Biomedical subjects

Christian D Muller

Publications and source records attributed to Christian D Muller.

12 recordsLinked to original sources

PubMed2005

The retinoblastoma gene and its product are targeted by ICBP90: a key mechanism in the G1/S transition during the cell cycle.

The retinoblastoma protein (pRB) is encoded by the RB1 gene whose promoter contains several putative binding sites for ICBP90 (Inverted CCAAT box Binding Protein of 90 kDa), a transcriptional regulator of the topoisomerase IIalpha gene. ICBP90 has two consensus binding sites for pRB in its primary sequence. Here, we show that pRB and ICBP90 co-immunoprecipitate in cell extracts of proliferating human lung fibroblasts and of proliferating or confluent Jurkat cells. GST pull-down assays and immunocytochemistry, after cell synchronization in late G1 phase, confirmed this interaction. Overexpression of ICBP90 induces downregulation of pRB expression in lung fibroblasts as a result of mRNA decrease. DNA chromatin immunoprecipitation experiment shows that ICBP90 binds to the RB1 gene promoter under its methylated status. Overexpression of ICBP90 increases the S and G2/M phase cell fractions of serum-starved lung fibroblasts as assessed by flow cytometry analysis and increases topoisomerase IIalpha expression. Together, these results show that ICBP90 regulates pRB at the protein and gene transcription levels, thus favoring the entry into the S phase of the cells. We propose that ICBP90 overexpression, found in cancer cells, is involved in the altered checkpoint controls occurring in cancerogenesis.

Blotting, Western↗

PubMed2005

Upregulation of proinflammatory proteins through NF-kappaB pathway by shed membrane microparticles results in vascular hyporeactivity.

OBJECTIVE: Microparticles are membrane vesicles with procoagulant and proinflammatory properties released during cell activation, including apoptosis. The present study was designed in dissecting the effects evoked by microparticles on vascular reactivity. METHODS AND RESULTS: Microparticles from either apoptotic T lymphocytic cells or from plasma of diabetic patients with vascular complications induced vascular hyporeactivity in response to vasoconstrictor agents in mouse aorta. Hyporeactivity was reversed by nitric oxide (NO) synthase plus cyclooxygenase-2 inhibitors, and associated with an increased production of vasodilatory products such as NO and prostacyclin. Microparticles induced an upregulation of proinflammatory protein expressions, inducible NO-synthase and cyclooxygenase-2, mainly in the medial layer of the vessels as evidenced by immunochemical staining. In addition, microparticles evoke NF-kappaB activation probably through the interaction with the Fas/Fas Ligand pathway. Finally, in vivo treatment of mice with lymphocyte-derived MPs induces vascular hyporeactivity, which was reversed by the combination of NO and cyclooxygenase-2 inhibitors. CONCLUSIONS: These data provide a rationale to explain the paracrine role of microparticles as vectors of transcellular exchange of message in promoting vascular dysfunction during inflammatory diseases.

6-Ketoprostaglandin F1 alpha↗

PubMed2005

TCR pathway involves ICBP90 gene down-regulation via E2F binding sites.

Antigen-induced cell death is essential for function, growth and differentiation of T-lymphocytes through legation of the T cell receptor. Since TCR-induced cell death occurs at late G1 checkpoint of the cell cycle and considering that ICBP90 is critical for G1/S transition, we studied the ICBP90 regulation through the TCR pathway in Jurkat cells. ICBP90 expression was strongly decreased after TCR triggering concomitantly to cyclin D3 and topoisomerase IIalpha expression decreases. Cell stimulation with PMA and/or calcium ionophore A23187 down-regulated ICBP90 expression. The decrease of ICBP90 protein and mRNA expressions was accompanied with cell growth arrest. A luciferase reporter assay demonstrated that activation of TCR pathways inhibit ICBP90 gene promoter activity. Three consensus E2F binding sites (called from E2F-a to E2F-c) were identified in the ICBP90 gene promoter and were subjected to mutations. The E2F-a, located in a highly active promoter fragment, shows a strong positive functional activity in proliferating cells. E2F-a and E2F-c binding sites are involved in the TCR-induced down-regulation of ICBP90 gene transcription. Altogether, our data demonstrate that TCR signaling pathways regulate ICBP90 gene expression through pRb/E2F complex. We propose that ICBP90 down-regulation is a key event in G1 arrest preceding T cell death.

Base Sequence↗

PubMed2005

Factors contributing to infectious diarrhea-associated pancreatic enzyme alterations.

