Biomedical subjects

B Gabriel

Publications and source records attributed to B Gabriel.

At least 19 recordsLinked to original sources

PubMed2006

[Thieno[3,2-c]quinoline-4-yl-amines--synthesis and investigation of activity against malaria].

Thieno[3,2-c]quinoline-4-yl-amines - synthesis and investigation of activity against malaria pH-Dependant reduction of the methyl 2-(2-nitrophenyl)thiophene-3-carboxylate 3, formed by Suzuki coupling of methyl 2-iodothiophene-3-carboxylate (2) with 2-nitrophenylboronic acid, yielded the cyclic hydroxamic acid 4 and the lactam 5, respectively. The 4-chlorothieno[3,2-c]quinoline 6 was formed from the lactam 5 by heating with POCI3/PCI5s. Melting of 6 with the novaldiamine base in phenol gave the chloroquine analogue 7, whereas the amodiaquine and the pyronaridine analogues 8 and 9 were obtained using phenol Mannich bases. The reaction of 6 with putrescine and N,N'-bis(3-aminopropyl)piperazine as spacer formed the bisquinoline derivatives 10 and 11 as well as the monosubstituted quinoline 12. In the same manner the isomeric 4-chlorothieno[2,3-c]quinoline 13 reacted to yield the quinoline-4-yl-amines 14-16. The compounds 7-12 and 14-16 were tested for in vitro growth inhibition of the malaria parasite Plasmodium falciparum. As most active compound the pyronaridine derivative 9 displayed an IC50 value of 210 nM with the chloroquine sensitive P. falciparum strain 3D7 and 750 nM with the chloroquine resistant P. falciparum strain Dd2. The N,N'-bis(3-aminopropyl)piperazine derivative 11 displayed in vivo activity in Plasmodium vinckei infected mice with an ED50 value of 30 mg/kg after i.p. administration.

Amines↗

PubMed2006

[Thieno[3,4-c]quinoline-4-yl-amines--synthesis and investigation of activity against malaria].

The 4-aryl derivative 3, obtained by Suzuki cross coupling of the methyl 4-bromothiophene-3-carboxylate (2) with 2-nitrophenylboronic acid cyclizes under reductive conditions pH-dependant to yield the tricyclic hydroxamic acid 4 or the lactam 5. The chlorothieno[3,4-c]quinoline 6 was formed by reaction of the lactam 5 with P,P-dichlorophenylphosphinoxide. The amines 7-14 were synthesized from the chloroimine 6. Compounds 7a,b, 8, 9, 10b, 11, 12 and 14a, b were tested for in vitro antimalarial activity using the chloroquine sensitive 3D7 and the chloroquine resistant Plasmodium falciparum strain Dd2. The highest activity were shown by 10b with IC50 values of 130 nM and 50 nM, respectively and by 11 with IC50 values of 190 nM and 44 nM, respectively.

Aminoquinolines↗

PubMed2005

Specific induction of pp125 focal adhesion kinase in human breast cancer.

The pp125 focal adhesion kinase (FAK) is involved in integrin-mediated cell signalling and overexpressed in a variety of solid tumours. Focal adhesion kinase expression has been correlated to invasion and metastasis, but the data on breast cancer are inconclusive. We analysed FAK mRNA, protein levels and expression patterns in primary breast cancer and normal breast tissue. FAK expression on the functional protein level and mRNA was determined in 55 matched pairs of breast cancer and corresponding normal tissue by Western blot, immunohistochemistry and RT-PCR. Using a score ranging from 0 to +5 for Western blots, we determined in normal breast tissue a score of 1.51+/-0.84 (mean+/-standard deviation), which was strongly induced to 2.91 (+/-1.22) in breast cancers (P<0.001). Overall, 45 out of 55 tissue pairs (81.8%) showed this upregulation of FAK protein in tumours in comparison to normal tissue. Immunohistochemistry confirmed these findings with a significant higher score for tumours vs physiological tissue (1.0+/-0.63 vs 2.27+/-0.91; P=0.001). Interestingly, no overall significant difference in the mRNA levels (P=0.359) was observed. In conclusion, expression levels of the FAK protein are specifically upregulated in breast cancer in comparison to matched normal breast tissue supporting its pivotal role in neoplastic signal transduction and representing a potential marker for malignant transformation.

