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M Anton

Publications and source records attributed to M Anton.

At least 55 records · Page 3Linked to original sources
PubMed1996

Phenylalanine 353 is a primary determinant for the positional specificity of mammalian 15-lipoxygenases.

Mammalian lipoxygenases are implicated in the biosynthesis of inflammatory mediators, in the pathogenesis of atherosclerosis and in the process of blood cell differentiation and maturation. With respect to their reaction specificity, three major types of mammalian lipoxygenases (15-lipoxygenases, 12-lipoxygenases and 5-lipoxygenases) may be classified. Although this nomenclature is commonly used, the mechanistic reasons for the positional specificity of lipoxygenases are not well understood. We investigated the structural reasons for lipoxygenase specificity by a combination of chimera formation and site-directed mutagenesis, and identified phenylalanine 353 as primary determinant for the positional specificity of rabbit reticulocyte 15-lipoxygenase. Modeling of the enzyme-substrate interaction suggested that the alignment of arachidonic acid at the active site appears to be influenced by this residue. According to the substrate orientation, the 15-lipoxygenase may be differentiated from two types of mammalian 12-lipoxygenases.

Amino Acid Sequence↗
PubMed1996

A helper-dependent adenovirus vector system: removal of helper virus by Cre-mediated excision of the viral packaging signal.

Adenoviruses are attractive vectors for the delivery of foreign genes into mammalian cells for gene therapy. However, current vectors retain many viral genes that, when expressed at low levels, contribute to the induction of a host immune response against transduced cells. We have developed a helper-dependent packaging system for production of vectors that have large regions of the genome deleted. Helper viruses were constructed with packaging signals flanked by loxP sites so that in 293 cells that stably express the Cre recombinase (293Cre), the packaging signal was efficiently excised, rendering the helper virus genome unpackageable. However, the helper virus DNA was replicated at normal levels and could thus express all of the functions necessary in trans for replication and packaging of a vector genome containing the appropriate cis-acting elements. Serial passage of the vector in helper virus-infected 293Cre cells resulted in an approximately 10-fold increase in vector titer per passage. The vector could be partially separated from residual helper virus by cesium chloride buoyant density centrifugation. Large scale preparations of vector yielded semi-purified stocks of approximately 10(10) transducing virions/ml, with < 0.01% contamination by the E1-deleted helper virus. This system should have great utility for the generation of adenovirus-based vectors with increased cloning capacity, increased safety and reduced immunogenicity.

Adenoviridae↗
PubMed1996

Production and characterization of human 293 cell lines expressing the site-specific recombinase Cre.

We have constructed 293 cell lines expressing the site-specific Cre recombinase from bacteriophage P1, that acts on a 34 bp target sequence called loxP. Stably transformed cells were obtained by transfection with a plasmid containing Cre and a selectable marker under the control of viral promoters. The resulting 293Cre cell lines could be used to induce expression from adenovirus vectors containing reporter genes under the control of a Cre responsive "molecular switch." High efficiency recombination was observed for Ad viral DNA containing loxP sites. The Cre expressing cell lines described here are likely to be useful for several purposes: For expression of toxic gene products from Cre inducible viral vectors, to induce recombination between loxP sites in transfected plasmids, and to induce deletions or rearrangements of genes defined by loxP sites in viral genomes.

Cell Line↗
PubMed1996

Microtubule-based peroxisome movement.

The association of peroxisomes with cytoskeletal structures was investigated both by electron microscopy and by kinetic analysis of peroxisome movement. The morphological studies indicated distinct interactions of peroxisomes with microtubules and frequently revealed multiple contact sites. The kinetic approach utilised microinjection and import of fluorescein-labeled luciferase in order to mark and track peroxisomes in vivo. Peroxisomal motility was analysed by time-lapse imaging and fluorescence microscopy. According to their movement peroxisomes were classified into two groups. Group 1 peroxisomes comprising the majority of organelles at 37 degrees C moved slowly with an average velocity of 0.024 +/- 0.012 micron/second whereas the movement of group 2 peroxisomes, 10-15% of the total population, was saltatory exhibiting an average velocity of 0.26 +/- 0.17 micron/second with maximal values of more than 2 microns/second. Saltations were completely abolished by the microtubule-depolymerising drug nocodazole and were slightly reduced by about 25% by cytochalasin D which disrupts the actin microfilament system. Double fluorescence labeling of both peroxisomes and microtubules revealed peroxisome saltations linked to distinct microtubule tracks. Cellular depletion of endogenous levels of NTPs as well as the use of 5'-adenylylimidodiphosphate, a nonhydrolysable ATP analog, applied to a permeabilised cell preparation both completely blocked peroxisomal movement. These data suggest an ATPase dependent, microtubule-based mechanism of peroxisome movement. Both the intact and the permeabilised cell system presented in this paper for the first time allow kinetic measurements on peroxisomal motility and thus will be extremely helpful in the biochemical characterisation of the motor proteins involved.

