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Biomedical subjects

M Philippe

Publications and source records attributed to M Philippe.

At least 37 records · Page 2Linked to original sources
PubMed1999

Molecular characterization of femA from Staphylococcus hominis and Staphylococcus saprophyticus, and femA-based discrimination of staphylococcal species.

The femA gene encodes a protein precursor which plays a role in peptidoglycan biosynthesis in Staphylococcus aureus and is also considered as a factor influencing the level of methicillin resistance. A femA homologous gene was recently characterized in S. epidermidis, entailing the possibility of femA phylogenetic conservation in staphylococcal species. Accordingly, we assessed the presence of femA homologous genes in S. hominis and S. saprophyticus. Strategy for identification relied upon alignment of S. aureus and D. epidermidis femA sequences and upon identification of potentially conserved regions. Amplifications of portions of the femA genes were performed under permissive annealing conditions, by using several sets of primers designed to match the consensus regions. DNA sequencing of overlapping PCR fragments led to the characterization of the entire femA genes of S. hominis and S. saprophyticus, and provided more precise information on the femA start codon for all five species. The genomic organization of all these femA genes appeared highly conserved, with alternance of homologous and variable regions. On this basis, a consensus sequence of the femA gene was defined and interspecies variations were exploited to design strategies for staphylococci species-specific identification, including multiplex PCR amplification and a reverse hybridization assay.

Amino Acid Sequence↗
PubMed1999

Use of image analysis and immunostaining of bone marrow trephine biopsy specimens to quantify residual disease in patients with B-cell chronic lymphocytic leukemia.

Marrow residual disease (RD) in patients with B-cell chronic lymphocytic leukemia (B-CLL) who are in complete remission (CR) after treatment with purine analogues is reported to have a prognostic value, but sample dilution, factors interfering with marrow aspiration, or undetectable immunoglobulin rearrangement can affect the assessment of RD by molecular or immunologic methods. As demonstrated for hairy cell leukemia and follicular lymphoma, bone marrow trephine biopsy specimen immunostaining (BMT/IS) can successfully detect residual malignant cells. The aim of this study was to use BMT/IS and computerized image analysis (CIMA) of bcl-2-positive cells to quantify RD in B-CLL patients in CR, after achievement of CR and more than 1 year later. This methodology was compared with other conventional techniques, i.e., cytologic, flow cytometric, cytogenetic, and molecular analysis. BMT/IS readily detected RD in every trephine biopsy specimen examined, either after CR or at distant follow-up. CIMA allowed an objective quantification of residual B-CLL cells, as evidenced by the correlation with semiquantitative polymerase chain reaction results. Both analyses indicated a progression of RD. This finding was also supported (but inconsistently) by the other techniques. CIMA with an interstitial labeling index, therefore, seems to be a reproducible and sensitive method to detect persistence and progression of RD in patients with B-CLL. This method could apply to other hematologic malignancies infiltrating the bone marrow.

Adult↗
PubMed1999

[Delivery logbook: pertinence of the collected data for scientific exploitation].

OBJECTIVES: To determine if the data recorded in the delivery logbook are relevant and complete compared to the data registered in the patient's medical chart. MATERIAL AND METHODS: Prospective study during one month recording all the data registered by the midwives or the medical doctors in the delivery room immediately after the birth. To compare them to the information collected in the patient's medical chart. Our delivery logbook has 55 headings for each patient. During October 1998, we had 156 births. RESULTS: We had 5.3% of errors divided by missing data and erroneous entries. More precisely, we recorded 9.5% of errors in the antepartum data, 3.2% in intrapartum or postpartum events and 5.8% in the newborn information. CONCLUSION: This study demonstrates that medical data used for scientific projects and recorded in the delivery logbook, have to be interpreted very cautiously. It seems to be reasonable that each obstetrical unit should standardize its registration of data and carry out internal audits.