Biomarkers, Tumor↗

PubMed2005

Evaluation of patients after extraperitoneal lymph node dissection for cervical cancer.

OBJECTIVE: The presence of nodal metastases is an important prognostic factor in patients with cervical cancer. To adjust our therapy to the anatomic extent of the disease, we performed a surgical staging with extraperitoneal lymph node dissection (EPLND). The goal of our study was to evaluate the clinical outcome and side effects of the combined treatment approach of EPLND and either radical hysterectomy in case of early stage cervical cancer (FIGO Ia/b and IIa) and negative nodes, or pelvic radiotherapy/extended field radiotherapy with concomitant chemotherapy in case of positive nodes or advanced stage cervical cancer (FIGO IIb, III, and IVa). PATIENTS AND METHODS: Fifty-nine patients with primarily diagnosed invasive cervical cancer underwent EPLND. The value of this procedure as a diagnostic tool for evaluating the extent of disease was determined. Additionally, treatment-related complications and clinical outcomes were monitored. RESULTS: A total of 983 lymph nodes were removed during EPLND (mean 16.7). According to the results of EPLND, radical hysterectomy was abandoned due to histopathologically confirmed lymph node involvement by frozen section in 11 out of 36 patients with early stage cervical cancer (31%). The most common adverse effects directly related to surgery in general (EPLND or combined EPLND and radical hysterectomy) were lymph cysts in seven patients (12%). Only in the group of patients who received EPLND followed by radical hysterectomy, 2 out of 25 patients (8%) developed a severe ileus postoperatively (WHO Grade 3 toxicity). The treatment approach of combined EPLND followed by radio- and chemotherapy was without major complications (WHO Grade 3 or 4 toxicity). After a mean follow up of 28 months (range 6-60), 44 out of 58 patients (one patient lost to follow up) are without evidence of disease (76%), 2 patients have progressive disease (3%), and 12 patients died of their disease (21%). Using Kaplan-Meier analysis, the estimated 5-year overall survival rate for all patients is 64% (SD +/- 9%). Performing the Cox proportional regression analysis, in contrast to clinical FIGO staging (P = 0.24; ns), lymph node involvement was the only significant independent predictor for overall survival (P = 0.04). CONCLUSION: Our data support the approach of pretherapeutic surgical staging by performing EPLND as a diagnostic tool with a low complication rate. This allows an individualized treatment for cervical cancer patients.

Adult↗

PubMed2004

Osmotically induced membrane tension facilitates the triggering of living cell electropermeabilization.

Very little is known about the molecular mechanisms supporting living cell membrane electropermeabilization. This concept is based on the local membrane permeability induced by cell exposure to brief and intense external electric field pulses. During the electric field application, an electro-induced membrane electric potential difference is created that is locally associated with the dielectric properties of the plasma membrane. When the new membrane electric potential difference locally reaches a critical value, a local alteration of the membrane structure is induced and leads to reversible permeabilization. In our study, we attempted to determine whether mechanical tension could modulate the triggering of membrane electropermeabilization. Change in lateral tension of Chinese Hamster Ovary cell membrane has been osmotically induced. Cell electropermeabilization was performed in the minute time range after the osmotic stress, i.e., before the regulatory volume decrease being activated by the cell. Living cell electropermeabilization was analyzed on cell population using flow cytometry. We observed that electropermeabilization triggering was significantly facilitated when the lateral membrane tension was increased. The main conclusion is that the critical value of transmembrane potential needed to trigger membrane electropermeabilization, is smaller when the membrane is under lateral mechanical constraint. This supports the hypothesis that both mechanical and electrical constraints play a key role in transient membrane destabilization.

