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M Philippe

Publications and source records attributed to M Philippe.

At least 73 records · Page 4Linked to original sources
PubMed1995

The concentrations of AcSDKP, a physiological inhibitor of cell proliferation, vary during oogenesis and early development in Xenopus laevis.

The endogenous tetrapeptide AcSer-Asp-Lys-Pro (AcSDKP) is present in the circulation and cells of mammals. Extracellular AcSDKP is a negative regulator of cell proliferation inhibiting the S phase entry of numerous cell types such as hematopoietic progenitors, hepatocytes, lymphocytes and several continuous cell lines. In contrast, the biological role of cellular AcSDKP remains unknown. We have recently reported a decrease of cellular AcSDKP concentrations by 50% accompanying the initiation of DNA synthesis in mitogen-induced proliferation of human lymphocytes. In the present study we measured the variations of cellular AcSDKP concentrations during oogenesis and early development of Xenopus. Cellular AcSDKP concentrations peaked at stage IV of oogenesis and then continuously decreased during the final oocyte maturation and during the initial 15-h period of exponential cell division of the embryo. Altogether, our present and previously published data suggest that cellular AcSDKP may be involved in the regulation of cell proliferation in eukaryotic species.

Amino Acid Sequence↗
PubMed1995

Overview of the regulation of S-phase in Xenopus egg extracts.

Recently, several components required for nuclear DNA replication and involved in the regulation of S-phase within the cell cycle have been identified. Though detailed models of these events are not available, we focus on recent developments arisen from analysis in Xenopus egg extracts.

Animals↗
PubMed1995

Early events in DNA replication require cyclin E and are blocked by p21CIP1.

Using immunodepletion of cyclin E and the inhibitor protein p21WAF/CIP1, we demonstrate that the cyclin E protein, in association with Cdk2, is required for the elongation phase of replication on single-stranded substrates. Although cyclin E/Cdk2 is likely to be the major target by which p21 inhibits the initiation of sperm DNA replication, p21 can inhibit single-stranded replication through a mechanism dependent on PCNA. While the cyclin E/Cdk2 complex appears to have a role in the initiation of DNA replication, another Cdk kinase, possibly cyclin A/Cdk, may be involved in a later step controlling the switch from initiation to elongation. The provision of a large maternal pool of cyclin E protein shows that regulators of replication are constitutively present, which explains the lack of a protein synthesis requirement for replication in the early embryonic cell cycle.

Amino Acid Sequence↗
PubMed1995

Left ventricular fibrosis in renovascular hypertensive rats. Effect of losartan and spironolactone.

Myocardial fibrosis resulting from arterial hypertension alters myocardial structure and function. Myocardial fibrosis is characterized by a pathological accumulation of types I and III collagens. We used an aldosterone antagonist (spironolactone) and an angiotensin II antagonist (losartan) to elucidate the respective role of these hormones and hypertension in the development of myocardial fibrosis in the Goldblatt model of two-kidney, one clip hypertension in the rat. Fibrosis was assessed by computer-assisted morphometry in the interstitial space, around coronary arteries, in microscar areas, and on left ventricular sections stained with Sirius red and by biochemical techniques. Morphometry was performed with both standard light and polarization microscopy; this latter method was used to quantify yellow-red and green collagen fibers. Concurrently, type I and type III collagen mRNAs were evaluated by a semiquantitative polymerase chain reaction method. The collagen content of the untreated two-kidney, one clip hypertensive rats increased mainly around the coronary arteries; the number and surface area of microscars also increased in chronic hypertension. Losartan treatment decreased systolic pressure and yellow-red collagen fiber content in all areas, whereas spironolactone treatment decreased green collagen fiber content without decreasing systolic pressure. mRNA levels for types I and III collagens showed profiles similar to those of yellow-red and green collagen fiber contents, respectively, suggesting that yellow-red collagen fibers are mainly type I collagen fibers and green collagen fibers are mainly type III collagen fibers. These results suggest that angiotensin II, possibly together with hypertension, and aldosterone, independently of hypertension, have a major influence on myocardial fibrosis, inducing type I and type III collagen deposits, respectively, mainly around coronary arteries.