Bias↗
PubMed1999

Kinetic analysis of cell population growth during cultivation in vitro.

XL-2 cells (Xenopus laevis) were used for kinetic analysis of cell population growth. The dependence of the time of cell duplication on the percentage of cells in the G0 phase of the cell cycle was studied and described by a mathematical expression. Possible causes of the changes in the ratio between the percentage of cells in the cell cycle and that in the G0 phase were analyzed. These are the decrease in the percentage of cells in the G0 phase due to the increase in the number of dividing cells, their position in the cell islets, the number of nuclei, the relative position of cells in the G0 phase. It was shown that the loss of the free edge by cells during their transition to the second layer of the cell islets without any changes in spreading led to a significant increase in the percentage of cells in the G0 phase. The percentage of cells in the G0 phase increased about five times for multinuclear cells. Analysis of the position of cells in the G0 phase showed that these cells were mostly in groups of two, three or four. Studies of a real cell culture in the logarithmic phase of growth (48-120 h of cultivation) showed that the percentage of cells in the G0 phase did not virtually change and all processes were equalized by one another. We propose a new method to determine the cell cycle duration under conditions from the time of cell culture duplication and the data on the percentage of cells in the G0 phase. This method can be used when traditional approaches using BrdU or [3H]]thymidine are difficult to implement or are unacceptable.

Animals↗
PubMed1999

Keratinocyte-melanocyte co-cultures and pigmented reconstructed human epidermis: models to study modulation of melanogenesis.

Normal human melanocytes were amplified and cultured in a new defined culture medium without phorbol esters or cholera toxin. The medium decreased considerably the doubling time and increased the possible passage number. Melanocytes were co-seeded with normal human keratinocytes into 24 well culture dishes to screen potentially active modulators of melanogenesis. For the assay, the co-cultures were exposed to the compounds under investigation in the presence of 14C-thiouracil and 3H-leucine. Control cultures contain L-tyrosine or kojic acid, modulators which served as internal calibration standards. Changes in the rate of melanin synthesis were measured on the basis of 14C-thiouracil incorporation into newly synthesized melanin. A reduction or increase in 3H-leucine incorporation was taken as an indication of cytotoxicity or induction of proliferation, respectively. The NHK-NHM co-culture screening assay provides a useful tool to compare the activity of known modulators of melanogenesis and to perform structure-activity studies with newly identified modulators to improve their activity. The efficacy of particularly interesting new compounds was further evaluated on reconstructed pigmented epidermis after repeated topical application. The same model was used to assess the anti-pigmenting effect of sunscreens on UV-induced pigmentation. Integration of melanocytes from different ethnic origin resulted in pigmented epidermis reflecting different skin phenotypes, Caucasian, Asian and African.

1-Methyl-3-isobutylxanthine↗
PubMed1998

The product of the mammalian orthologue of the Saccharomyces cerevisiae HBS1 gene is phylogenetically related to eukaryotic release factor 3 (eRF3) but does not carry eRF3-like activity.

We describe here the cloning and sequencing of human and mouse cDNAs encoding a putative GTP binding protein. Sequence comparison shows that these cDNAs (named eRFS) are likely to represent the orthologues of the yeast Saccharomyces cerevisiae HBS1 gene and that the C-terminal domains of the encoded proteins share structural features with eukaryotic elongation factor eEF-1A and release factor 3 (eRF3) families. The phylogenetic analysis suggests that eRFS proteins and Hbs1p form a cluster of orthologous sequences branching with the eRF3 family. Nevertheless, in yeast, the human eRFS protein and Hbs1p do not complement eRF3/Sup35p thermosensitive mutation and do not interact with eRF1.

Amino Acid Sequence↗
PubMed1998

Cell cycle analysis and synchronization of the Xenopus cell line XL2.