Animals↗

PubMed2004

Optical imaging of in vivo gene expression: a critical assessment of the methodology and associated technologies.

Following and quantifying the expression of reporter gene expression in vivo is very important to monitor the expression of therapeutic genes in targeted tissues in disease models and/or to assess the effectiveness of systems of gene therapy delivery. Gene expression of luminescent or fluorescent proteins can be detected directly on living animals by simply observing the associated optical signals by means of a cooled charged-coupled device camera. More accurate resolution can be obtained with more sophisticated technologies. Time-course and quasi-quantitative monitoring of the expression can be obtained on a given animal and followed on a large time window. The present paper describes the physical and technological methodologies and associated problems of in vivo optical imaging. Several examples of in vivo detection of gene delivery are described.

Animals↗

PubMed2004

[Thieno[2,3-c]quinolines - synthesis and biological investigation].

pH-Dependant reduction of the methyl 3-(2-nitrophenyl)thiophene-2-carboxylate (3), obtained by Suzuki cross-coupling of the methyl 3-iodothiophene-2-carboxylate with 2-nitrophenyl boronic acid yields the cyclic hydroxamic acid 4 and the lactam 5, respectively. The lactam 5 is also formed by reacting the compound 2 with pinacolato 2-aminophenylboronate. The 4-chlorothieno[2,3-c]quinoline 6 is formed from the lactam 5 by heating with POCl3/PCl5. Melting of 6 with the novaldiamine base in phenol gives the chloroquine analogue 7, whereas the amodiaquine and the cycloquine analogues 8 and 9 are obtained using phenol Mannich bases. The hydroxamic acid 4 has a moderate effect on eicosanoid biosynthesis in human whole blood. The growth of the chloroquine resistent Plasmodium falciparum strain Dd2 is inhibited by the pyronaridine derivative 9 with an IC50 value of 650 nM.

Animals↗

PubMed2003

[Calcium and electropermeabilized cells].

Trains of short and intense electric pulses may induce a reversible local permeabilization on the membrane of the treated cells. Hydrophilic species can then almost freely cross the envelope and either enter or escape from the cytoplasm. The purpose of the present study was to investigate the possibility of introducing well defined amounts of Ca2+ ions within the cell. Chinese hamster ovary cells were used as a model system. When the pulsing buffer contained high levels of free Ca2+, the survival of cells was strongly affected. A 1 mM level was well tolerated. When cells were pulsed under moderated field conditions, it was observed that Ca2+ entered cells very rapidly (second time range). But the basic cytoplasmic level was set back spontaneously within a few minutes. The perspectives of this electrical injection are discussed for basic cell biology and high-throughput biotechnology.

Animals↗

PubMed2002

Molecular mechanisms in signal transduction: new targets for the therapy of gynecologic malignancies.

Growth factor-mediated signal transduction pathways in gynecologic malignancies are summarized. Targeting critical tyrosine kinase pathways may lead to more specific anticancer regimens in gynecologic oncology. During the past 10 years significant progress in cancer treatment has been made through the discovery of potent specific and well-tolerated inhibitors of signal transduction. Improved understanding of molecules involved in signal transduction pathways will undoubtedly result in an increasing number of compounds under clinical investigation. Some of the described molecular therapeutic approaches are suitable to enrich the conventional chemotherapy.

Animals↗

PubMed2002

Factors controlling electropermeabilisation of cell membranes.

