Molecular detection of circulating prostate cells in cancer II: Comparison of prostate epithelial cells isolation procedures.
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Cell Separation↗
Biomedical subjects
M Philippe
Publications and source records attributed to M Philippe.
Detection of intracellular antigens by flow cytometry: comparison of two chemical methods and microwave heating.
Detection of intracellular antigens by flow cytometry requires effective fixation and permeabilization of the cell membrane. This study compares three fixation/permeabilization techniques: two commercial chemical reagents, the ORTHOPermeaFix (OPF) and the FIX&PERM Cell Permeabilization Kit (F&P), and a novel method based on microwave heating (MWH). They have been applied to the detection of two nuclear (p53 and rb/p105) and two cytoplasmic (bcl-2 and mdr-1/gp-170) antigens, using positive- and negative-control cell lines and peripheral blood mononuclear cells. Western blotting was performed as a control of protein expression. For the four antigens assessed, cellular morphology, discrimination between intact cells and debris, percentage of positive cells, and mean fluorescence intensity were examined. For this last parameter, the assessment of the MWH technique was performed using SD and a graphical approach inspired by the concepts described by Bland and Altman (Lancet 1986;346: 1085-7) as well as Petersen et al. (Clin Chem 1997;43: 2039-46). The statistical analysis shows that MWH is comparable to the commercial methods and that its reproducibility is also equivalent to OPF and F&P. As assessed for some of the most clinically relevant intracytoplasmic and intranuclear antigens, the MWH method appears to be a valuable and inexpensive alternative. It is worth noting that, unlike commercial reagents, MWH altered surface antigens. Interestingly, this feature, which would prevent cell selection on the basis of combined membrane and intracellular epitopes, is associated with a decrease of nonspecific background fluorescence.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Eukaryotic release factor 1 (eRF1) abolishes readthrough and competes with suppressor tRNAs at all three termination codons in messenger RNA.
It is known from experiments with bacteria and eukaryotic viruses that readthrough of termination codons located within the open reading frame (ORF) of mRNAs depends on the availability of suppressor tRNA(s) and the efficiency of termination in cells. Consequently, the yield of readthrough products can be used as a measure of the activity of polypeptide chain release factor(s) (RF), key components of the translation termination machinery. Readthrough of the UAG codon located at the end of the ORF encoding the coat protein of beet necrotic yellow vein furovirus is required for virus replication. Constructs harbouring this suppressible UAG codon and derivatives containing a UGA or UAA codon in place of the UAG codon have been used in translation experiments in vitro in the absence or presence of human suppressor tRNAs. Readthrough can be virtually abolished by addition of bacterially-expressed eukaryotic RF1 (eRF1). Thus, eRF1 is functional towards all three termination codons located in a natural mRNA and efficiently competes in vitro with endogenous and exogenous suppressor tRNA(s) at the ribosomal A site. These results are consistent with a crucial role of eRF1 in translation termination and forms the essence of an in vitro assay for RF activity based on the abolishment of readthrough by eRF1.
Animals↗
Prevalence of activated protein C resistance and analysis of clinical profile in thromboembolic patients. A Belgian prospective study.
OBJECTIVES: To assess the prevalence of activated protein C resistance (APC-R) among healthy subjects and thromboembolic patients and to determine the clinical characteristics associated with APC-R. DESIGN: A prospective study. SETTING: One academic medical centre. SUBJECTS: 91 health controls and 126 thromboembolic patients. MEASUREMENTS: Patients and control were genotyped for the factor V Leiden (VaQ506) mutation. The anticoagulant response of the patient's plasma to activated protein C was also determined. RESULTS: The frequency of APC-R was 3.3% among healthy control subjects and 22% among thrombotic patients of whom 18% were heterozygous and 4% were homozygous. The mean age at the first thrombotic event and the severity of thrombotic disease including the proportion of proximal deep vein thrombosis and the frequency of lung embolism were identical among APC-R positive and negative patients. A family history of thromboembolic disease was elicited more frequently in APC-R positive than in APC-R negative patients (57% vs. 22%, P < 0.001). The recurrence rate was higher for APCR-R positive patients (57% vs. 34%, P < 0.05). The percentage of cases with a factor predisposing to thrombosis was very similar in APC-R positive (57%) and negative (68%) patients. CONCLUSIONS: A familial history of thromboembolic disease and recurrences are significantly more frequent among APC-R positive than APC-R negative patients.
Adolescent↗
Beta-thalassaemia in indigenous Belgians: an update.