We have determined the length of the cell cycle and its different phases in a permanent Xenopus tadpole cell line, XL2. Following BrdU labeling, the total length of the cell cycle was estimated as 28 h. The different phases of the cell cycle, G1, S, G2, and M were, respectively, 14 h, 10 h 45 min, 2 h 30 min, and 54 min. Knowing these parameters, we were able to develop methods that selectively enrich cells in different phases of the cycle. Treatment with aphidicolin resulted in a S phase block in which more than 85% of the cells showed S phase chromosomes. Almost 60% of the cells were arrested in mitosis after a double block with aphidicolin/nocodazole or aphidicolin/ALLN (acetyl-leucyl-leucyl-norleucinal) treatment. This synchronization protocol will greatly facilitate studies of biochemical events associated with specific gene regulation through the cell cycle. Our synchronization protocol does not disturb cell metabolism as the expression of cyclin B2 during the cell cycle is in agreement with the results obtained with mammalian cells.

Animals↗
PubMed1998

The small G-proteins Rap 1 as potential targets of vasoactive intestinal peptide effects in the human clonic cancer cells HT29.

We recently reported that the vasoactive intestinal peptide (VIP) potently inhibited proliferation and induced in parallel a strong cAMP rise, in the human colonic cancer cell line HT29. In this study, we investigated whether Rap 1 proteins could be potential targets of VIP effects in HT29 cells. These Ras-related proteins in which activity was demonstrated to be regulated by PKA phosphorylation, are considered as potential modulators of the Ras / Raf / MAP kinases cascade that governs cell growth control. Our data revealed that the Rap 1a isoform is highly expressed in HT29 cells and mainly localized in a late endosomal compartment. In these cells, VIP induces Rap 1 phosphorylation and a yet unidentified modification that leads to their acidification. This latter Rap 1 acidification seems to be, at least partially, cAMP-dependent. It is concluded that in HT29 cells, Rap 1 proteins may be part of a VIP-induced signaling cascade.

Antibodies↗
PubMed1998

Introduction of a normal retinoblastoma (Rb) gene into Rb-deficient lymphoblastoid cells delays tumorigenicity in immunodefective mice.

Inactivation of the Rb susceptibility gene occurs in various human cancers and has been associated with tumorigenicity. Rb gene is inactivated in 30% of acute leukaemias. The effect of Rb protein expression was assessed in the lymphoblastoid cell line IM-9 defective for Rb protein, after stable transfection with a wild-type Rb gene. The Rb transgene was under the control of the MoMuLv-LTR. Protein expression by the transduced cells was confirmed by Western blot and flow cytometry analyses. Compared to the parental cell line, growth rate remained unchanged in the Rb transfected clones. In SCID mice however, tumor formation originating from these clones was delayed. The current data suggest therefore that, in this Rb-defective haematopoietic cell line, Rb expression correlates with reduced tumorigenicity but not with reduced growth rate.

Animals↗
PubMed1998

Cardiac troponin I as an early marker of myocardial damage after coronary bypass surgery.