OBJECTIVES: Several pathogens have been involved as etiologic agents of acute pancreatitis. We studied 59 patients presenting acute infectious diarrhea in order to determine the incidence as well as to identify factors which may contribute to the occurrence of pancreatic enzyme alteration or true acute pancreatitis. METHODS: Patients were evaluated for serum lipase and amylase, and 24-hours urinary amylase. Clinical and biological parameters were noted. Abdominal sonography and rectosigmoidoscopy were performed. RESULTS: Pancreatic enzyme alteration was found in 24% of patients. Twelve had salmonellosis and 2 Campylobacter jejuni infection. They had more prolonged diarrhea, more frequent renal impairment and increased triglyceridemia. Triglyceridemia was correlated to blood amylase, inflammatory syndrome and renal impairment. Serum amylase was linked to serum urea and creatinine and to biological markers of inflammation. Three patients had true acute pancreatitis. CONCLUSION: Patients presenting dysentery-like infectious diarrhea and upper abdominal pain should be investigated for concomitant pancreatic reaction or acute pancreatitis which seems more frequent in patients with enterocolitis due to enteroinvasive microbes, mostly non-typhoidal Salmonella. Pancreatic disturbances are related to the severity of these infections. However, overt infectious diarrhea-associated pancreatitis is a rare event.

Abdominal Pain↗

PubMed2005

Extended half-life upon binding of destabilized intrabodies allows specific detection of antigen in mammalian cells.

The ectopic expression of antibody fragments inside mammalian cells (intrabodies) is a challenging approach for probing and modulating target activities. We previously described the shuttling activity of intracellularly expressed Escherichia coli beta-galactosidase conferred by the single-chain Fv (scFv) fragment 13R4 equipped with nuclear import/export signals. Here, by appending to scFvs the proteolytic PEST signal sequence (a protein region rich in proline, glutamic acid, serine and threonine) of mouse ornithine decarboxylase, we tested whether short-lived or destabilized intrabodies could affect the steady-state level of target by redirecting it to the proteasomes. In the absence of antigen, the half-life of the modified scFv 13R4, relative to untagged molecules, was considerably reduced in vivo. However, after coexpression with either cytoplasmic or nuclear antigen, the destabilized 13R4 fragments were readily maintained in the cell and strictly colocalized with beta-galactosidase. Analysis of destabilized site-directed mutants, that were as soluble as 13R4 in the intracellular context, demonstrated that binding to antigen was essential for survival under these conditions. This unique property allowed specific detection of beta-galactosidase, even when expressed at low level in stably transformed cells, and permitted isolation by flow cytometry from a transfected cell mixture of those living cells specifically labeled with bound intrabody. Altogether, we show that PEST-tagged intrabodies of sufficient affinity and solubility are powerful tools for imaging the presence and likely the dynamics of protein antigens that are resistant to proteasomal degradation in animal cells.

Active Transport, Cell Nucleus↗

PubMed2005

Targeted gene delivery to cancer cells with nanometric DNA particles enveloped with folic acid using a polymerisable anchor.

Progress in the design of gene delivery system is vital for cancer gene therapy since many physiological and intracellular barriers remain. We have developed a technology for condensing genes into nanometric delivery systems. In this paper, we present a novel strategy for decorating 30 nm DNA particles with folic acid for cancer cell recognition. Physicochemical and biological experiments show that these DNA complexes selectively bind to cells expressing the corresponding folic acid receptor.

Carrier Proteins↗

PubMed2005

Reverse pharmacognosy: application of selnergy, a new tool for lead discovery. The example of epsilon-viniferin.

The aim of reverse pharmacognosy is to find new biological targets for natural compounds by virtual or real screening and identify natural resources that contain the active molecules. To demonstrate the applicability of this concept, we report here a study on epsilon-viniferin, an active ingredient for cosmetic development. Nevertheless, this natural substance is weakly defined in terms of biological properties. SELNERGY, an inverse docking computer software, was used to identify putative binding biological targets for epsilon-viniferin. Among the 400 screened proteins two targets were retained. For cosmetic application, cyclic nucleotide phosphodiesterase 4 (PDE4) was the most interesting candidate. Moreover, other PDE subtypes (1, 2, 3, 5 and 6) were not retained, indicating a selectivity for PDE4. The experimental binding tests on the 6 subtypes of PDE revealed a significant selectivity of epsilon-viniferin for the PDE4 subtype. This selectivity was confirmed by evaluation of epsilon-viniferin on the secretion of TNF-alpha and Interleukin-8. Our data demonstrated that epsilon-viniferin possesses anti-inflammatory properties by inhibiting PDE4 subtype. In conclusion, reverse pharmacognosy and its inverse docking component cannot only be integrated into a program for new lead discovery but is also a useful approach to find new applications for identified compounds.

Benzofurans↗

PubMed2004

Celastrol inhibits pro-inflammatory cytokine secretion in Crohn's disease biopsies.

Crohn's disease is a chronic intestinal inflammatory process. In modern therapy, TNF-alpha inhibition is the main goal. The aim here is to characterize the effects of Celastrol, a pentacyclic-triterpene, on the secretion of inflammatory cytokines by LPS-activated human cells. Celastrol dose-dependently inhibited the secretion of all tested pro-inflammatory cytokines with IC(50) in the nanomolar range. Effect not related to glucocorticoid receptor activity is shown by competition experiments with the steroid antagonist RU486. Celastrol inhibited the pro-inflammatory cytokine secretion from mucosal inflammatory biopsies from Crohn's disease patients. Cytometry emphasized that for all tested pro-inflammatory cytokines, CD33(+) cells are the most sensitive. Quantitative-PCR and confocal analysis on a human monocytic cell line indicated that Celastrol acts at the transcriptional level by inhibiting LPS-induced NF-kappaB translocation. Celastrol might be a putative anti-inflammatory drug in the treatment of inflammatory diseases, given its inhibition of cytokine production by intestinal biopsies from Crohn's disease patients.