Beta-thalassaemia, a widespread autosomal recessive disorder, occurs sporadically in Northern and Western European countries. Molecular analysis of the beta-globin gene has been carried out in 30 members of 15 unrelated indigenous Belgian families which presented with non sideropenic hypochromic and microcytic anaemia. For all of them, extensive search failed to find an ancestor at risk for the disease. The beta-globin genes were first screened for frequent beta-thalassemic mutations by dot-blot hybridization with specific radiolabeled oligonucleotide probes. Direct automated fluorescence-based DNA sequencing and, in one case, Southern blotting were also used. All the 30 patients were found to be heterozygous for a beta-thalassemic mutation. Eight different mutations were identified. Among these, four are commonly found in the Mediterraneans: codon 8 (-AA), IVS-I-1 (G-->A), IVS-1-6 (T-->C) and codon 39 (C-->T); three have occasionally been reported: initiation codon (T-->C) and codon 35 (C-->A) and a rare deletion of 12.6 kb which removes all the beta-globin gene and its flanking regions. A new mutation, a -CC deletion at codon 38/39 was found in one family. These results both at the biological and molecular level show that beta-thalassaemia exist in indigenous Belgian families with no known ancestor a risk for the disease. They also show that clinicians and biologists should keep in mind the existence of beta-thalassaemia in indigenous Belgian families when investigating hypochromic and microcytic anaemia in patients whom the past familial history does not evocate a risk for the disease.
Adenine↗
Overexpression of human release factor 1 alone has an antisuppressor effect in human cells.
Two eukaryotic proteins involved in translation termination have recently been characterized in in vitro experiments. Eukaryotic release factor 1 (eRF1) catalyzes the release of the polypeptide chain without any stop codon specificity. The GTP-binding protein eRF3 confers GTP dependence to the termination process and stimulates eRF1 activity. We used tRNA-mediated nonsense suppression at different stop codons in a cat reporter gene to analyze the polypeptide chain release factor activities of the human eRF1 and eRF3 proteins overexpressed in human cells. In a chloramphenicol acetyltransferase assay, we measured the competition between the suppressor tRNA and the human release factors when a stop codon was present in the ribosomal A site. Whatever the stop codon (UAA, UAG, or UGA) present in the cat open reading frame, the overexpression of human eRF1 alone markedly decreased translational readthrough by suppressor tRNA. Thus, like the procaryotic release factors RF1 and RF2 in Escherichia coli, eRF1 seems to have an intrinsic antisuppressor activity in human cells. Levels of antisuppression of overexpression of both eRF3 and eRF1 were almost the same as those of overexpression of eRF1 alone, suggesting that eRF1-eRF3 complex-mediated termination may be controlled by the expression level of eRF1. Surprisingly, when overexpressed alone, eRF3 had an inhibitory effect on cat gene expression. The results of cat mRNA stability studies suggest that eRF3 inhibits gene expression at the transcriptional level. This indicates that in vivo, eRF3 may perform other functions, including the stimulation of eRF1 activity.
Blotting, Northern↗
A panel of antibodies for the immunostaining of Bouin's fixed bone marrow trephine biopsies.
AIMS: To assess a panel of antibodies on Bouin's fixed bone marrow trephine (BMT) biopsies. These biopsies are widely used in routine diagnosis of various haematological malignancies and may be the sole material available in many centres; however, information regarding the immunostaining of this material is lacking. METHODS: Biopsies were taken from 72 patients presenting with various haematological malignancies (leukaemia, 38; lymphoma, 14; multiple myeloma, 20). A panel of antibodies was assessed on Bouin's fixed BMT biopsies by the alkaline phosphatase-antialkaline phosphatase method. RESULTS: Three B (MB2, LN-2, Ki-B5) and two T cell lineage antibodies (UCHL-1, CD3-r) reliably identified lymphoid cells, while MPO-r, Leu-M1/CD15, and KP-1/CD68 recognised cells from the myeloid or histiocytic/macrophage series. Reed-Sternberg cells were stained by LN-2, Leu-M1, and CD30. Antibodies specific for plasma cells (VS38) and hairy cells (DBA.44) gave a variable pattern of staining. Among the proliferation markers, proliferative cell nuclear antigen but not Ki-67 related antibodies were effective. CONCLUSION: This study presents a panel of antibodies with reactivity not restricted to common fixatives that are also suitable for Bouin's fixed BMT biopsies.
Antibodies↗
Circulating and cellular markers of endothelial dysfunction with aging in rats.
