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Biomedical subjects

P Juin

Publications and source records attributed to P Juin.

At least 19 recordsLinked to original sources
PubMed2006

TOM22, a core component of the mitochondria outer membrane protein translocation pore, is a mitochondrial receptor for the proapoptotic protein Bax.

The association of Bax with mitochondria is an essential step in the implementation of apoptosis. By using a bacterial two-hybrid assay and crosslinking strategies, we have identified TOM22, a component of the translocase of the outer mitochondrial membrane (TOM), as a mitochondrial receptor of Bax. Peptide mapping showed that the interaction of Bax with TOM22 involved the first alpha helix of Bax and possibly two central alpha helices, which are homologous to the pore forming domains of some toxins. Antibodies directed against TOM22 or an antisense knockdown of the expression of TOM22 specifically inhibited the association of Bax with mitochondria and prevented Bax-dependent apoptosis. In yeast, a haploid strain for TOM22 exhibited a decreased expression of TOM22 and mitochondrial association of ectopically expressed human Bax. Our data provide a new perspective on the mechanism of association of Bax with mitochondria as it involves a classical import pathway.

Animals↗
PubMed2004

Dose-finding study of weekly 24-h continuous infusion of 5-fluorouracil associated with alternating oxaliplatin or irinotecan in advanced colorectal cancer patients.

PURPOSE: To determine maximum tolerated dose, safety and efficacy of weekly 24 h infusional 5-fluorouracil (5-FU) combined alternately with oxaliplatin and irinotecan. PATIENTS AND METHODS: Advanced colorectal carcinoma patients in first- or second-line chemotherapy received increasing doses of 5-FU (weekly 24 h continuous intravenous infusion without leucovorin) on days 1, 8, 15 and 22, irinotecan days 1 and 15; and oxaliplatin days 8 and 22, every 35 days. RESULTS: Thirty-four patients received 175 cycles. The median age was 64 years (range 47-78). Eighteen per cent of patients had the primary tumor in the rectum, with a median of one disease site (range one to three), and liver involvement in 88% and lung in 38%. Six (18%) patients had chemotherapy for prior advanced disease. The most frequent grade 3-4 toxicity was neutropenia (41% of patients), but the regimen was well tolerated clinically, with febrile neutropenia in two patients and grade 4 neutropenia lasting >7 days in one; grade 3-4 diarrhea, nausea and vomiting in 6% of patients; grade 3-4 peripheral neuropathy in 9% of patients. Seventeen patients had a partial response (50%; 95% confidence interval 33%-67%), 13 had stable disease and one had progressive disease. Five patients underwent metastatic surgical resection after tumor shrinkage. Median response duration was 14 months (range 4.7-29.2+) and median time to progression was 11.3 months (range 1.1+-30.7+). CONCLUSIONS: This combination three-drug regimen is feasible and well tolerated without toxicity overlap. Preliminary antitumor activity compares well with standard double combinations, with an unusually long median time to progression. The recommended dose is 5-FU 3000 mg/m(2), weekly for 4 weeks, irinotecan 100 mg/m(2) days 1 and 15, oxaliplatin 80 mg/m(2) days 8 and 22. Further assessment of antitumor activity and safety is warranted.

Aged↗
PubMed2004

Weekly combination of docetaxel and vinorelbine in metastatic breast cancer: a phase I/II study.

OBJECTIVE: A phase I/II study was carried out to determine the recommended dose (RD) and to assess the efficacy and safety of a weekly docetaxel-vinorelbine combination in advanced breast cancer (ABC) patients. METHODS: Twenty-four female patients with histologically proven ABC received intravenous vinorelbine (20 min) followed by intravenous docetaxel (1 h) on days 1, 8 and 15 of a 4-week cycle. Starting doses were 20 mg/m2 docetaxel and 15 mg/m2 vinorelbine. RESULTS: Patients had a median age of 62 years (range 38-74 years), and 92% had performance status 0-1. The most common sites of metastases were the lungs (32%), liver (29%) and bone (14%). Seventy-one percent of patients had received prior chemotherapy. The RDs of docetaxel and vinorelbine were 20 and 15 mg/m2, respectively. Dose-limiting toxicities were neutropenia-induced dose delay and febrile neutropenia. The response rate at the RD was 43%. All responses were seen in non-pretreated patients. Grade 3-4 neutropenia occurred in 80% of patients, 3 of whom experienced febrile neutropenia and died as a possible consequence of neutropenia. CONCLUSION: This docetaxel-vinorelbine combination as first-line therapy yields a response rate similar to that of single-agent docetaxel as second-line therapy. However, given the high rate of myelotoxicity, higher doses are not feasible.

