Biomedical subjects

O Bernard

Publications and source records attributed to O Bernard.

At least 37 records · Page 2Linked to original sources

PubMed2001

Cytoskeletal changes regulated by the PAK4 serine/threonine kinase are mediated by LIM kinase 1 and cofilin.

PAK4 is the most recently identified member of the PAK family of serine/threonine kinases. PAK4 differs from other members of the PAK family in sequence and in many of its functions. Previously, we have shown that an important function of this kinase is to mediate the induction of filopodia in response to the Rho GTPase Cdc42. Here we show that PAK4 also regulates the activity of the protein kinase LIM kinase 1 (LIMK1). PAK4 was shown to interact specifically with LIMK1 in binding assays. Immune complex kinase assays revealed that both wild-type and constitutively active PAK4 phosphorylated LIMK1 even more strongly than PAK1, and activated PAK4 stimulated LIMK1's ability to phosphorylate cofilin. Immunofluorescence experiments revealed that PAK4 and LIMK1 cooperate to induce cytoskeletal changes in C2C12 cells. Furthermore, dominant negative LIMK1 and a mutant cofilin inhibited the specific cytoskeletal and cell shape changes that were induced in response to a recently characterized constitutively activated PAK4 mutant.

Actin Depolymerizing Factors↗

PubMed2001

Involvement of the NF-kappaB pathway in the transforming properties of the TEL-Jak2 leukemogenic fusion protein.

Constitutively active tyrosine kinases are frequently expressed in various types of human leukemias as the result of chromosomal translocations. The TEL-Jak2 fusion oncoprotein possesses transforming properties in both animal and cellular models, that are tightly dependent on Stat5 activation. In the IL-3-independent TEL-Jak2-transformed Ba/F3 cells, activation of the PI-3K/Akt pathway appears essential to cell proliferation. Here we report a sustained activation of NF-kappaB factors in Ba/F3 cells, which inhibition dramatically impairs cell viability, indicating that NF-kappaB signaling exerts a major role in the anti-apoptotic activities of TEL-Jak2 oncoprotein.

Animals↗

PubMed2001

Constitutively active STAT5 variants induce growth and survival of hematopoietic cells through a PI 3-kinase/Akt dependent pathway.

Signal Transducer and Activator of Transcription (STATs) are important mediators of cytokine and growth factor-induced signal transduction. STAT5A and STAT5B have been shown to play a role in survival and proliferation of hematopoietic cells both in vitro and in vivo and to contribute to the growth and viability of cells transformed by the TEL-JAK2 oncoprotein. In this study, we investigated the molecular mechanisms by which constitutively active STAT5 proteins induce cell proliferation and survival of Ba/F3 cell lines expressing either dominant positive STAT5A or STAT5B variants or TEL-JAK2 or TEL-ABL fusion proteins. Our results showed that active STAT5 constitutively interacted with p85, the regulatory subunit of the PI 3-kinase. A constitutive activity of the PI 3-kinase/Akt pathway was observed in these cells and required for their cell cycle progression. In contrast, while activity of the PI 3-kinase/Akt pathway was required for survival of Ba/F3 cells expressing the constitutively active forms of STAT5A or STAT5B, it was dispensable for cells transformed by TEL-JAK2 or TEL-ABL fusion proteins, suggesting that additional survival pathways take place in these transformed cells.

Animals↗

PubMed2001

Orthotopic liver transplantation for mitochondrial respiratory chain disorders: a study of 5 children.

BACKGROUND: Liver involvement in mitochondrial respiratory chain disorders (MRCD) frequently ends in liver failure and death. Because of the high risk of extrahepatic, particularly neuromuscular, manifestations of the disease, the indication of orthotopic liver transplantation (OLT) in these patients remains controversial. We report on 5 such children in whom OLT was carried out, in an attempt to help clarify the matter. PATIENTS: Patients 1 and 2 presented with fulminant liver failure at ages 7 and 6 months respectively. Emergency liver transplantation was performed before etiological investigations were completed. Retrospective examination of the explanted livers showed defects in complexes I, III and IV. In patient 1, severe neurological deterioration occurred 2 months after OLT with fatal outcome 9 months later. Patient 2 is alive 22 months after OLT with moderate motor impairment. Patients 3, 4 and 5 presented with progressive liver failure before 6 months of age. Surgical liver biopsies displayed a 50% defect in complex IV (patient 3), a defect in complexes I, IV (patient 4) and in complexes I, III, IV (patient 5). Because there was no clinical extrahepatic involvement on investigations, OLT was carried out in these patients. Patient 3 died of multiple organ failure soon after OLT, patients 4 and 5 are alive respectively 21 months and 12 months after OLT with normal neurological examination. CONCLUSION: OLT may be a valid therapeutic option in infants with delayed liver cell failure due to MRCD, only after performing in emergency a thorough inves tigation to exclude clinically significant extrahepatic, especially neuromuscular, involvement.