The influence of age on endothelial functional markers was investigated in rats. Angiotensin I converting enzyme (ACE) activity and nitric oxide synthase (NOS) mRNA expressions were examined in the lung and aorta of 10-, 20-, and 30-mo-old normotensive rats. These data were extended by the measurement of circulating endothelial cells. ACE activity was significantly decreased in plasma (P < 0.01) and lungs (P < 0.01) at 30 mo, whereas it was significantly increased in the aorta (P < 0.001) at this age. Conversely, ACE mRNA levels decreased with age in the lung (P < 0.05). The level of constitutive endothelial NOS (eNOS) mRNA was significantly reduced in the aorta of 30-mo-old rats (P < 0.05), but no changes were observed in the lungs. The level of inducible NOS (iNOS) mRNA in the aorta was significantly decreased in 20- and 30-mo-old rats (P < 0.01), whereas it was significantly increased in the lung at 30 mo (P < 0.01). Interestingly, eNOS was expressed approximately 30 times more (P < 0.001) in the aorta than iNOS, whereas in the lung it was only slightly higher than iNOS (35%; P < 0.001). Neuronal NOS mRNA expression was not modified with aging. In the aorta, guanosine 3',5'-cyclic monophosphate concentration followed NOS expressions and showed a significant decrease at 30 mo (P < 0.001). An increase in the number of circulating endothelial cells was observed in the oldest rats, possibly reflecting an increase in endothelial cell turnover with aging. The present results demonstrate that aging modifies the expression of endothelial markers implicated in the regulation of vasomotor tone. This age-dependent impairment of endothelial functions could contribute to the increased risk of pathological processes within the arterial wall associated with aging.
Aging↗
Identification of a new HLA-DPB1*6501 allele in a Caucasian individual.
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Alleles↗
Beta-thalassaemia in indigenous Belgian families: identification of a novel mutation.
The molecular basis of beta-thalassemia was investigated at the DNA level in 28 Belgians from 14 unrelated families. All the patients were heterozygous for beta-thalassaemia. Seven different mutations were identified using a combination of dot-blot hybridization with allele-specific oligonucleotide probes and direct automated fluorescence-based DNA sequencing. Among these mutations, four are commonly found in the Mediterraneans - codon 8 (-AA), IVS-I-1 (G --> A), IVS-I-6 (T --> C) and codon 39 (C --> T)-and two have occasionally been reported-initiation codon (T --> C) and codon 35 (C --> A). The last mutation, a -CC deletion at codons 38/39, appears to be a novel mutation and can routinely be investigated by AvaII restriction on amplified DNA. We report our findings, discuss the diversity of the mutations found in Belgium and show the usefulness of direct DNA sequencing in a population in which the molecular defects of beta-thalassaemia have yet to be characterized and in which screening is hampered by the wide range of potential mutations.
Belgium↗
Improved survival in rats administered NG-nitro L-arginine methyl ester due to converting enzyme inhibition.
Blockade of the renin-angiotensin system (RAS) prevents the increase in blood pressure (BP) induced by chronic administration of NG-nitro L-arginine methyl ester (L-NAME) in rats. In the present study, we showed how a converting enzyme inhibitor can prevent the end-stage tissue damage due to chronic nitric oxide (NO) synthase blockade and thus improve the survival rate. Three experiments were performed. In the first, rats (n = 10) were given L-NAME (50 mg/kg) and 10 other rats were given L-NAME plus quinapril (10 mg/kg) starting 1 month after L-NAME administration. Ten untreated rats were used as controls. Rats were killed after 2 months, and the RAS, renal function, and renal morphology were analyzed. In the second experiment, a similar protocol was used, and function and morphological damage in renal slices and cervical medullary tissue were assessed after 4 months of L-NAME and 3 months of quinapril + L-NAME. In the third experiment, a similar protocol was used, but to establish survival curves, the animals were not killed. L-NAME significantly increased BP without causing any significnat changes in plasma renin activity (PRA) at 2 months. The aortic wall cyclic GMP content was significantly decreased, and the angiotensin-converting enzyme (ACE) activity was increased by L-NAME. Quinapril significantly reversed the high BP induced by L-NAME without changing the decrease in the aortic wall cyclic GMP. Two-month L-NAME treatment decreased renal function and damaged renal tissue. Quinapril prevented both proteinuria and morphological damage. Four-month L-NAME treatment induced renal end-stage damage and infarctions of the cervical medulla. Quinapril prevented this end-stage damage in the kidney and cervical medulla. Quinapril therefore prevented the increased mortality due to L-NAME. Hence, inhibition of ACE, despite its lack of effect on arterial wall cyclic GMP, does reverse the hypertension and prevent end-stage vascular damage induced by chronic L-NAME in target organs.