Actins↗
PubMed1996

Concentrations of polychlorinated biphenyls and chlorinated pesticides in human breast milk--a case study.

Concentrations of chlorinated pesticides (p,p'-DDE, lindane), hexachlorobenzene (HCB), sum of polychlorinated biphenyls (PCBs), and PCB congeners in breast milk during lactation and the distribution of the chlorinated compounds in fat tissue, blood serum and breast milk were pursued in five primi- or secundipara from the hinterland of a clinic of gynaecology and obstetrics in Brno in 1993. Capillary gas chromatography with ECD detection was used for the determination of the residues. The concentrations of HCB, lindane, p,p'-DDe, and sum of PCBs ranged from 42.5 to 238.4, from < 1.0 to 7.4 from 231.4 to 557.6, and from 661.3 to 2888.9 micrograms.kg-1 of milk fat, respectively. Seventeen PCB congeners were screened, of which 11 were identified in most of the milk samples (IUPAC numbers 28, 101, 118, 126, 128, 138, 153, 156, 170, 180, 194). Congeners 138, 153, 170, and 180 were the most abundant and their concentrations in microgram.kg-1 of milk fat ranged from 122.9 to 501.0 for 153, from 103.4 to 372.1 for 138, from 66.1 to 407.9 for 180, from 26.8 to 183.1 for 170, and from 10.0 to 41.7 for 118.

Adipose Tissue↗
PubMed1995

High frequency recombination between loxP sites in human chromosomes mediated by an adenovirus vector expressing Cre recombinase.

An adenovirus vector (AdCre1) expressing Cre recombinase has been used to induce recombination between loxP sites in human chromosomes. G418 resistant cells with one loxP site, generated by transfection with a plasmid containing loXp between the SV40 promoter and the G418 resistance (neo) gene, were infected with AdCre1 and transfected with a plasmid containing loxP adjacent to a promoterless hisD gene. This resulted in integration of hisD downstream of the SV40 promoter with gain of histidinol and loss of G418 resistance. Since AdCre1 is non-replicating and Cre expression transient, histidinol resistant cells containing the hisD gene flanked by loxP sites were stable. Reinfection of these cells with AdCre1 induced excision of hisD in over 90% of infected cells. This high efficiency of site-specific recombination suggests that AdCre1 may be exploited for temporal and tissue-specific regulation of gene expression and for chromosome engineering in vitro and in animals.

Adenoviridae↗
PubMed1995

Site-specific recombination mediated by an adenovirus vector expressing the Cre recombinase protein: a molecular switch for control of gene expression.

We have constructed replication-defective human adenovirus (Ad) type 5 vectors containing the gene for the Cre recombinase from bacteriophage P1 under control of the human cytomegalovirus immediate-early promoter (AdCre). Expression of the protein was detected in replication-permissive (293) and in nonpermissive (MRC5) cell lines, and its biochemical activity was demonstrated in a cell-free recombination assay using a plasmid containing two loxP sites. To study Cre-mediated recombination in an intracellular system, we constructed an Ad vector (AdMA19) containing the luciferase cDNA under control of the human cytomegalovirus promoter but separated from it by an extraneous spacer sequence flanked by loxP sites which blocked luciferase expression. Upon coinfection of 293 or MRC5 cells with AdMA19 and AdCre, luciferase expression was specifically induced by Cre-mediated excision of the intervening sequence. The use of Ad vectors combined with the Cre-loxP system for regulation of gene expression and other possible applications is discussed.

Adenoviruses, Human↗
PubMed1995

Overexpression of p53 and MDM2 proteins in cervical neoplasia.

Sixteen methacarn-fixed and paraffin-embedded cervical squamous lesions were examined using monoclonal antibodies to p53 and MDM2 in order to compare the expression of both proteins in cervical neoplasia. Standard avidin-biotin or streptavidin-biotin immunoperoxidase techniques were employed. The results show that the overexpression of both proteins takes place in a meaningful proportion of cervical neoplastic lesions. The expression of either protein is not very frequent in CIN 1 and 2. The overexpression of either p53 (9/12) or MDM2 (4/10) proteins was recorded in the group of more advanced lesions, their level fluctuating from 10% to 60% positive cells. The results suggest the possibility of an interaction of the p53 and MDM2 proteins in some cases of cervical neoplasia.

Acetates↗
PubMed1995

[Respiratory allergies among bakers and pastry cooks: epidemiologic survey done in 1991 by the occupational physicians of the Loire-Atlantique].