Child↗

PubMed2001

The TEL-Jak2 oncoprotein induces Socs1 expression and altered cytokine response in Ba/F3 cells.

The leukemia-associated TEL-Jak2 fusion protein possesses a constitutive tyrosine kinase activity and transforming properties in hematopoietic cell lines and animal models. In the murine pro-B Ba/F3 cell line, this fusion constitutively activates the Signal Transducer and Activator of Transcription 5 (Stat5) factors and, as a consequence, induces the sustained expression of various Stat5-target genes including the Cytokine Inducible SH2-containing protein (Cis) gene, which codes for a member of the Suppressor of Cytokine Signaling (Socs) protein family. In TEL-Jak2-transformed Ba/F3 cells, we also observed the upregulation of the Socs1 gene, whose product has been reported to negatively regulate the Jak kinase activity. In transient transfection experiments, Socs1 physically interacts with TEL-Jak2 and interferes with the TEL-Jak2-induced phosphorylation and activation of Stat5 factors, probably through the Socs1-induced proteasome-mediated degradation of the fusion protein. Interestingly, TEL-Jak2-expressing Ba/F3 cells were found to be resistant to the anti-proliferative activities of gamma interferon (IFN-gamma) seemingly as a consequence of Socs1 constitutive expression. These results indicate that the Socs1-dependent cytokine feedback loop, although active, is bypassed by the TEL-Jak2 fusion, but may play a role in the leukemogenic process by altering the cytokine responses of the leukemic cells. Our results also suggest that Socs1 plays a role in shutting down the signaling from the normally activated Jak2 kinase by inducing its proteasome-dependent degradation.

Animals↗

PubMed2001

Dominant-negative c-Jun promotes neuronal survival by reducing BIM expression and inhibiting mitochondrial cytochrome c release.

Sympathetic neurons require nerve growth factor for survival and die by apoptosis in its absence. Key steps in the death pathway include c-Jun activation, mitochondrial cytochrome c release, and caspase activation. Here, we show that neurons rescued from NGF withdrawal-induced apoptosis by expression of dominant-negative c-Jun do not release cytochrome c from their mitochondria. Furthermore, we find that the mRNA for BIM(EL), a proapoptotic BCL-2 family member, increases in level after NGF withdrawal and that this is reduced by dominant-negative c-Jun. Finally, overexpression of BIM(EL) in neurons induces cytochrome c redistribution and apoptosis in the presence of NGF, and neurons injected with Bim antisense oligonucleotides or isolated from Bim(-/-) knockout mice die more slowly after NGF withdrawal.

Adenoviridae↗

PubMed2001

The wide spectrum of multidrug resistance 3 deficiency: from neonatal cholestasis to cirrhosis of adulthood.

BACKGROUND & AIMS: We have specified the features of progressive familial intrahepatic cholestasis type 3 and investigated in 31 patients whether a defect of the multidrug resistance 3 gene (MDR3) underlies this phenotype. METHODS: MDR3 sequencing, liver MDR3 immunohistochemistry, and biliary phospholipid dosage were performed. RESULTS: Liver histology showed a pattern of biliary cirrhosis with patency of the biliary tree. Age at presentation ranged from the neonatal period to early adulthood. Sequence analysis revealed 16 different mutations in 17 patients. Mutations were identified on both alleles in 12 patients and only on 1 allele in 5. Four mutations lead to a frame shift, 2 are nonsense, and 10 are missense. An additional missense mutation probably representing a polymorphism was found in 5 patients. MDR3 mutations were associated with abnormal MDR3 canalicular staining and a low proportion of biliary phospholipids. Gallstones or episodes of cholestasis of pregnancy were found in patients or parents. Children with missense mutations had a less severe disease and more often a beneficial effect of ursodeoxycholic acid therapy. CONCLUSIONS: At least one third of the patients with a progressive familial intrahepatic cholestasis type 3 phenotype have a proven defect of MDR3. This gene defect should also be considered in adult liver diseases.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed2001

Outcome of liver disease in children with Alagille syndrome: a study of 163 patients.