Electric field pulses are a new approach for drug and gene delivery for cancer therapy. They induce a localized structural alteration of cell membranes. The associated physical mechanisms are well explained and can be safely controlled. A position dependent modulation of the membrane potential difference is induced when an electric field is applied to a cell. Electric field pulses with an overcritical intensity evoke a local membrane alteration. A free exchange of hydrophilic low molecular weight molecules takes place across the membrane. A leakage of cytosolic metabolites and a loading of polar drugs into the cytoplasm are obtained. The fraction of the cell surface which is competent for exchange is a function of the field intensity. The level of local exchange is strongly controlled by the pulse duration and the number of successive pulses. The permeabilised state is long lived. Its lifetime is under the control of the cumulated pulse duration. Cell viability can be preserved. Gene transfer is obtained but its mechanism is not a free diffusion. Plasmids are electrophoretically accumulated against the permeabilised cell surface and form aggregates due to the field effect. After the pulses, several steps follow: translocation to the cytoplasm, traffic to the nucleus and expression. Molecular structural and metabolic changes in cells remain mostly poorly understood. Nevertheless, while most studies were established on cells in culture (in vitro), recent experiments show that similar effects are obtained on tissue (in vivo). Transfer remains controlled by the physical parameters of the electrical treatment.

Animals↗

PubMed2001

[Calcium oscillations induced by lindane in peritoneal macrophages of mice: control by the maturation stage of the macrophage].

Mouse resident peritoneal macrophages loaded with Fluo-3 were examined for changes in cytosolic calcium concentration ([Ca2+]i) after stimulation with gamma-hexachlorocyclohexane (Lindane or gamma-HCCH). These studies, realized on macrophage populations, or single cells, by digital imaging microscopy, sought to determine the role of calcium influx on cyclical changes according to maturation stages of macrophages. Single cell analysis of [Ca2+]i changes in macrophages, after gamma-HCCH exposure in 600 microM extracellular calcium, demonstrated that: 1) these [Ca2+]i variations were asynchronous oscillations with the same frequency (1.7 min-1), and 2) these [Ca2+]i variations in macrophages were not at the same [Ca2+]i level. This heterogeneity could be correlated to a cell size partition of the macrophage population (10.1 +/- 0.44 and 11.45 +/- 0.43 microns). In the presence of 100 microM calcium, gamma-HCCH induced a calcium influx into the two subpopulations, but the calcium oscillations appeared only in small macrophages. In the largest ones, [Ca2+]i slowly decreased back down to the basal level. The cell size variation could be correlated to a phenotypic heterogeneity, linked to the differenciation stage of the cell. Peroxydase activity showed that small macrophages were in fact exudate macrophages and the largest ones were resident macrophages. Inhibition of the oscillatory patterns by a decrease in the extracellular calcium concentration ([Ca2+]ext) or by lanthanum chloride (LaCl3) addition is indicative of the important role of calcium influx in the triggering of oscillations. The calcium influx was transient and induced inositol phosphate (InsP3) production in macrophages. The maintainance of these calcium oscillations depended on calcium mobilization from intracellular calcium stores by InsP3, since neomycin and 8-(diethylamino) octyl 3,4,5-trimethoxybenzoate (TMB-8) abolished the oscillations. gamma-HCCH induced a transient calcium entry which triggered phospholipase C activation and the associated [Ca2+]i oscillations. However, we showed that differences in cell responses were observed in relationship with the differentiation stage of the mouse peritoneal macrophages, and with the extracellular calcium concentration.

Aniline Compounds↗

PubMed1999

The generation of reactive-oxygen species associated with long-lasting pulse-induced electropermeabilisation of mammalian cells is based on a non-destructive alteration of the plasma membrane.

Chinese hamster ovary (CHO) cells in suspension were subjected to pulsed electric fields suitable for electrically mediated gene transfer (pulse duration longer than 1 ms). Using the chemiluminescence probe lucigenin, we showed that a generation of reactive-oxygen species (oxidative jump) was present when the cells were electropermeabilised using millisecond pulses. The oxidative jump yield was controlled by the extent of alterations allowing permeabilisation within the electrically affected cell area, but showed a saturating dependence on the pulse duration over 1 ms. Cell electropulsation induced reversible and irreversible alterations of the membrane assembly. The oxidative stress was only present when the membrane permeabilisation was reversible. Irreversible electrical membrane disruption inhibited the oxidative jump. The oxidative jump was not a simple feedback effect of membrane electropermeabilisation. It strongly controlled long-term cell survival. This had to be associated with the cell-damaging action of reactive-oxygen species. However, for millisecond-cumulated pulse duration, an accumulation of a large number of short pulses (microsecond) was extremely lethal for cells, while no correlation with an increased oxidative jump was found. Cell responses, such as the production of free radicals, were present during electropermeabilisation of living cells and controlled partially the long-term behaviour of the pulsed cell.