Angiotensin-Converting Enzyme Inhibitors↗
Sustained increase in aortic endothelial nitric oxide synthase expression in vivo in a model of chronic high blood flow.
Physiological adaptation of normal blood vessels to acute or chronic changes in blood flow is endothelium dependent. In vitro studies have shown that, among other genes, NO synthase (NOS) 3 mRNA and protein expression is enhanced by acute elevation of shear stress in endothelial cells. We have investigated the effect of chronic high blood flow on NOS3 mRNA and protein expression in rat aorta. NOS3 mRNA levels were measured by quantitative polymerase chain reaction (PCR) in the aortas of 12 rats with arteriovenous fistulas and 9 sham-operated control rats. The PCR assay indicated that NOS3 mRNA levels were significantly enhanced (twofold) during high blood flow. Western blots showed that immunoreactive NOS3 levels were also increased to a similar extent. Furthermore, the Ca(2+)-dependent NOS activity, measured by the L-arginine to L-citrulline conversion assay, and the cGMP content were also significantly increased in the proximal aortic wall submitted to the arteriovenous shunt. These results indicate that NOS3 mRNA and protein expression is enhanced in vivo during chronic high blood flow.
Animals↗
HLA class II-associated genetic susceptibility in idiopathic progressive sensorineural hearing loss.
To investigate the association between genes in the major histocompatibility complex and inner ear disease susceptibility at the DNA level, high-resolution genotyping for HLA class II (HLA-DR, -DQ, -DP) was performed by polymerase chain reaction-sequence-specific oligonucleotide reverse dot blot and polymerase chain reaction-restriction fragment length polymorphism analysis in 34 patients with idiopathic progressive sensorineural hearing loss (PSHL) and in 214 controls. The frequencies of DRB1*0301, DRB3*0101, DQB1*0201, and DPB1*0401 were significantly increased in patients with idiopathic PSHL compared with controls. The DQB1*0301 allele was significantly decreased in the patients. A linkage disequilibrium was probably responsible for the concomitant increase of both DRB1*0301 and DRB3*0101 alleles in patients. The increase of DQB1*0201 in patients was associated with the DRB1*0301 allele. In addition, the telomeric DPB1*0401 allele may act as an independent risk factor. The DQB1*0301 allele may have a protective role in the pathogenesis of idiopathic PSHL. These results suggest that the specific HLA class II gene products may confer susceptibility or resistance to idiopathic PSHL.
Adult↗
Xenopus cyclin E, a nuclear phosphoprotein, accumulates when oocytes gain the ability to initiate DNA replication.
The capacity to initiate DNA replication appears during oocyte maturation in Xenopus. Initiation of S phase is driven by several components which include active cyclin/cdk complexes. We have identified three Xenopus cyclin E clones showing 59% amino acid identity with human cyclin E. The recruitment of cyclin E mRNA, like cdk2 mRNA, into the polysomal fraction during oocyte maturation, results in the accumulation of the corresponding proteins in unfertilized eggs. Cyclin E mRNA remains polyadenylated during cleavage and anti-cyclin E antibodies detect Xlcyclin E in embryonic nuclei at this time. Cdk2 protein is necessary for the phosphorylation of radiolabelled cyclin E added to egg extracts. Radiolabelled Xlcyclin E enters interphase nuclei and, though stable through interphase and mitosis, is not associated with condensed mitotic chromatin. In egg extracts, endogenous Xlcyclin E rapidly associates with nuclei before S phase and remains nuclear throughout interphase, becoming nucleoplasmic in G2/prophase. Under conditions where initiation of replication is limiting in extracts, Xlcyclin E associates only with those nuclei that undergo S phase. These features are entirely consistent with the view that Xlcyclin E is required for initiation of S phase.
Amino Acid Sequence↗
Eukaryotic polypeptide chain release factor eRF3 is an eRF1- and ribosome-dependent guanosine triphosphatase.
Termination of translation in eukaryotes is governed by two polypeptide chain release factors, eRF1 and eRF3 on the ribosome. eRF1 promotes stop-codon-dependent hydrolysis of peptidyl-tRNA, and eRF3 interacts with eRF1 and stimulates eRF1 activity in the presence of GTP. Here, we have demonstrated that eRF3 is a GTP-binding protein endowed with a negligible, if any, intrinsic GTPase activity that is profoundly stimulated by the joint action of eRF1 and the ribosome. Separately, neither eRF1 nor the ribosome display this effect. Thus, eRF3 functions as a GTPase in the quaternary complex with ribosome, eRF1, and GTP. From the in vitro uncoupling of the peptidyl-tRNA and GTP hydrolyses achieved in this work, we conclude that in ribosomes both hydrolytic reactions are mediated by the formation of the ternary eRF1-eRF3-GTP complex. eRF1 and the ribosome form a composite GTPase-activating protein (GAP) as described for other G proteins. A dual role for the revealed GTPase complex is proposed: in " GTP state," it controls the positioning of eRF1 toward stop codon and peptidyl-tRNA, whereas in "GDP state," it promotes release of eRFs from the ribosome. The initiation, elongation, and termination steps of protein synthesis seem to be similar with respect to GTPase cycles.