BACKGROUND AND AIMS: Various opinions have been expressed as to the long term prognosis of liver disease associated with Alagille syndrome (AGS). PATIENTS AND METHODS: We reviewed the outcome of 163 children with AGS and liver involvement, investigated from 1960 to 2000, the end point of the study (median age 10 years (range 2 months to 44 years)) being death, liver transplantation, or the last visit. RESULTS: At the study end point, of the 132 patients who presented with neonatal cholestatic jaundice, 102 remained jaundiced, 112 had poorly controlled pruritus, and 40 had xanthomas; cirrhosis was found in 35/76 livers, varices in 25/71 patients, and liver transplantation had been carried out in 44 patients (33%). Forty eight patients died, 17 related to complications of liver disease. Of 31 patients who did not present with neonatal cholestatic jaundice, five were jaundiced at the study end point, 17 had well controlled pruritus, and none had xanthomas; cirrhosis was found in 6/18 patients, varices in 4/11, and none underwent liver transplantation. Nine patients died, two of liver disease. In the whole series, actuarial survival rates with native liver were 51% and 38% at 10 and 20 years, respectively, and overall survival rates were 68% and 62%, respectively. Neonatal cholestatic jaundice was associated with poorer survival with native liver (p=0.0004). CONCLUSIONS: The prognosis of liver disease in AGS is worse in children who present with neonatal cholestatic jaundice. However, severe liver complications are possible even after late onset of liver disease, demanding follow up throughout life.

Adolescent↗

PubMed2001

Hybrid modelling of anaerobic wastewater treatment processes.

This paper presents a hybrid approach for the modelling of an anaerobic digestion process. The hybrid model combines a feed-forward network, describing the bacterial kinetics, and the a priori knowledge based on the mass balances of the process components. We have considered an architecture which incorporates the neural network as a static model of unmeasured process parameters (kinetic growth rate) and an integrator for the dynamic representation of the process using a set of dynamic differential equations. The paper contains a description of the neural network component training procedure. The performance of this approach is illustrated with experimental data.

Bacteria, Anaerobic↗

PubMed2001

Hybrid neural modelling of an anaerobic digester with respect to biological constraints.

A hybrid model for an anaerobic digestion process is proposed. The fermentation is assumed to be performed in two steps, acidogenesis and methanogenesis, by two bacterial populations. The model is based on mass balance equations, and the bacterial growth rates are represented by neural networks. In order to guarantee the biological meaning of the hybrid model (positivity of the concentrations, boundedness, saturation or inhibition of the growth rates) outside the training data set, a method that imposes constraints in the neural network is proposed. The method is applied to experimental data from a fixed bed reactor.

Anaerobiosis↗

PubMed2001

Advanced monitoring and control of anaerobic wastewater treatment plants: software sensors and controllers for an anaerobic digester.

A mass balanced based model representing the dynamical behaviour of anaerobic digester has served as a basis for the design of software sensors for the concentration of inorganic carbon, alkalinity and volatile fatty acids. The predictions of the sensors are close to the actual off-line measurements. The model has also been used to design a model-based adaptive linearizing controller and a fuzzy controller whose objective is to regulate the ratio of the intermediate alkalinity over the total alkalinity below some desired value (0.3) under which the process is assumed to remain in stable conditions and avoid VFA accumulation. Both controllers were calibrated via extensive numerical simulations and implemented. The controllers proved successful in maintaining the ratio of TA over PA below 0.3, even in presence of large variations of the organic load.

Anaerobiosis↗

PubMed2000

Phosphorylation of ADF/cofilin abolishes EGF-induced actin nucleation at the leading edge and subsequent lamellipod extension.

In metastatic rat mammary adenocarcinoma cells, cell motility can be induced by epidermal growth factor. One of the early events in this process is the massive generation of actin barbed ends, which elongate to form filaments immediately adjacent to the plasma membrane at the tip of the leading edge. As a result, the membrane moves outward and forms a protrusion. To test the involvement of ADF/cofilin in the stimulus-induced barbed end generation at the leading edge, we inhibited ADF/cofilin's activity in vivo by increasing its phosphorylation level using the kinase domain of LIM-kinase 1 (GFP-K). We report here that expression of GFP-K in rat cells results in the near total phosphorylation of ADF/cofilin, without changing either the G/F-actin ratio or signaling from the EGF receptor in vivo. Phosphorylation of ADF/cofilin is sufficient to completely inhibit the appearance of barbed ends and lamellipod protrusion, even in the continued presence of abundant G-actin. Coexpression of GFP-K, together with an active, nonphosphorylatable mutant of cofilin (S3A cofilin), rescues barbed end formation and lamellipod protrusion, indicating that the effects of kinase expression are caused by the phosphorylation of ADF/cofilin. These results indicate a direct role for ADF/cofilin in the generation of the barbed ends that are required for lamellipod extension in response to EGF stimulation.