Angiotensin II↗
PubMed1995

Both cdc2 and cdk2 promote S phase initiation in Xenopus egg extracts.

Xenopus egg extracts induce S phase DNA replication in added sperm pronuclei in a highly regulated manner, similar to events in vivo. Removal of cyclin-dependant kinases (cdks) or cdk2 from these extracts using affinity matrices severely inhibits initiation of S phase. We have used p13suc1 beads to remove both cdk2 and cdc2 proteins from egg extracts and developed a method to replace either protein alone to assess their capacity to initiate DNA replication. Re-addition of either cdk2 or cdc2 proteins to depleted extracts, through translation of their respective mRNAs, restimulated replication, judged by both total synthesis and labelling index. An ATP-binding-site mutant cdk2 mRNA (cdk2.R33) failed to stimulate replication and inhibited S phase initiation in mock-depleted extracts. Both human and Xenopus cdc2 mRNAs rescued replication in this system. Human mutant mRNAs have been used to show that the stimulation induced requires cdc2 catalytic activity, though not its mitotically active form. Rescue of replication by p34cdc2 is also observed in extracts depleted of cdks with a cdk2 antibody, which still retain much of their endogenous cdc2 protein. We conclude that newly synthesised p34cdc2, but not the inherited 'old' form, can induce S phase and in this form may overlap in function with p33cdk2.

Animals↗
PubMed1995

Cardiac angiotensin converting enzyme overproduction indicates interstitial activation in renovascular hypertension.

OBJECTIVES: Angiotensin converting enzyme (ACE) activity in the plasma does not change significantly with hypertension in two-kidney, one-clip hypertensive (2K-1C) rats. However, heart ACE activity and mRNA increase with hypertension. We measured the ACE activity and mRNA in hypertrophied hearts at different times after clipping, and determined the cellular distribution of its increase in the left ventricle of 2K-1C hypertensive rats. METHODS: Cardiac ACE activity was quantified in left and right ventricles using a radiolabeled synthetic ACE substrate, and ACE mRNA steady-state level was quantified by ribonuclease protection assay. Tissue localization of ACE in normal and hypertrophied hearts was determined by measuring ACE activity in isolated ventricular cells. In situ hybridization with a rat ACE cDNA and immunohistochemistry with a monoclonal anti-ACE antibody were used to identify tissue compartments producing ACE mRNA and protein. RESULTS: The left ventricle was hypertrophied 2 weeks after clipping and remained hypertrophied at 12 weeks. Left ventricular ACE activity was significantly increased 2 and 4 weeks (3.2 +/- 0.3 in 2K-1C vs. 1.7 +/- 0.1 pmol/mg prot/min in sham-operated rat) after renal artery clipping, but not at 12 weeks. The right ventricle was slightly hypertrophied 4 weeks after clipping and remained hypertrophied at 12 weeks. Right ventricular ACE activity was significantly increased at 4 (6.7 +/- 0.6 in 2K-1C vs. 3.1 +/- 0.3 pmol/mg prot/min in sham-operated rat) and 12 weeks. ACE activity was not detectable in cardiomyocytes isolated by Percoll gradient. Neither was ACE mRNA detected in isolated cardiomyocytes, even after ACE mRNA amplification by RT-PCR. In contrast, ACE activity and mRNA were detected in pooled non-cardiomyocytic cells. Thus the increase in cardiac ACE activity associated with hypertension must be due to an increase in ACE expression by non-cardiomyocytic cells. In situ hybridization showed an autoradiographic signal for ACE mRNA over the endothelial cells of coronary arteries and over the interstitial spaces including pericoronary and fibrosis areas. Immunohistochemistry confirmed these data, showing ACE on endothelial cells and in pericoronary spaces with an increased signal in pericoronary and fibrosed areas in hypertensive hypertrophied left ventricle. CONCLUSION: Besides its usual endothelial expression, ACE is absent from cardiomyocytes and present in interstitial tissue, in the pericoronary spaces in normal tissue and more markedly in hypertrophied ventricles.