Acridines↗

PubMed1999

Electropermeabilization of cell membranes.

A position dependent modulation of the membrane potential difference is induced when an electric field is applied to a cell. When cells are submitted to short lived electric field pulses with an overcritical intensity, a local membrane alteration is induced, which may reseal. Its molecular definition remains unknown. A free exchange of hydrophilic molecules takes place across the membrane. A leakage of cytosolic metabolites is present. However, a loading of polar drugs into the cytoplasm is obtained. A short description of the processes affecting the cell membrane organization is given. Lipids appear as the primary target of the field effect as in the case of liposomes. Nevertheless membrane proteins appear to be affected by a direct or by a back effect. The permeabilized state is long lived. The cell metabolism plays indeed a critical role in the recovery. The cell viability can be nevertheless preserved.

Journal Article↗

PubMed1999

Time courses of mammalian cell electropermeabilization observed by millisecond imaging of membrane property changes during the pulse.

Time courses of electropermeabilization were analyzed during the electric field application using a rapid fluorescent imaging system. Exchanges of calcium ions through electropermeabilized membrane of Chinese hamster ovary cells were found to be asymmetrical. Entry of calcium ions during a millisecond pulse occurred on the anode-facing cell hemisphere. Entry through the region facing the cathode was observed only after the pulse. Leakage of intracellular calcium ions from electropermeabilized cell in low-calcium content medium was observed only from the anode-facing side. The exchanges during the pulse were mostly due to diffusion-driven processes, i.e., governed by the concentration gradient. Interaction of propidium iodide, a dye sensitive to the structural alteration of membrane, with cell membrane was asymmetrical during electropermeabilization. Localized enhancement of the dye fluorescence was observed during and after the pulsation on the cell surface. Specific staining of a limited anode-facing part of the membrane was observed as soon as the pulse was applied. The membrane fluorescence level increased during and immediately after the pulse whereas the geometry of the staining was unchanged. The membrane regions stained by propidium iodide were the same as those where calcium exchanges occurred. The fraction of the membrane on which structural alterations occurred was defined by the field strength. The density of defects was governed by the pulse duration. Electropermeabilization is a localized but asymmetrical process. The membrane defects are created unequally on the two cell sides during the pulse, implying a vectorial effect of the electric field on the membrane.

Animals↗

PubMed1999

N-linked oligosaccharides can protect target cells from the lysis mediated by NK cells but not by cytotoxic T lymphocytes: role of NKG2-A.

We have previously shown that glycophorin A (GPA), inserted by electropulsation into the membrane of K562 cells, protected them from natural killer (NK) cell-mediated cytotoxicity and the unique N-linked oligosaccharide of GPA was essential for resistance to occur. The present study demonstrates that the protection level conferred by GPA is similar to the resistance induced by HLA-Cw3 expressed by transfected K562 cells. A monoclonal antibody against NKG2-A, an NK inhibitory receptor interacting with HLA class I antigens and belonging to the C-type lectin receptor, was able to restore the ability of NK cells to lyse K562 cells expressing HLA-Cw3 at the cell membrane but not electroinserted-GPA, suggesting that the N-linked oligosaccharide of GPA cannot be a ligand for NKG2-A. GPA was then electroinserted into the membrane of two lymphoblastoid B-cell lines: one was sensitive to NK cell-mediated lysis, the other was susceptible to cytotoxic CD8+ T-lymphocyte (CTL)-mediated cytotoxicity. The electroinserted GPA protected the target cells from NK-mediated cytotoxicity, whereas it did not modify the cell susceptibility to lysis by CTL. Endoglycosidase F treatment abolished the resistance towards NK cell-mediated lysis, suggesting that N-linked glycans could inhibit mechanisms used by NK cells to exert their cytotoxic function in agreement with our previous results.