Actin Depolymerizing Factors↗

PubMed2000

TEL-JAK2 transgenic mice develop T-cell leukemia.

We previously reported a fusion between TEL and JAK2 in a t(9;12)(p24;p13) chromosomal translocation in childhood acute T-cell leukemia. This fusion gene encodes a TEL-JAK2 chimeric protein in which the 336 amino-terminal residues of TEL, including its specific self-association domain, are fused to the kinase domain of JAK2. TEL-JAK2 exhibits constitutive activation of its tyrosine kinase activity which, in turn, confers growth factor-independent proliferation to the interleukin-3-dependent Ba/F3 hematopoietic cell line. To elucidate the properties of TEL-JAK2 in primary cells and to create an animal model for TEL-JAK2-induced leukemia, we generated transgenic mice in which the TEL-JAK2 complementary DNA was placed under the transcriptional control of the EmuSRalpha enhancer/promoter. TEL-JAK2 founder mice and their transgenic progeny developed fatal leukemia at 4 to 22 weeks of age. Selective amplification of CD8-positive T cells was observed in blood, lymph nodes, thymus, spleen, and bone marrow. Expression of a tyrosine-phosphorylated TEL-JAK2 protein and activation of STAT1 and STAT5 (signal transducer and activator of transcription) were detected in leukemic tissues. TEL-JAK2 diseased mice also displayed invasion of nonhematopoietic organs, including liver, brain, lung, and kidney, by leukemic T cells. Leukemic organs of founder and transgenic progeny contained a monoclonal/oligoclonal T-cell population as analyzed by the rearrangement of the TCRbeta locus. Transplantation of TEL-JAK2 leukemic cells in nude mice confirmed their invasive nature. We conclude that the TEL-JAK2 fusion is an oncogene in vivo and that its expression in lymphoid cells results in the preferential expansion of CD8-positive T cells. (Blood. 2000;95:3891-3899)

Animals↗

PubMed2000

Design of a fungal bioprocess for vanillin production from vanillic acid at scalable level by Pycnoporus cinnabarinus.

The biotechnological process of vanillin production from vanillic acid by Pycnoporus cinnabarinus was scaled-up at the laboratory level. Vanillin production was studied in two types of bioreactors, a mechanically agitated and an air-lift bioreactor. In the mechanically agitated bioreactor where vanillin was produced in greater quantities, oxygen availability was studied during the growth and production phases. A maximal aeration rate (90l/h equivalent to 0.83 volume of air/volume of medium/min or vvm) during the growth phase and a minimal aeration rate (30 l/h equivalent to 0.28 vvm) during the production phase were necessary to increase vanillin production to 1260 mg/l. Vanillic acid bioconversion to vanillin occurred under the conditions of reduced dissolved oxygen concentration, gentle agitation, high carbon dioxide production and low specific growth rate. However, under these conditions, vanillin production was accompanied by a significant amount of methoxyhydroquinone. Vanillin over a concentration of 1000 mg/l was shown to be highly toxic to the growth of P. cinnabarinus on agar medium. The application of selective XAD-2 resin led to a reduction of vanillin concentration in the medium, thus limiting its toxicity towards the fungal biomass as well as the formation of unwanted by-products such as methoxyhydroquinone and allowed the concentration of vanillin produced to reach 1575 mg/l.

Journal Article↗

PubMed2000

Software sensors to monitor the dynamics of microbial communities: application to anaerobic digestion.

A mass balance based model has been derived to represent the dynamical behavior of the ecosystem contained in an anaerobic digester. The model considers two bacterial populations: acidogenic and methanogenic bacteria. It forms the basis for the design of a software sensor considering both a model of the biological system and on-line gaseous measurements. The software sensor computes the concentration of inorganic carbon and volatile fatty acids (VFA) in the digester. Another software sensor is dedicated to the estimation of the bacterial biomasses. The predictions of the software sensors for a real experiment are very close to the actual off-line measurements. The software sensors monitor the accumulation of VFA and thus very early detect a destabilization of the digester due to overloading. The presented methodology demonstrates the usefulness of advanced monitoring techniques for an improved understanding of the internal working of a biological system.

Bacteria, Anaerobic↗

PubMed2000

Implication of Bcl-2 and Caspase-3 in age-related Purkinje cell death in murine organotypic culture: an in vitro model to study apoptosis.