Angiotensin II↗
PubMed1995

[Demonstration of molecular forms of Rap 1 in cells or tissues deriving from normal or pathological human colonic epithelium].

Mutations of the cK-ras gene which confer oncogenic properties to the corresponding encoded small G protein, occur in 30 to 50% of the human colonic adenocarcinomas. Overexpression of Rap 1A, a member of the Ras family, in K-ras transformed fibroblasts, reverts the transforming properties of the oncogene. This indicates that Rap 1A may exert antagonistic properties towards the K-Ras protein. In this respect, we have been interested in comparing Rap 1 expression in the human adenocarcinoma cell line HT29 and in safe or pathological tissues deriving from the human colonic epithelium. In the human adenocarcinoma cells HT29, several immunoreactive forms of Rap 1 of 71 kDa, 47 kDa, 40 kDa and 24 kDa are detected. Extraction in Triton X-114 allows separation of HT29 cell proteins on the basis of their differential hydrophobic properties. Parallelly, proteins were separated in crude cytosolic or membrane fractions. Most of the immunoreactive material corresponding to the 24 kDa band may contain hydrophobic and membrane associated components. The molecular nature of the higher size components is also discussed here. In the tissues, the 47 kDa form which is common in all of the safe and pathological samples considered, appears to be specifically expressed in the colon. Besides, the 71, 68 and 24 kDa were found in pathological tissues. High expression of Rap 1 was demonstrated to be correlated with cell differentiation in the safe colonic epithelium and in the adenomas. Cloning of the Rap 1A cDNA is now in progress in the laboratory, using PCR detection in an HT29 expression library.

Adenocarcinoma↗
PubMed1995

HLA class II genotyping: two assay systems compared.

In the last few years, a variety of DNA-based human leukocyte antigen (HLA) typing methods have emerged, revealing the extreme polymorphism of HLA genes. This polymorphism makes it difficult for a clinical laboratory to establish the best HLA typing strategy. In this study we have compared two techniques for performing HLA-DRB typing: a commercial rapid assay based on the polymerase chain reaction (PCR) followed by reverse dot-blot hybridization of the PCR products (the Inno-LiPA assay), and a method based on PCR followed by restriction fragment length polymorphism analysis. We found that both methods provide reliable results with a high rate of concordance (97%) and that Inno-LiPA is convenient for large-scale routine typing. However, if a high-resolution allelic typing is required, each method lacks accuracy but using them in association improves the accuracy of the results.

Alleles↗
PubMed1994

Cloning of the Xenopus laevis cdk2 promoter and functional analysis in oocytes and during early development.

CDK2 (cyclin-dependent kinase 2) is a serine/threonine kinase which is involved in regulating S-phase entry in higher eukaryotes. To investigate the transcriptional control of this gene, a 13-kb Xenopus laevis genomic clone containing the 5' flanking sequences was isolated. A 2.7-kb fragment containing the promoter region was sequenced and the transcription start point (tsp) was determined by primer extension. Several putative regulatory elements, such as the E2F-binding site, Y box and octamer-binding site, were localized in this region, but no TATA box was found. When fused to cat, a reporter gene encoding chloramphenicol acetyltransferase, the 5' flanking sequences were shown to function in oocytes and an enhancer activity was found in this region. During early embryogenesis, cdk2 promoter activity was tested and de novo transcription was detected at the mid-blastula transition.

Animals↗
PubMed1994

A highly conserved eukaryotic protein family possessing properties of polypeptide chain release factor.