The aim was to study the prevalence of respiratory allergy (rhinitis and asthma) in a population of bakers and pastrycooks. In 1991, 485 bakers and pastry cooks were examined by 27 work-physicians of Loire-Atlantic. The investigation was composed of a standardised questionnaire (signs of respiratory function, atopic history, smoking of tobacco ...), a clinical examination, and tests of respiratory function. An allergy assessment was made of all subjects with symptoms. 14.4% of subjects had rhinitis and 6.4% asthma. Development of these pathologies was clearly job-related for 2/3 of those with rhinitis and more than half of the asthmatics (55%). Occupational rhinitis and asthma were significantly more frequent in bakers than in pastrycooks and were linked to atopic history. Occupational asthma was associated with length of exposure to flour and with occupational rhinitis. In conclusion, these findings are comparable with or a little less than those that have been reported in occupational literature. They under-estimate the importance of the problem because of the occupational selection effect that is associated with these pathologies. Rhinitis and asthma are 1.5 to 3 time more common in bakers than in pastrycooks.

Adult↗
PubMed1995

[Respiratory allergies among symptomatic bakers and pastry cooks: initial results of a prevalence study].

A survey was carried out on respiratory symptoms and skin prick response to common allergens, storage mite and occupational allergens. Among 178 symptomatics bakers and pastry workers from small businesses in western France, only 65 people underwent skin prick and specific-IgE. 12 (18%) workers were skin positive to at least one common or occupational allergens. The more often skin positive were D. Ptero. mite 36 (57%); Alpha amylase 23 (35%); wheat flour 17 (26%); saccharomyces cerevisiae 16 (25%); Ephestia 15 (24%). The sensitivity of skin test was better than specific IgE for D. Ptero. Mite 36 (57%); and Alpha amylase 23 (35%). The sensitivity of specific IgE was better than skin test for wheat flour 26 (45%) and rye flour 23 (40%). Occurrence of skin positive to occupational allergen among symptomatics with rhinitis and asthma is much more frequent in workers with skin positive to common allergens (40/36) than in workers with skin negative (8/20). Atopy must be regarded as an important predisposing factor for skin sensitisation to occupational allergens. We conclude in the necessity of a standardised allergologic exploration to be done in symptomatics bakers.

Allergens↗
PubMed1995

Energy aspects for elastic and viscous shoe soles and playing surfaces.

The purpose of this project was to determine the effect of changes in stiffness and viscosity of the foot ground interface on the work performed during locomotion. The estimation of the work during locomotion was derived from a mathematical two segment model, representing the foot and the rest of the body. The typical passive elements between the foot and the rest of the body were replaced by a strategic formulation of how a resultant force, F, representing the net effect of all the muscles between the foot and the rest of the body, has to evolve over time in a running situation. The calculations were performed under the assumption that the force F is selected so that the mechanical work performed by F is minimal. The estimations of the work required during a step cycle is generally higher for softer than for harder springs and for low damping compared with high damping. The model calculations demonstrate that specific combinations of material properties may be advantageous or disadvantageous from an energy point of view.

Algorithms↗
PubMed1993

Investigation of the oxygenation of phospholipids by the porcine leukocyte and human platelet arachidonate 12-lipoxygenases.

When arachidonate 12-lipoxygenase purified from porcine leukocytes was incubated aerobically with 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine, the phospholipid reacted at up to 30% of the rate of a free fatty acid substrate; the esterified arachidonic acid was oxygenated predominantly to the (12S)-12-hydroperoxy product. The porcine leukocyte enzyme was also capable of metabolizing phosphatidylcholine containing esterified (15S)-15-hydroperoxy-5,8,11,13-eicosatetraenoic acid; oxygenation occurred predominantly at the 14R position. Reaction with mitochondrial and endoplasmic membranes of rat liver produced esterified (12S)-12-hydroperoxy-5,8,10,14-eicosatetraenoic acid and (13S)-13-hydroperoxy-9,11-octadecadienoic acid as major oxygenation products. Thus, porcine leukocyte 12-lipoxygenase is capable of oxygenating not only free polyenoic fatty acids but also more complex substrates such as phospholipids and biomembranes. In contrast, the human platelet 12-lipoxygenase is almost inactive with these esterified polyenoic fatty acids. In regard to the function of these enzymes, the leukocyte-type of 12-lipoxygenase has similar catalytic activities to the mammalian 15-lipoxygenase and its physiological function may include the structural modification of membrane lipids.

Animals↗
PubMed1993

Cloning and sequencing of the Klebsiella pneumoniae tonB gene and characterization of Escherichia coli-K. pneumoniae TonB hybrid proteins.

