neocortical pathology in PSP, as revealed by the presence of pathological proteins, is more extended than thought so far. Our biochemical approach appears to be more sensitive than the immunohistochemical one and can clearly differentiates between two types of neurofibrillary pathology, the Alzheimer type with a triplet of abnormal Tau proteins (Tau 55, 64 and 69) and the PSP type with a characteristic doublet (Tau 64 and 69).

Aged↗
PubMed1994

Comparison of ribosomal DNA length and restriction site polymorphisms in Gremmeniella and Ascocalyx isolates.

The small subunit (SSU) and the internal transcribed spacer (ITS) of nuclear ribosomal DNA genes from 27 specimens of the fungal genera Gremmeniella and Ascocalyx were amplified by PCR. Length polymorphisms were observed in the SSU and allowed the differentiation of four groups among the isolates tested: (i) Ascocalyx abietis; (ii) Gremmeniella isolates from Picea spp.; (iii) Gremmeniella isolates from Abies balsamea; and (iv) Gremmeniella isolates from Abies sacchalinensis, Larix spp., and Pinus spp. The amplified ITS was the same length for all Gremmeniella specimens and was 60 bp longer in A. abietis. Phylogenetic analysis of length polymorphisms and of 24 restriction sites in the SSU and ITS showed that Gremmeniella isolates were more related to each other than to the Ascocalyx isolate. Furthermore, seven groups were evident within the genus Gremmeniella. Our results confirm that Gremmeniella and Ascocalyx should be kept as different taxa and suggest that the taxonomy of the former could be revised to consider isolates from Abies balsamea and from Picea spp. to be two different varieties while incorporating Gremmeniella laricina into G. abietina, as a new variety.

Base Sequence↗