The termination of protein synthesis in ribosomes is governed by termination (stop) codons in messenger RNAs and by polypeptide chain release factors (RFs). Although the primary structure of prokaryotic RFs and yeast mitochrondrial RF is established, that of the only known eukaryotic RF (eRF) remains obscure. Here we report the assignment of a family of tightly related proteins (designated eRF1) from lower and higher eukaryotes which are structurally and functionally similar to rabbit eRF. Two of these proteins, one from human and the other from Xenopus laevis, have been expressed in yeast and Escherichia coli, respectively, purified and shown to be active in the in vitro RF assay. The other protein of this family, sup45 (sup1) of Saccharomyces cerevisiae, is involved in omnipotent suppression during translation. The amino-acid sequence of the eRF1 family is highly conserved. We conclude that the eRF1 proteins are directly implicated in the termination of translation in eukaryotes.

Amino Acid Sequence↗
PubMed1994

Promoter function of the human glucose-6-phosphate dehydrogenase gene depends on two GC boxes that are cell specifically controlled.

Human glucose-6-phosphate dehydrogenase is expressed in all cells by a housekeeping gene whose regulatory 5'-flanking sequence includes at least nine GC boxes. By transient transfection of HeLa and HepG2 cells with constructs containing glucose-6-phosphate dehydrogenase gene regions linked to a reporter gene, we have now delineated the core promoter and have located upstream stimulatory and inhibitory sequences. By mutational analysis, we demonstrate that the activity of the core promoter requires two out of seven GC boxes. We show that stimulatory protein 1 (Sp1)-related factors and activator protein 2 (AP-2)-related proteins bind to these two boxes in band-shift experiments. One point mutation that affects the binding of only the Sp1-related factors to one or both boxes causes a marked decrease of promoter activity in HepG2 cells but not in HeLa cells. We conclude that (a) two out of many seemingly redundant GC boxes are necessary to drive a G+C-rich housekeeping promoter; (b) factors that bind to GC boxes may exert cell-type-specific regulation of housekeeping gene promoter activity; (c) point mutations in the promoter of the glucose-6-phosphate dehydrogenase gene can inhibit its transcription.

Base Sequence↗
PubMed1994

Stage and tissue-specific expression of the alcohol dehydrogenase 1 (Adh-1) gene during mouse development.

The Adh-1 gene product, ADH-A2, the only known murine class I alcohol dehydrogenase, is able to oxidize retinol (vitamin A) into retinaldehyde, the first enzymatic step in the conversion of retinol into its biologically active metabolite retinoic acid. We have investigated the developmental expression pattern of Adh-1 transcripts by in situ hybridization. Transcripts were first detected by embryonic day 10.5 in the mesonephros mesenchyme. During the following gestational days, Adh-1 transcripts were detected in several mesenchymal areas, such as nasal, laterocervical, and genital regions. Adh-1 transcripts were also detected in a small ectodermal domain at the anterior margins of both forelimbs and hindlimbs. During late fetal development. Adh-1 transcripts were found essentially in the epidermis and in a number of tissues which continue to express the gene after birth, such as liver, kidney, gut epithelium, adrenal cortex, testis interstitium, and ovarian stroma. In contrast, a strong expression of Adh-1 was found in the mesenchyme of developing lungs, but not in the adult organ. This highly regulated expression of Adh-1 is discussed with respect to the local synthesis of retinoic acid during development. Although the promoter of the human counterpart of Adh-1 contains a retinoic acid response element (Duester et al. [1991] Mol. Cell. Biol. 11:1638-1646), we report that this element is not conserved in the murine gene. Consistently, Adh-1 promoter-containing reporter constructs were not retinoic acid-inducible in cotransfections assays with RARs and/or RXRs, suggesting that retinoic acid regulation of Adh-1 differs from that of the human gene.

Adrenal Cortex↗
PubMed1994

The kinesin-related protein Eg5 associates with both interphase and spindle microtubules during Xenopus early development.