Adult↗
PubMed2000

The coordinate release of cytochrome c during apoptosis is rapid, complete and kinetically invariant.

Release of cytochrome c from mitochondria triggers activation of caspase proteases and death of a cell by apoptosis. However, the mechanism and kinetics of cytochrome c release remain unknown. Here we study this event by using green fluorescent protein (GFP)-tagged cytochrome c, and find that the release of cytochrome-c-GFP always precedes exposure of phosphatidylserine and the loss of plasma-membrane integrity - characteristics of apoptotic cells. Once initiated, the release of cytochrome- c-GFP continues until all of the protein is released from all mitochondria in individual cells, within about 5 minutes, regardless of the type or strength of stimulus or the time elapsed since the stimulus was applied. Temperatures ranging from 24 degrees C to 37 degrees C do not change the duration of release, and nor does the addition of caspase inhibitors. Further, we find that the electron-transport chain can maintain the mitochondrial transmembrane potential even after cytochrome c has been released.

Adenosine Triphosphate↗
PubMed1999

The C-terminus of bax is not a membrane addressing/anchoring signal.

It has been suggested that BCL-2 family members associate with certain organelles through their hydrophobic C-terminus which in the case of bcl-2, appears to play a key role in the regulation of apoptosis. We have investigated the association of bax with microsomal, nuclear and mitochondrial membranes using a cell-free system and found, contrary to bcl-2, that bax binds poorly to these organelles. Deletion of the C-terminal of bax (baxDeltaC) or exchanging the C-terminal ends of bax and bcl-XL suggests that the bax C-terminus is not an addressing/anchoring signal. In agreement with this observation, HL-60 cells transfected with either bax or baxDeltaC show no difference in sensitivity to an apoptotic signal. In the cell-free system, at low pH, bax becomes associated with mitochondria after a change of conformation, a result consistant with its structural homology with certain bacterial toxins. In HL-60 cells, as observed in the cell-free system, bax acquired a protease resistant conformation upon its translocation from the cytosol to the mitochondria after the induction of apoptosis.

Amino Acid Sequence↗
PubMed1999

c-Myc-induced sensitization to apoptosis is mediated through cytochrome c release.

Expression of c-Myc sensitizes cells to a wide range of pro-apoptotic stimuli. We here show that this pro-apoptotic effect is mediated through release of mitochondrial holocytochrome c into the cytosol. First, activation of c-Myc triggers release of cytochrome c from mitochondria. This release is caspase-independent and blocked by the survival factor IGF-1. Second, c-Myc-induced apoptosis is blocked by microinjection of anticytochrome c antibody. In addition, we show that microinjection of holocytochrome c mimics the effect of c-Myc activation, sensitizing cells to DNA damage and to the CD95 pathway. Both p53 and CD95/Fas signaling have been implicated in c-Myc-induced apoptosis but neither was required for c-Myc-induced cytochrome c release. Nonetheless, inhibition of CD95 signaling in fibroblasts did prevent c-Myc-induced apoptosis, apparently by obstructing the ability of cytosolic cytochrome c to activate caspases. We conclude that c-Myc promotes apoptosis by causing the release of cytochrome c, but the ability of cytochrome c to activate apoptosis is critically dependent upon other signals.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Alternating chemotherapy and accelerated split-course irradiation in locally advanced nonsmall cell lung carcinoma.