Base Sequence↗
Is there a human [psi]?
The yeast Sup35p protein which is responsible for the [psi] phenotype, is a GTP-binding protein involved in translation termination. It was suggested recently that the [psi] determinant has prion-like properties that were localized in the 114 N-terminal amino acids of the protein. In this study, we show that the 5' end of the human SUP35 gene open reading frame is longer than previously reported by 138 codons. This N-terminal sequence presents similarities with the N-terminus of S. cerevisiae Sup35p protein, involved in [psi] maintenance. By transfection of human cells and Western blotting, we demonstrate that translation is initiated at the first AUG encountered at the 5' end of the human SUP35 gene. The longest form of the protein, which contains the N-terminal extension, is the major form of Sup35p protein in non transfected cells. Moreover, an analog of the long form of Sup35p protein is found in various mouse tissues. We suggest that the protein encoded by SUP35 gene could have, at least in human, the properties described for the yeast [psi] element.
Amino Acid Sequence↗
Termination of translation in eukaryotes is governed by two interacting polypeptide chain release factors, eRF1 and eRF3.
Termination of translation in higher organisms is a GTP-dependent process. However, in the structure of the single polypeptide chain release factor known so far (eRF1) there are no GTP binding motifs. Moreover, in prokaryotes, a GTP binding protein, RF3, stimulates translation termination. From these observations we proposed that a second eRF should exist, conferring GTP dependence for translation termination. Here, we have shown that the newly sequenced GTP binding Sup35-like protein from Xenopus laevis, termed eRF3, exhibits in vitro three important functional properties: (i) although being inactive as an eRF on its own, it greatly stimulates eRF1 activity in the presence of GTP and low concentrations of stop codons, resembling the properties of prokaryotic RF3; (ii) it binds and probably hydrolyses GTP; and (iii) it binds to eRF1. The structure of the C-domain of the X.laevis eRF3 protein is highly conserved with other Sup35-like proteins, as was also shown earlier for the eRF1 protein family. From these and our previous data, we propose that yeast Sup45 and Sup35 proteins belonging to eRF1 and eRF3 protein families respectively are also yeast termination factors. The absence of structural resemblance of eRF1 and eRF3 to prokaryotic RF1/2 and RF3 respectively, may point to the different evolutionary origin of the translation termination machinery in eukaryotes and prokaryotes. It is proposed that a quaternary complex composed of eRF1, eRF3, GTP and a stop codon of the mRNA is involved in termination of polypeptide synthesis in ribosomes.
Amino Acid Sequence↗
Cyclin B/p34cdc2 triggers phosphorylation of DNA ligase I during Xenopus laevis oocyte maturation.
Phosphorylation of DNA ligase I has been analyzed during Xenopus laevis early development. The enzyme, which is involved in DNA replication and DNA repair events, is accumulated during oogenesis to reach a maximum in the stage VI oocyte, and remains at a constant level during maturation. When maturation of the oocyte is induced (in vivo or in vitro), this leads to a post-translational modification of the protein. In stage VI oocytes, a DNA ligase I of apparent molecular mass 180 kDa is detected immunologically whereas a 190-kDa form is found in unfertilized eggs and persists until the tadpole stage. This modification is due to phosphorylation performed by a protein kinase that is turned on 3-4 h after induction of the maturation. Activation of the kinase requires protein synthesis, and appearance of phosphorylated DNA ligase coincides with activation of histone H1 kinase activity. Induction of DNA ligase I modification and maturation are induced in the absence of protein synthesis following injection of maturation promoting factor into oocytes. Immunoprecipitated oocyte DNA ligase I is phosphorylated and its molecular mass modified by purified cyclin B/p34cdc2 in vitro. DNA ligase I phosphorylation is not induced in oocyte extract where only mitogen-activated-protein kinase is induced. Phosphorylation of DNA ligase I induced by cdc2 kinase occurs at the time new DNA replication and recombination activities appear in eggs.
Animals↗