We have examined the changing abundance and distribution of the kinesin-related protein Eg5 during oogenesis and early development in Xenopus laevis. Antibodies raised against proteins synthesized from parts of a novel Eg5 gene expressed in eggs were used for Western blotting and immunofluorescence. Eg5 protein was highly enriched in oocytes and eggs compared with other adult tissues. It accumulated during the latter stages of oogenesis and increased a further threefold during oocyte maturation. Its level then gradually declined during early development. In oocytes, eggs, and early embryos, Eg5 protein could be detected throughout the cytoplasm and in subcortical aggregates. Eg5 staining was found concentrated in meiotic and mitotic spindles, mainly toward the poles. Some Eg5 staining colocalized with microtubules in interphase cells, including the aligned subcortical microtubules in fertilized eggs implicated in the cortical rotation that specifies the dorsoventral axis. Interphase association of Eg5 with microtubules during early development was confirmed by copelleting the protein with microtubules from egg homogenates. In tadpoles and tissue culture cells, Eg5 colocalized with spindle microtubules throughout mitosis but not with interphase microtubules. These results suggest that the Eg5 microtubule motor may function in meiosis, mitosis, and interphase during early development.

Amino Acid Sequence↗
PubMed1994

High expression of bcl-2 is the rule in acute lymphoblastic leukemia, except in Burkitt subtype at presentation, and is not correlated with the prognosis.

Bcl-2 expression and its prognostic value were evaluated using the APAAP technique in 40 ALL patients. Because of a possible synergy between c-myc and bcl-2 proteins in cell lines, we compared 40 ALL of either T or B lineage with seven Burkitt's ALL or sporadic Burkitt's lymphomas (BL). We found the same high expression of bcl-2 in adult (Ad) and childhood (Ch) malignancies examined at presentation, regardless of the T or B phenotype. No bcl-2 expression was detectable in BL cases, except for two investigated at relapse. Bcl-2 staining intensity was also not related to the stage of blast differentiation, except in Ad T-ALL, or to the relapse rate. Ki-67, a marker of proliferation, was used to investigate the possible correlation between bcl-2 expression and the proliferative activity. An inverse correlation was found only in BL at presentation. We confirm that bcl-2 expression is the rule in ALL, regardless of the immunophenotypic characteristics at presentation, and that high expression is not correlated with a bad prognosis. Sporadic BL may represent a particular type of tumor, with bcl-2 expression in relapse, but not at presentation.

Adolescent↗
PubMed1994

Effect of chronic ANG I-converting enzyme inhibition on aging processes. II. Large arteries.

The consequences of hypertension and aging on cardiovascular structure and function are reputed to be similar, suggesting that blood pressure plays a role in the aging process. However, the exact relationship between aging, blood pressure, and the arterial structure-function relationship has not been demonstrated. To test the effects of aging, renin-angiotensin system, and pressure on the arterial wall, 20 normotensive male WAG/Rij rats were killed at 6, 12, 24, and 30 mo of age and compared with similar groups treated with an angiotensin (ANG)-converting enzyme inhibitor (ACEI), perindopril. Arterial function was determined by a systemic hemodynamic study and by in situ measurement of carotid compliance. Arterial wall structure was determined by histomorphometric and biochemical methods. Aging did not significantly modify blood pressure, but ACE inhibition decreased blood pressure significantly from 6 to 30 mo. Plasma renin activity decreased with age and increased with ACEI. Plasma atrial natriuretic factor increased with age and was significantly decreased with ACEI. Absolute and relative left ventricular weight increased with age, and ACEI delayed these increases. Arterial wall stiffness increased with age, as shown by a significant decrease in systemic and local arterial compliance and by an increase in aortic characteristic impedance. The increase in carotid wall compliance after poisoning of smooth muscle contractile function (KCN) was greater in young (6- and 12-mo old) than in old (24- and 30-mo old) rats. Chronic ACEI treatment increased basal carotid compliance values slightly and did not change KCN carotid compliance. The aortic and carotid luminal size increased regularly with age. Aging was associated without any change in absolute elastin content. In contrast, collagen content increased with aging. Aging was also associated with an increase in medial thickness. Medial thickening was mainly due to smooth muscle hypertrophy. Aging was associated with intimal proliferation, which became progressively thicker and collagen rich. ACEI treatment did not prevent aortic lumen enlargement but significantly postponed the increase in medial and intimal thickening. Biochemical determinations of the aortic wall components confirmed the morphometric data. In conclusion, the age-dependent large artery enlargement and stiffening were observed both in normotensive rats and in those rats whose blood pressure was lowered by ACEI. This suggests that aging and blood pressure affect arterial wall structure and function by different mechanisms.