STUDY OBJECTIVE: To evaluate the performance of cardiac specific markers, cardiac troponin I (cTnI) and CK-MB by mass assay (CK-MB mass), for the early diagnosis of myocardial ischemia and/or infarction after coronary bypass surgery. METHODS: Prospective clinical, electrocardiograpic and biologic follow-up of 117 patients undergoing isolated coronary surgery with the use of intermittent anterograde normothermic blood cardioplegia. Blood samples for biochemical analysis were drawn before surgery (T0) and at 2 (T1), 6 (T2), 10 (T3) and 20 h (T4) after aortic cross-clamp release. Without knowledge of the biochemical data, patients were classified according to the electrocardiographic evolution into two groups: group 1, uneventful recovery and group 2, evidence of ischemia/infarction based on continuous ST-T segment monitoring and 12-lead ECG. RESULTS: No patients had abnormal markers at T0. At T1, although both markers were elevated, no difference was noted between the two groups. At T2, 6 h after surgery, cTnI and CK-MB mass levels were significantly higher in group 2 than in group 1 (median = 17 microg/l, Interquartile Range (IR): 14.7-27.3 vs. 3.1 microg/l, IR 1.9-5.3 for cTnI and median 42.5 microg/l, IR: 27.1-95.7 vs. 13.6 microg/l, IR: 9.5-18.5 for CK-MB mass). A receiver operating characteristic (ROC) curve analysis shows that a cTnI value of 13.1 microg/ml has 100% specificity and 90% sensitivity to separate both groups, whereas a value of 33.2 microg/ml for CK-MB mass has a specificity of 100% and a sensitivity of 73%. At T3 and T4, the same difference was noted between the groups. cTnI values in all six patients with a Q-wave infarction were > or = 20 ng/ml, whereas only one of five patients with prolonged ischemia had cTnI level > 20 ng/ml. CONCLUSION: As soon as 6 h postoperatively, cTnI and CK-MB by mass assay were able to separate those patients with an uneventful recovery from those with significant ischemia. This is particularly useful in frequent cases when the ECG is difficult to interpret.

Biomarkers↗
PubMed1998

The prothrombin gene G20210A variant in an unselected thromboembolic population. A Belgian prospective clinical study.

The presence of the 20210A allele of the prothrombin gene has recently been shown to be a risk factor of venous thromboembolism, probably mediated through increased prothrombin levels. The aim of the study was to determine the frequency of the prothrombin 20210A allele in 193 consecutive unselected patients with venous thromboembolism and 100 healthy controls and to analyze the clinical profile associated with this new inherited thrombophilic factor. In agreement with previous reports, we found a frequency of 7.3% of heterozygous carriers of the 20210A allele among patients and 1% among controls. We confirm that plasma prothrombin levels are more elevated in the individuals bearing the prothrombin 20210A allele compared with those who do not. We did not find any relationship between the presence of the prothrombin 20210A allele and either a family history of thromboembolism, the rate of recurrences or the age at disease onset. However, the co-inheritance in the same individual of both prothrombin 20210A allele and factor V Leiden was associated with a significantly lower age at disease onset suggesting a synergistic contribution of both abnormalities.

Adult↗
PubMed1998

Functional interaction between the bovine papillomavirus virus type 1 replicative helicase E1 and cyclin E-Cdk2.

We have found that the replicative helicase E1 of bovine papillomavirus type 1 (BPV-1) interacts with a key cell cycle regulator of S phase, the cyclin E-Cdk2 kinase. The E1 helicase, which interacts with cyclin E and not with Cdk2, presents the highest affinity for catalytically active kinase complexes. In addition, E1, cyclin E, and Cdk2 expressed in Xenopus egg extracts are quantitatively coimmunoprecipitated from crude extracts by either anti-Cdk2 or anti-E1 antibodies. E1 protein is also a substrate of the cyclin E-Cdk2 kinase in vitro. Using the viral components required for in vitro BPV-1 replication and free-membrane cytosol from Xenopus eggs, we show that efficient replication of BPV plasmids is dependent on the addition of E1-cyclin E-Cdk2 complexes. Thus, the BPV initiator of replication and cyclin E-Cdk2 are likely to function together as a protein complex which may be the key to the cell cycle regulation of papillomavirus replication.

Amino Acid Sequence↗
PubMed1998

Angiotensin I-converting enzyme genotype influences arterial response to injury in normotensive rats.