BACKGROUND: The prognosis of patients with locally advanced nonsmall cell lung carcinoma (NSCLC), which is usually unresectable, is very poor, and patient survival rarely reaches 1 year. However, prolonged survival correlated with objective responses has been observed among patients with intrathoracic disease treated with a combination of cytotoxic drugs and local irradiation despite the lack of consensus regarding the schedule of such combined therapy. From October 1989 to November 1993, a Phase II study was conducted to evaluate the tolerability and efficacy of alternating chemotherapy and accelerated split-course radiotherapy in the treatment of patients with locally advanced NSCLC. METHODS: Sixty-three consecutive patients with unresectable Stage III NSCLC entered this study. The treatment was composed of 3 cycles of combined chemotherapy and radiotherapy at 4-week intervals. Chemotherapy with cisplatin (30 mg/m2/day) and etoposide (100 mg/m2/day) was delivered intravenously on Days 1, 2, and 3, followed by radiotherapy on Days 4-8. A course of radiotherapy consisted of 1.5 gray (Gy) per fraction twice a day (3 Gy per day) for 5 consecutive days, for a total dose of 15 Gy. Response was assessed after 3 courses, for a total irradiation dose of 45 Gy. In cases of objective antitumoral response with operable tumor, surgery was performed. A fourth course of chemotherapy and radiotherapy, for a total dose up to 60 Gy in 12 weeks, was administered to all patients. Two additional courses of chemotherapy were given, for a total of six. RESULTS: Of the 63 patients, 62 were evaluable for response. Six had a complete remission and 36 a partial response, resulting in an overall response rate of 67.7%. Nine patients underwent surgery (pneumonectomy for seven patients and lobectomy for two patients), and the complete disappearance of any residual tumor was documented histologically in four. Of the 290 chemotherapy courses delivered, there were 31 of Grade 3-4 toxicity, mainly leukopenia and vomiting. The median times of freedom from disease progression and overall survival were 8 months (confidence interval [CI], 7-9.5) and 14 months (CI, 10-22), respectively. The 1-, 2-, and 5-year survival rates of the 62 patients were 54%, 35%, and 21%, respectively. Patients who responded had a significantly longer median survival (16 months) than nonresponders (7 months) (P = 0.02). However, there was no difference in the survival of resected and nonresected patients. CONCLUSIONS: This treatment schedule resulted in a high response rate with prolonged survival. However, the toxicity of this approach was not negligible, even though it did not preclude this strategy. This combined modality must be compared with other combinations of alternating or sequential chemoradiotherapy.

Aged↗
PubMed1998

Potentiation of apoptosis by mitochondria in a cell-free system.

Using a cell-free system, we show that rat liver mitochondria, but not mitochondrial extracts, potentiated apoptosis triggered by cytosols derived from apoptotic cells. Apoptosis potentiated by mitochondria appeared to be inhibited by caspase 3 but not by caspase 1 inhibitors. A cytosolic caspase-3-like activity was increased by the addition of mitochondria to apoptotic cytosols; the latter activation was inhibited by the addition of bcl-2. Chelation of calcium by EGTA significantly and specifically inhibited the apoptosis potentiated by mitochondria as well as the increase of caspase-3-like activity. The incubation of mitochondria with apoptotic cytosols led to the release of cytochrome c, this latter phenomenon being inhibited by EGTA. Calcium or cytochrome c and dATP, however, did not reproduce the mitochondrial potentiation in the absence of the organelle. Thus, mitochondria can initiate and potentiate apoptosis through similar but not identical mechanisms.

Animals↗
PubMed1998

The isolated complex of the translocase of the outer membrane of mitochondria. Characterization of the cation-selective and voltage-gated preprotein-conducting pore.

The complex of the translocase mitochondrial outer membrane (TOM), mediates recognition, unfolding, and translocation of preproteins. We have used a combination of biochemical and electrophysiological methods to study the properties of the preprotein-conducting pore of the purified TOM complex. The pore is cation-selective and voltage-gated. It shows three main conductance levels with characteristic slow and fast kinetics transitions to states of lower conductance following application of transmembrane voltages. These electrical properties distinguish it from the mitochondrial voltage-dependent anion channel (porin) and are identical to those of the previously described peptide-sensitive channel. Binding of antibodies to the C terminus of Tom40 on the intermembrane space side of the outer membrane modifies the channel properties and allows determination of the orientation of the channel within the lipid bilayer. Mitochondrial presequence peptides specifically interact with the pore and decrease the ion flow through the channel in a voltage-dependent manner. We propose that the presequence-induced closures of the pore are related to structural alterations of the TOM complex observed during the various stages of preprotein movement across the mitochondrial outer membrane.

Biological Transport↗
PubMed1998

Induction of a caspase-3-like activity by calcium in normal cytosolic extracts triggers nuclear apoptosis in a cell-free system.