The tonB gene of Klebsiella pneumoniae was cloned on a 1.4-kb EcoRV fragment and expressed in Escherichia coli. Determination of the nucleotide (nt) sequence of the cloned fragment identified tonB as an open reading frame (ORF) of 729 nt. The TonB protein consists of 243 amino acids (aa) and is highly homologous to Enterobacter aerogenes TonB. The genetic context of K. pneumoniae and En. aerogenes tonB is the same, but is different from that of E. coli, Salmonella typhimurium and Serratia marcescens. K. pneumoniae tonB complemented an E. coli tonB mutant as efficiently as E. coli tonB when cloned on the same type of plasmid. Replacement of the highly hydrophobic C terminus of E. coli TonB by the weakly hydrophobic C terminus of K. pneumoniae TonB yielded a hybrid protein of the same activity as E. coli wild-type TonB. The result shows that the strong hydrophobicity of the C-terminal end is not necessary for the function of E. coli TonB.

Amino Acid Sequence↗
PubMed1993

The wild-type allele of tonB in Escherichia coli is dominant over the tonB1 allele, encoding TonBQ160K, which suppresses the btuB451 mutation.

The entire coding sequence of the tonB gene, except for nine codons at the 3' end, was deleted from the chromosome of Escherichia coli. Introduction of the btuB451 suppressor mutant tonB1 into the chromosome of such a tonB deletion strain showed that the tonB1 allele was active as a suppressor in a single copy at 37 degrees C and 42 degrees C but not at 28 degrees C. No temperature dependence was seen when FepA- or FhuA-dependent activities of the tonB1 gene product (TonBQ160K) were tested. The btuB451 suppressor activity of tonB1 was inhibited by the simultaneous presence within the cells of the tonB+ allele on a multicopy plasmid. This represents the first case of dominance among different tonB alleles. Inhibition of suppression was abolished by overexpression of the btuB451-encoded receptor protein. Competition for binding of TonB+ and TonBQ150K to ExbB was excluded as the cause of dominance. Based on our data we conclude that competition for binding of TonB+ and TonBQ160K to the btuB451 gene product is the reason for the observed dominance. The implications of these findings for the mechanism of btuB451 suppression by tonB1 are discussed.

Alleles↗
PubMed1993

Pharmacological modulation of the spasmogenic response to methylxanthines in guinea-pig trachea.

The spasmogenic activity of methylxanthines was evaluated in guinea-pig isolated trachea treated with indomethacin (2.8 microM) and cooled to 20 degrees C. The contraction elicited by caffeine or theophylline (10 mM) was reduced in the presence of ouabain (10 microM), amiloride (100 microM), staurosporine (1 microM), H-7 (50 microM), polymyxin B (500 microM), K(+)-free solution, low Na+ (25 mM) medium or Ca(2+)-free (EGTA 0.1 mM) solution but was unaltered in the presence of verapamil (10 microM) or vanadate (10-100 microM). These results suggest that tracheal spasm to methylxanthines predominantly involves Ca2+ release from intracellular stores with a minor component due to extracellular Ca2+ entry through verapamil-insensitive pathways. A Na+/Ca2+ exchange process and the activation of protein kinase C may be also involved.

Animals↗
PubMed1992

[Histiocytosis X and the orbit in children].

Histiocytosis X, a clinical entity which was not defined and classified exactly so far, is manifested by a varied range of clinical manifestations. In rare instances the affection of the orbit is the only and first symptom of the disease. The authors present an account of three children. In one isolated affection of the orbit with a non-inflammatory swelling of the external portion of the upper eyelid was the first symptom of the disease. In the other two patients with the acute multifocal form of histiocytosis X exophthalmos developed only 3 and 7 months resp. after establishment of the diagnosis. In the discussion of symptoms, the prognosis and treatment the authors are inclined to believe that at present the prognosis of multifocal form of the disease is not always adverse. In the frequently difficult differential diagnosis of orbital lesions histiocytosis X should be considered as one of the possible causes of affections of the orbit in children.

Child, Preschool↗
PubMed1992

[DNA flow cytometry: perspective prognostic parameters in carcinoma of the uterine cervix].

We studied the relation between the 5-year disease free interval and the flow cytometric DNA content in a group of 55 patients treated by radiation for squamous cell carcinoma of the uterine cervix, stages Ib-IIIb (FIGO). The diploid DNA content was associated with a better prognosis, while prognostically unfavourable tumours tended to be aneuploid. The relation was statistically significant in the whole group (p = 0.016), in stage II disease (p = 0.003) and in the subgroup formed by the combination of stages I and II (p = 0.000). In stage III we did not prove the relation. Analysis of the survival function revealed also a better prognosis of diploid tumours (p = 0.041) in the whole group. The division into clearly diploid and non-diploid tumours seems to be more suitable for evaluation (p = 0.012). The difference between the prognostically favourable and unfavourable groups is expressed more clearly. We consider the flow cytometric DNA content a perspective prognostic parameter in squamous cell carcinoma of the uterine cervix. Its significance is apparent especially in patients treated by radiotherapy, because the size of the tumour cannot be assessed reliably in these cases.

Carcinoma, Squamous Cell↗