Two normotensive strains of rat, the Lou and Brown Norway (BN) strains, have contrasting levels of plasma angiotensin-converting enzyme (ACE). To investigate the degree of genetic determination of ACE expression, a polymorphic marker of the ACE gene was analyzed in inbred rats of the two strains. The two inbred strains were shown to bear different alleles for a polymorphic marker at the ACE gene. The segregation of the alleles of this marker and the plasma ACE levels were studied in a group of F2 rats issued from a cross between Lou and BN rats. The degree of genetic determination of plasma ACE activity was estimated to be 94% in the F2 cohort. The ACE locus accounts for 74% of total plasma ACE variance. ACE activity and mRNA expression in lungs were also genetically determined. The difference observed in ACE mRNA accumulation in the lungs between the two strains was due to a difference in the transcriptional rate of the ACE gene, as shown in nuclear run-on experiments. No differences were observed in arterial blood pressure of homozygous F2 progeny. In these animals, ACE genotype did not interfere with the pressor or the depressor responses to ACE-dependent vasoactive peptides. There was a significant effect of strain on constitutive or inducible membrane or soluble ACE activity in primary cultures of vascular cells. Neointima formation in the carotid artery 14 days after balloon injury was also influenced by the genotype in F2 homozygous progeny, whereas the medial area was not. These results demonstrate that there is a close relationship between the genetically determined ACE expression and the inducibility of the ACE gene. The degree of genetic determination of ACE expression in inbred rat strains offers a unique opportunity to study the interaction between genetic and environmental determinants of ACE expression and its involvement in response to experimental cardiovascular and renal injury.

Animals↗
PubMed1998

Chronic blockade of NO synthase activity induces a proinflammatory phenotype in the arterial wall: prevention by angiotensin II antagonism.

Chronic blockade of NO production induces hypertension and early occlusive and fibrotic end-stage organ damage owing to vascular lesions in the brain, kidney, and heart. In this study, we evaluated the inflammatory phenotypic changes induced in the arterial wall by chronic N(G)-nitro-L-arginine methyl ester (L-NAME) administration and the effect of an angiotensin II receptor (AT1) antagonist, irbesartan, on these changes. For this purpose, 2 groups of rats received L-NAME in the drinking water (50 mg x kg(-1) x d(-1)) for 2 months. One group received no other treatment and the other was treated with irbesartan (10 mg x kg(-1) x d(-1)). A third group (controls) received neither L-NAME nor irbesartan. After 8 weeks, plasma, aortas, and left ventricles were sampled from all 3 groups. Expression of inducible NO synthase (iNOS) was evaluated at both the mRNA (quantitative reverse transcription-polymerase chain reaction) and the protein (Western blot and immunohistochemistry) level in the aorta. Expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) was evaluated by reverse transcription-polymerase chain reaction, Western immunoblotting, and immunohistochemistry; inflammatory cell infiltration by immunohistochemistry; and fibrosis by Sirius red staining. Chronic L-NAME administration induced the expression of iNOS in the aorta, which was localized in smooth muscle cells as shown by immunohistochemistry and NADPH diaphorase activity. ICAM-1 and VCAM-1 expression was also increased in aortas of L-NAME-treated rats. These phenotypic changes of the vascular wall were associated with inflammatory cell infiltration and fibrosis in the heart. All of these pathological phenomena were prevented by the angiotensin II antagonist irbesartan. The proinflammatory phenotypic changes of the vascular wall induced by blockade of NOS activity could be involved in the interaction between endothelial dysfunction and the development of arteriosclerosis.

Angiotensin II↗
PubMed1998

Expression of plasminogen activator inhibitors type-1 and type-2 in the mouse lung after administration of crystalline silica.