Calcium is involved in several steps of the apoptotic process. In nuclei, endonucleases are presumed to be the main targets of calcium; however, little is known about its role during the cytosolic phase of apoptosis. We used a cell-free system to address this question. Our results show that CaCl2 triggered nuclear apoptosis (i.e. typical morphological change and DNA fragmentation) at concentrations of 5 mM. This concentration was lowered 10-fold by the co-incubation with cytosolic extracts from nonapoptotic cells. Apoptotic changes induced by the incubation of nuclei with CaCl2 in the presence of these cytosols were strongly reduced in the presence of an inhibitor of caspase-3 and to a lesser extent by an inhibitor of caspase-1. We also show that calcium-induced apoptosis is affected by protease inhibitors such as N-tosyl-L-phenylalanine chloromethyl ketone, but not by calpain or several lysosomal protease inhibitors. The addition of CaCl2 to the cell-free system increased a caspase-3 activity in nonapoptotic cytosols as shown by specific antibodies and an enzymatic assay. No activation of a caspase-3-like activity by the addition of cytochrome c was observed in these extracts under similar conditions. The enhanced caspase-3 activity induced by calcium was inhibited by protease inhibitors affecting morphological nuclear apoptosis except for those responsible for the degradation of lamin A. These results suggest that CaCl2 could trigger, in normal cells, an apoptotic cascade through the activation of cytosolic caspase-3 activity.

Amino Acid Sequence↗
PubMed1998

Treatment of carcinoma of the uterine cervix with concomitant cisplatin, 5-fluorouracil and split course hyperfractionated radiotherapy.

To improve local and systemic control of bulky (>4 cm) and/or advanced primary cancer of the uterine cervix, 35 patients were treated with concomitant cisplatin (CDDP), 5-fluorouracil (5-FU) and split course hyperfractionated radiotherapy. Radiation was administered to the pelvis in five-day courses at a dose of 1.5 Gy twice daily every 21 days until a median dose of 45 Gy was reached. 15 Gy more were administered to involved parametrium or central tumor by external radiotherapy or brachytherapy. The irradiated zone was extended to include paraaortic lymph nodes if necessary. CDDP was administered at a dose of 20 mg m(-2) and 5 FU at a dose of 500 mg m(-2) from day one to day five of each course. The median number of combined treatment courses per patient was four (1-6). Local responses were obtained in 19 out of 24 patients in whom evaluation was feasible (i.e. who did not undergo surgery prior to combined therapy). Median survival was not attained with a median follow up of 33 months, three year overall survival was 62% and 52% in patients with local control and in the whole population respectively. Several patients with stage III and IV tumors achieved a very long survival. Acute toxicity was manageable but three patients required surgical repair of late radiation complications. This combined chemotherapy and radiotherapy resulted in good local control and did not rule out surgery.

Adult↗
PubMed1997

Relationship between the peptide-sensitive channel and the mitochondrial outer membrane protein translocation machinery.

The peptide-sensitive channel (PSC), a cationic channel of the mitochondrial outer membrane, is blocked by synthetic mitochondrial presequences and by nonmitochondrial basic peptides such as dynorphin B(1-13). Both types of peptides are imported into mitochondria. However, the import of dynorphin B(1-13) had to be further characterized since its properties differed from those of the general import pathway used by mitochondrial peptides. Cross-linking experiments with iodinated dynorphin B(1-13) led to the labeling of TOM 40/ISP 42, a component of the protein import machinery of the outer membrane. Accordingly, dynorphin B(1-13) could also be used as a presequence to direct the import of a cytosolic protein into the mitochondria. Pretreatment of intact mitochondria by trypsin removed components capable of discriminating between true mitochondrial presequences and other basic peptides active on the PSC. After proteolysis, both types of peptides appeared to cross the outer membrane through the same pathway. Involvement of the PSC in the translocation complex was shown by immunoprecipitation of the PSC activity by anti-ISP 42 antibodies. Taken together, the present data reinforce the hypothesis that the PSC is the pore responsible for the translocation of protein through the outer membrane.

Animals↗
PubMed1996

Characterization and function of the mitochondrial outer membrane peptide-sensitive channel.