Carbohydrate Conformation↗

PubMed1998

Design of benzamidine-type inhibitors of factor Xa.

A series of derivatives of rac-benzenesulfonyl-glycyl-phenylalanine or its ethyl ester with a combination of thioamido/amidino or amidino/amidino substituents in the benzene rings was synthesized as potential inhibitors of factor Xa (fXa). Among these, the racemic 4'-amidinobenzenesulfonyl-glycyl-4-amidinophenylalanine ethyl ester was found to exhibit the highest affinity for fXa despite the unfavored location of the amidino substituent in the para position. X-ray structural analysis of the trypsin complex with this bis-benzamidine compound revealed a retro-binding mode if compared to those of similar compounds, so far analyzed in complexes with trypsin or fXa. This noncanonical binding mode as well as its slow plasma clearance rates in rats, if compared to those of other benzamidine derivatives, suggests this compound as an interesting new lead structure for the design of fXa inhibitors.

Animals↗

PubMed1998

Focal adhesion kinase pp125FAK and the beta 1 integrin subunit are constitutively complexed in HaCaT cells.

Binding of integrins to the extracellular matrix (ECM) activates various signal transduction pathways and regulates gene expression in many cell types. Integrin-dependent cytoplasmic protein/protein interactions are necessary for activation of those signal transduction cascades. In our studies we investigated a possible association of pp125FAK, an adhesion involved tyrosine kinase, with the integrin beta 1 subunit. Further we wanted to know to which extent protein tyrosine phosphorylation affects cell adhesion to the ECM and the possible beta 1 integrin/pp125FAK complex. We were able to show that in HaCaT cells (a human keratinocyte derived cell line) the integrin beta 1 subunit is associated with tyrosine kinase pp125FAK. This association was observed in ECM-adherent cells and nonadherent cells and is independent of tyrosine phosphorylation. However, cell adhesion of HaCaT cells to specific substrates requires tyrosine phosphorylation since genistein treatment that blocks phosphorylation of many cellular proteins as pp125FAK led to a reduced substrate adhesion.

Carcinoma, Squamous Cell↗

PubMed1997

Direct observation in the millisecond time range of fluorescent molecule asymmetrical interaction with the electropermeabilized cell membrane.

Interaction of two stains (propidium iodide and ethidium bromide) with electropermeabilized living Chinese hamster ovary cells is observed using an ultrafast fluorescence image acquisition system. The computing process is linked to an ultra-low-light intensifying camera working with a very short time resolution (3.33 ms per image). Altered parts of the cell membrane were identified via the enhancement in fluorescence intensity of the dyes. They reflect the electropermeabilized part of the membrane in which free flow of dye occurred. Images of the fluorescence interaction patterns of the two dyes, in a maximum 20-ms time lag after pulsation, reveal asymmetrical permeabilization of the cell membrane. For electric field intensities higher than a first threshold value, permeabilization is always observed on the anode-facing side of the cell. For electric field intensities over a second higher threshold value, the two electrode-facing hemispheres of the cell are permeabilized, the hemisphere facing the anode being most permeable. These data support the conclusion that electropermeabilization of living cell membrane is affected by its resting potential. The asymmetrical pattern of the dye interaction is not dependent on the nature or concentration of the dye, the ionic strength of the pulsing buffer, or the duration of the pulse. The field intensity determines the fraction of the membrane in which molecular alterations can occur. The extent of alteration in this localized region is determined by the duration of the pulse when a single pulse in the millisecond time range is applied.

Animals↗