Cell Line↗

PubMed2004

The cyclin-dependent kinase inhibitor p21(cip1/waf1) enhances the cytotoxicity of ganciclovir in HSV-tk transfected ovarian carcinoma cells.

Suicide gene therapy could be an attractive addition to the treatment of ovarian carcinomas, for which acquired chemoresistance frequently results in treatment failure. Here we show that transfection of the HSV-tk gene, followed by incubation with up to 1 mM ganciclovir fails to induce cell death in SKOV3 chemoresistant human ovarian carcinoma cells. However, co-transfection of HSV-tk with Cip1/Waf1 encoding the p21(cip1/waf1) inhibitor of cdks, allows 100 microM ganciclovir to eradicate the population of tumor cells. Potentiation of a drug by co-transfer of HSV-tk with Cip1/Waf1could thus represent another therapeutic approach for tumours that are resistant to conventional therapy.

Adenocarcinoma↗

PubMed2004

Phosphorylation of ICBP90 by protein kinase A enhances topoisomerase IIalpha expression.

Inverted CCAAT box binding protein of 90kDa (ICBP90) is a nuclear protein involved in the topoisomerase IIalpha (TopoIIalpha) gene expression. It belongs to a family of E3 ligases of the RING finger type and its expression is deregulated in cancer cells. Previous studies have shown that high expression of ICBP90 may impair the control of G1/S transition of the cell cycle in various cancer cell lines. Since PKA signaling pathway is involved in G1/S transition of the cell cycle, the aim of the present study was to investigate whether cAMP signaling pathways involve phosphorylation of ICBP90. Here, we show that phosphorylation of ICBP90 through the cAMP signaling pathway accelerates exit of forskolin-treated cells from the G1 phase and increases binding of ICBP90 to the ICB2 element of the TopoIIalpha gene promoter with a subsequent increase of TopoIIalpha expression. We identify S298 of ICBP90 as target for PKA. We propose that cAMP signaling pathway enhances TopoIIalpha expression through ICBP90 phosphorylation, which may be one of the major events involved in the G1/S transition.

Amino Acid Sequence↗

PubMed2004

In vitro modulation of inflammatory cytokine production by three lipid emulsions with different fatty acid compositions.

BACKGROUND AND AIMS: Studies have suggested that 100% long-chain triacylglycerols (LCTs) lipid emulsions exhibit immunosuppressive effects, sometimes suspected to favor infectious complications in patients receiving parenteral nutrition. Newer emulsions, in particular olive oil-based emulsions, seem to have lesser immunosuppressive effects. We studied the in vitro effect of 100% LCTs (Intralipide), 50% LCTs-50% medium chain triacylglycerols (Medialipide), and 80% olive oil-based lipid emulsions (ClinOleic) on inflammatory cytokines production by peripheral blood mononuclear cells (PBMCs). METHODS: PBMCs separated by gradient centrifugation, or whole blood, were incubated with 0.001%, 0.01%, 0.1% and 1% of the three tested lipid emulsions during 24h in the presence or absence of activation by lipopolysaccharide and phytohemagglutinin. Then, supernatants were collected and cytokines measured (ELISA). RESULTS: The three lipid emulsions reduced basal TNF-alpha and IL-1beta production in PBMCs and whole blood cultures. However, ClinOleic was significantly less powerful in TNF-alpha and IL-1beta inhibition by isolated PBMCs than Intralipide and Medialipide. Basal TNF-alpha production was equally inhibited by the three emulsions in whole blood, but IL-1beta production was not significantly modified by ClinOleic. Interleukin-6 and -8 were not affected. After cell activation, lipid emulsions exhibit no effect on cytokines production. CONCLUSION: ClinOleic induces a significantly lower in vitro inhibition of TNF-alpha and IL-1beta production by PBMCs than 100% LCTs or 50% LCTs-50% MCTs emulsions, and therefore might be more immune neutral. These effects vary from one subject to another, and disappeared after cell activation. Therefore, caution must be taken before extrapolation in vivo. Provided information should be taken into account for future design of clinical trials studying the immune modulating properties of lipid emulsions.

Adult↗

PubMed2003

ICBP90 expression is downregulated in apoptosis-induced Jurkat cells.

Inhibition of apoptosis, resulting from an increase in anti-apoptotic protein, plays a fundamental role in carcinogenesis. Because ICBP90 gene expression is deregulated in cancer cells, we studied its expression in Jurkat cells under apoptotic conditions to see whether ICBP90 is involved in the regulation of apoptosis. We found that ICBP90 expression and the percentage of living cells were dose-dependently decreased in PHA and ionophore A23187-stimulated Jurkat cells, but not in THP-1 cells. These results suggest that apoptosis is dependent upon ICBP90 expression downregulation and that ICBP90 exhibits anti-apoptotic properties.

Apoptosis↗