Neuronal cell death is an essential feature of nervous system development and neurodegenerative diseases. Most Purkinje cells in murine cerebellar organotypic culture die when taken from 1-5-day-old mice (P1-P5), whereas they survive when taken before or after these ages. Using DNA gel electrophoresis, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) and electron microscopic analyses, we were able to show that this massive Purkinje cell death is apoptotic in nature and reaches a peak at P3. From the several endogenous genes known to be involved in the apoptotic process, we have focused on two: the bcl-2 and the caspase-3 that encode for anti-apoptotic and pro-apoptotic proteins, respectively. Immunostaining for activated Caspase-3 correlated with Purkinje cell death. A better survival of Purkinje cells was observed in P3 slices taken from hu-bcl-2 transgenic mice, and in slices treated with z-DEVD.fmk (an inhibitor of numerous caspases). Thus, these two genes are implicated in the age-related Purkinje cell apoptosis in organotypic culture. As Purkinje cell death in vitro takes place at the same age as Purkinje cells engaged in intense synaptogenesis and dendritic remodeling in vivo, we propose that this apoptosis reflects a naturally occurring Purkinje cell death during this critical period.

Animals↗

PubMed2000

Human Bcl-2 protects against AMPA receptor-mediated apoptosis.

Dysfunctions of the (S)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) subtype of ionotropic receptor for the brain's major excitatory neurotransmitter, L-glutamate, occur in various neurological conditions. We have previously demonstrated that AMPA receptor-mediated excitotoxicity occurs by apoptosis and here examined the influence of the expression of cell death repressor gene Bcl-2 on this excitotoxic insult. Using neuronal cortical cultures prepared from transgenic mice expressing the human Bcl-2 gene, the influence of Bcl-2 on AMPA receptor-mediated neuronal death was compared with that seen with staurosporine and H2O2. At day 6 cultures were exposed to AMPA (0.1-100 microM), and cellular injury was analyzed 48 h after insult using phase-contrast microscopy, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide viability assay, and DNA staining with 4,6-diamidino-2-phenylindole and Sytox Green. AMPA produced a concentration-dependent increase in cell death that was significantly attenuated by human Bcl-2. AMPA (3 microM) increased the number of apoptotic nuclei to 60% of control in wild-type cultures, and human Bcl-2 significantly decreased the number of apoptotic nuclei to 30% of AMPA-treated cultures. Human Bcl-2 only provided significant neuroprotection against neuronal injury induced by low concentrations of staurosporine (1-10 nM) and H2O2 (0.1-30 microM) and where neuronal death was by apoptosis, but not against H2O2-induced necrosis. Our findings indicate that overexpression of Bcl-2 in primary cultured neurons protects in an insult-dependent manner against AMPA receptor-mediated apoptosis, whereas protection was not seen against more traumatic insults. This study provides new insights into the molecular therapeutics of neurodegenerative conditions.

Animals↗

PubMed2000

Unusual early presentation of Gilbert syndrome in pediatric recipients of liver transplantation.

BACKGROUND: Gilbert syndrome as a rule becomes manifest in adolescence or in early adulthood; it may be transferred by the donor to orthotopic liver transplant (OLT) recipients. METHODS: We examined the frequency of Gilbert syndrome in 46 OLT pediatric recipients who had a follow-up of 1 year or more. Diagnostic criteria included unexplained chronic or recurrent unconjugated hyperbilirubinemia; its increase after reduced caloric intake plus prolonged fasting, without changes of the proportion of conjugated bilirubin; and high relative amounts of serum unconjugated bilirubin IXa and prevalence of the monoglucuronide over the diglucuronide. RESULTS: Of the 46 patients, 42 had normal bilirubin values. Only four otherwise healthy OLT recipients showed hyperbilirubinemia and normal conjugated fractions. Liver donors had been four men. Hyperbilirubinemia persisted with a fluctuating pattern for the whole follow-up after OLT in all. Total bilirubin level in blood samples obtained after reduced caloric intake and prolonged fasting became notably higher than basal values, whereas the proportion of conjugated bilirubin remained stable. High relative amounts of unconjugated bilirubin IXa and prevalence of the monoglucuronide over the diglucuronide were found. Finally, DNA from liver donors' lymphocytes was available for one jaundiced and two nonjaundiced patients: tests for abnormalities in the promoter region of the gene for the enzyme bilirubin uridine diphospho-glucuronosyltransferase were in agreement with a diagnosis of GS in the former one, CONCLUSIONS: Gilbert syndrome may have an unusual early presentation in pediatric OLT recipients.

Age Factors↗