Aging↗
PubMed1993

Long-term follow-up of residual disease in acute lymphoblastic leukemia patients in complete remission using clonogeneic IgH probes and the polymerase chain reaction.

We sequentially studied bone marrow (BM) samples of 25 patients in complete remission of an acute lymphoblastic leukemia (ALL) using a simplified polymerase chain reaction (PCR) strategy (direct use of the PCR product as a clonogenic probe recognizing rearranged Ig heavy chain sequences) as a first approach. BM aspirates were serially investigated after obtention of a complete response. When sensitivity was less than 1:10(4), the PCR fragment was sequenced and a specific oligonucleotide was synthetized and used as a probe (five cases). Cases in which minimal residual disease (MRD) became undetectable were cross-controlled using either TCR delta rearrangement or a specific translocation to circumvent the problem of false-negative results arising from clonal evolution. The median follow-up was 30 months (3 to 51 months). Within the first 3 months of complete remission, MRD was detectable in 22 of 23 investigated patients and remained so in 19 of 21 patients examined at 6 months, regardless of the long-term clinical outcome. In patients remaining in complete remission at 30 months or more, two patterns of MRD emerged during the follow-up. Either it continuously decreased to ultimately become undetectable (five patients) or remained detectable (five patients) with an increase after discontinuation of treatment in two. In the eight patients who relapsed, MRD persisted throughout the clinical course, and eventually increased 3 to 12 months before relapse was clinically detectable. In one case, clonal evolution of the VDJ heavy chain region was observed and recurrence of MRD shown by the use of TCR delta rearrangement as a control. We conclude that the use of this simplified methodology is a valuable tool for the follow-up of MRD in a majority of ALL patients, though in a few cases, sequencing needs to be performed to achieve a relevant sensitivity. The possibility of clonal evolution requires a cross-control of any sample becoming negative whatever the initial rearrangement used to generate a probe. In patients on therapy, sequential search for MRD seems to be a good tool for predicting the long-term outcome. In addition, patients remaining positive at the time treatment is discontinued or with a high tumor burden after a few months therapy may be at a higher risk of subsequent relapse, although a longer follow-up is needed to answer this question.

Adolescent↗
PubMed1993

Retrovirally transduced antisense sequences stably suppress P210BCR-ABL expression and inhibit the proliferation of BCR/ABL-containing cell lines.

There is now strong evidence that the BCR-ABL gene product of the Philadelphia chromosome (P210) plays a crucial role in the pathogenesis of chronic myeloid leukemia (CML). We have previously shown that introduction of antisense oligonucleotides into K562 cells could transiently block the expression of P210 and specifically inhibit cellular growth in culture. In this report, we describe the use of a retroviral vector to introduce selected antisense and sense sequences, first into murine B10 cells, previously rendered interleukin-3 (IL-3) independent by transfection of BCR-ABL sequences, and second into K562 cells. The antisense transcripts generated under the control of MoMLV promoter specifically killed B10 cells in the absence of IL-3 and inhibited P210 expression almost completely. In K562 cells, the antisense sequences led to a dramatic reduction of P210 expression and increased their doubling time by more than twofold. This effect was not reversed by the addition of exogenous IL-3 to the culture medium. Control HeLa or HL60 cells infected with the same constructs did not show any change in proliferation rate, despite abrogation of the normal BCR gene products. Rather unexpectedly, P210 suppression was not lethal in K562 cells, showing that such a cell line does not rely entirely on the expression of P210 for surviving, but depends on it as far as growth properties are concerned. We conclude that this approach can successfully achieve stable suppression of the oncogenic protein P210 and may be used to study further the mechanisms by which P210 is transforming cells. The effect on fresh CML cells in bone marrow cultures remains to be assessed before we can tell whether this technique may be used for selective suppression of leukemic hematopoiesis in vitro.

3T3 Cells↗