The PSC (peptide-sensitive Channel), a cationic channel of large conductance, has been characterized in yeast and mammalian mitochondria by three different methods, "tip-dip," patch clamp of giant liposomes, and planar bilayers. The yeast and mammalian PSC share the common property to be blocked by basic peptides such as pCyt OX IV (1-12)Y which contains the first 12 residues of the presequence of cytochrome C oxidase subunit IV. The electrophysiological data are consistent with a translocation of the peptide through the pore. Analysis of the frequency of observation of the PSC in different fractions indicates that the channel is located in the outer mitochondrial membrane. Uptake measurements of iodinated peptides by intact mitochondria from a porin-less mutant show that the peptides are translocated through the outer membrane, presumably at the level of PSC. Among the peptides active on PSC, several, such as pCyt OX IV (1-22) and the reduced form of the mast cell degranulating peptide, induce an alteration of the voltage dependence or of the inactivation rate subsisting after washing and which is eliminated only by proteolysis of the interacting peptide. These irreversible effects may account for the variability of the properties of the PSC which would interact with cytosolic or intermembrane cations, peptides, or proteins, thus modulating the channel permeability. Finally, several lines of evidence suggest the participation of the PSC in protein translocation and some interaction with the general insertion pore of the outer membrane translocation machinery.

Amino Acid Sequence↗
PubMed1995

Involvement of the peptide sensitive channel in the translocation of basic peptides into mitochondria.

The Peptide Sensitive Channel (PSC), a cationic channel of the mitochondrial outer membrane, is blocked by several highly basic peptides. Among these peptides, the most active are pCOX IV (1-12)Y, a mitochondrial addressing peptide and dynorphin B (1-13), a peptide unrelated to mitochondrial physiology. The voltage-dependent characteristics of the block duration of the PSC induced by these peptides and the fact that these peptides are imported into mitochondria in an in vitro assay suggest the involvement of the PSC in peptide translocation into mitochondria. We have analyzed the interaction of Mast Cell Degranulating peptide (MCD), a disulfide rich basic peptide, with yeast and mammalian mitochondria. Electrophysiological experiments with native and reduced forms of this peptide (nMCD and rMCD) showed an interaction of both forms with the yeast PSC. On the other hand, only rMCD blocked the electrical activity of the bovine adrenal cortex PSC. Similarly, although both forms inhibited the import of dynorphin B (1-13) into yeast mitochondria, only rMCD inhibited this import in bovine mitochondria. The correlation between electrophysiological and biochemical data strongly suggest that dynorphin B is translocated across the outer membrane at the level of the PSC.

Adrenal Cortex↗
PubMed1994

Basic peptides can be imported into yeast mitochondria by two distinct targeting pathways. Involvement of the peptide-sensitive channel of the outer membrane.

The interaction of several basic peptides with yeast mitochondria has been analyzed. The peptides were selected for their ability to block a cationic channel of the outer membrane, the peptide-sensitive channel. These peptides possess common characteristics, such as a net positive charge superior to 2 and the capacity to form amphiphilic structures. They can be divided into two classes as follows: peptides of class I derived from mitochondrial signal peptides, such as the presequence of cytochrome c oxidase subunit IV, e.g. pCyt OX IV (1-12) Y; and peptides of class II unrelated to the mitochondria, such as dynorphin B (1-13). Class I peptides inhibited the translocation of a chimeric protein, cytochrome b2-DHFR, into the mitochondrial matrix, whereas peptides of class II failed to inhibit this import. Experiments with iodinated pCyt OX IV (1-12) Y and dynorphin B (1-13) showed, however, that both types of peptides were imported into yeast mitochondria in vitro and subsequently degraded. At 30 degrees C, two import mechanisms could be distinguished; the mitochondrial presequences (class I) were translocated into the matrix in a temperature- and potential-sensitive manner, probably along the general import pathway, while class II dynorphin B (1-13) was imported into the intermembrane space by a process that was neither temperature- nor potential-sensitive. At 0 degree C, both peptides were imported in a class II manner. The class II characteristics suggested the existence of a direct pathway into the intermembrane space, which may be associated with the peptide-sensitive channel. This hypothesis is substantiated by the competition for the import into the mitochondria between peptides of the two classes. The import of pCyt OX (1-12) Y was inhibited at 30 degrees C only by peptides of class I, IV whereas, at 0 degree C, this import was also inhibited by peptides of class II. Import of peptides of the latter class was inhibited by peptides of the two classes both at 0 degree C and 30 degrees C.

Amino Acid Sequence↗