Altered expression of plasminogen activator inhibitors (PAIs) is of potential relevance to the process of lung fibrosis. To clarify the involvement of PAIs in interstitial lung diseases, we examined whether alterations in PAI-1 and PAI-2 were induced in response to a single intratracheal administration of a fibrosing dose of crystalline silica in mice (5 mg x animal(-1)). The time course of changes in PAI activity and PAI-1 protein were characterized in bronchoalveolar lavage fluid (BALF) and changes in PAI-1 and PAI-2 messenger ribonucleic acid (mRNAs) were monitored by reverse transcriptase polymerase chain reaction (RT-PCR) in BALF cells and lung tissue up to the fibrotic stage of the disease. Substantial levels of PAI activity were found in BALF of control animals, whereas no PAI-1 protein was detected. In response to silica treatment, we observed an acute increase of PAI activity and PAI-1 protein levels in BALF (day 1), associated with an induction of PAI-1 and PAI-2 mRNA levels in lung tissue. In alveolar macrophages, silica treatment induced a persistent upregulation of PAI-2 mRNA only. One month after silica treatment, PAI activity was undetectable in BALF while substantial PAI activity was still present in controls. At the same time point, sustained upregulation of PAI-1 and PAI- 2 mRNAs was, however, noted in lung tissue of animals treated with silica. These findings support the possible implication of PAIs in the remodelling process induced by silica in the lung.

Animals↗
PubMed1998

The Xenopus protein kinase pEg2 associates with the centrosome in a cell cycle-dependent manner, binds to the spindle microtubules and is involved in bipolar mitotic spindle assembly.

By differential screening of a Xenopus laevis egg cDNA library, we have isolated a 2,111 bp cDNA which corresponds to a maternal mRNA specifically deadenylated after fertilisation. This cDNA, called Eg2, encodes a 407 amino acid protein kinase. The pEg2 sequence shows significant identity with members of a new protein kinase sub-family which includes Aurora from Drosophila and Ipl1 (increase in ploidy-1) from budding yeast, enzymes involved in centrosome migration and chromosome segregation, respectively. A single 46 kDa polypeptide, which corresponds to the deduced molecular mass of pEg2, is immunodetected in Xenopus oocyte and egg extracts, as well as in lysates of Xenopus XL2 cultured cells. In XL2 cells, pEg2 is immunodetected only in S, G2 and M phases of the cell cycle, where it always localises to the centrosomal region of the cell. In addition, pEg2 'invades' the microtubules at the poles of the mitotic spindle in metaphase and anaphase. Immunoelectron microscopy experiments show that pEg2 is located precisely around the pericentriolar material in prophase and on the spindle microtubules in anaphase. We also demonstrate that pEg2 binds directly to taxol stabilised microtubules in vitro. In addition, we show that the presence of microtubules during mitosis is not necessary for an association between pEg2 and the centrosome. Finally we show that a catalytically inactive pEg2 kinase stops the assembly of bipolar mitotic spindles in Xenopus egg extracts.

Amino Acid Sequence↗
PubMed1998

Expression of prostate-specific antigen and prostate-specific membrane antigen transcripts in blood cells: implications for the detection of hematogenous prostate cells and standardization.

Circulating prostate cells can be detected in cancer patients by using reverse transcriptase-PCR (RT-PCR) assay for prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSM) mRNA. A quality-control study involving a conventional RT-PCR assay was performed and, surprisingly, detected both transcripts in many negative control cell lines and in normal blood samples. The existence of an illegitimate transcription of the PSA and PSM genes was evidenced by sequence analysis of several PSM and PSA-PCR products. Sequencing indeed demonstrated the presence of a PSA or PSM polymorphism in some but not all the cell lines and patient samples, as well as a heterozygous mutation (G to A; Asp to Asn) in the Jurkat cell line. Moreover, the amount of PSA transcript in MCF-7, a PSA-negative breast line, increased after incubation with cycloheximide. Interestingly, the frequency of positivity was as high as 12% in male samples if only tested once, but dropped to 3% upon multiple testing of the same cDNA. This highlights the stochastic effects in RT-PCR results at high sensitivity, hence the importance of repetitive testing in clinical samples. Decreasing the number of cycles avoided the amplification of illegitimate transcripts but also affected the limit of detection, as evidenced with PSA and PSM cDNA containing plasmids, mixing of LNCap with normal blood samples, and the PSA-PSM-negative K562 cell line. The current data raise the need for a multicentric standardization of the RT-PCR methodology used to amplify PSA and PSM transcripts.

Adult↗