Biomedical subjects

E Rousseau

Publications and source records attributed to E Rousseau.

At least 19 recordsLinked to original sources

PubMed2005

Microarray profiling of isolated abdominal subcutaneous adipocytes from obese vs non-obese Pima Indians: increased expression of inflammation-related genes.

AIMS/HYPOTHESIS: Obesity increases the risk of developing major diseases such as diabetes and cardiovascular disease. Adipose tissue, particularly adipocytes, may play a major role in the development of obesity and its comorbidities. The aim of this study was to characterise, in adipocytes from obese people, the most differentially expressed genes that might be relevant to the development of obesity. METHODS: We carried out microarray gene profiling of isolated abdominal subcutaneous adipocytes from 20 non-obese (BMI 25+/-3 kg/m2) and 19 obese (BMI 55+/-8 kg/m2) non-diabetic Pima Indians using Affymetrix HG-U95 GeneChip arrays. After data analyses, we measured the transcript levels of selected genes based on their biological functions and chromosomal positions using quantitative real-time PCR. RESULTS: The most differentially expressed genes in adipocytes of obese individuals consisted of 433 upregulated and 244 downregulated genes. Of these, 410 genes could be classified into 20 functional Gene Ontology categories. The analyses indicated that the inflammation/immune response category was over-represented, and that most inflammation-related genes were upregulated in adipocytes of obese subjects. Quantitative real-time PCR confirmed the transcriptional upregulation of representative inflammation-related genes (CCL2 and CCL3) encoding the chemokines monocyte chemoattractant protein-1 and macrophage inflammatory protein 1alpha. The differential expression levels of eight positional candidate genes, including inflammation-related THY1 and C1QTNF5, were also confirmed. These genes are located on chromosome 11q22-q24, a region with linkage to obesity in the Pima Indians. CONCLUSIONS/INTERPRETATION: This study provides evidence supporting the active role of mature adipocytes in obesity-related inflammation. It also provides potential candidate genes for susceptibility to obesity.

Abdomen↗

PubMed2005

Increased expression of inflammation-related genes in cultured preadipocytes/stromal vascular cells from obese compared with non-obese Pima Indians.

AIMS/HYPOTHESIS: The specific contributions made by the various cell types in adipose tissue to obesity, particularly obesity-related inflammation, need to be clarified. The aim of this study was to elucidate the potential role of adipocyte precursor cells (preadipocytes/stromal vascular cells [SVC]). METHODS: We performed Affymetrix oligonucleotide microarray expression profiling of cultured abdominal subcutaneous preadipocytes/SVC isolated from the adipose tissue of 14 non-obese (BMI 25+/-4 kg/m2) and 14 obese (55+/-8 kg/m2) non-diabetic Pima Indian subjects. Quantitative real-time PCR (RT-PCR) was used to verify the differential expression of several genes in an independent group of subjects. RESULTS: We identified 218 differentially expressed genes with p values less than 0.01. Microarray expression profiling revealed that the expression of inflammation-related genes was significantly upregulated in preadipocytes/SVC of obese individuals. Quantitative RT-PCR confirmed the upregulation of IL8, CTSS, ITGB2, HLA-DRA, CD53, PLA2G7 and MMP9 in preadipocytes/SVC of obese subjects. CONCLUSIONS/INTERPRETATION: The upregulation of inflammation-related genes in preadipocytes/SVC of obese subjects may increase the recruitment of immune cells into adipose tissue and may also result in changes in the extracellular matrix (tissue remodelling) to accommodate adipose tissue expansion in obesity.

Adipocytes↗

PubMed2004

Capsazepine, a vanilloid antagonist, abolishes tonic responses induced by 20-HETE on guinea pig airway smooth muscle.

The aim of this study was to delineate the mode of action of 20-hydroxy-eicosatetraenoic acid (20-HETE) in airway smooth muscle (ASM) cells. ASM metabolizes arachidonic acid by various enzymatic pathways, including the cytochrome P-450 (CYP-450) omega-hydroxylase, which leads to the production of 20-HETE, a bronchoconstrictive eicosanoid. The present study demonstrated that 20-HETE induced concentration-dependent tonic responses in ASM, whereas transient responses were recorded in Ca2+-free solution, suggesting an intracellular Ca2+ release process. 20-HETE inotropic responses were abolished by 36 microM 2-aminoethoxydiphenyl borate or 1 microM thapsigargin but were insensitive to 10 microM ryanodine, indicating that inositol triphosphate receptors likely control the release of intracellular Ca2+. Sustained tension, which required Ca2+ entry, was partially blocked by 1 microM nifedipine (an L-type) and 100 microM Gd3+ (a nonselective cationic channel blocker). Moreover, in the absence of selective 20-HETE receptor antagonists, 20-HETE tonic responses were inhibited in a concentration-dependent manner (0.1-10 microM) by capsazepine, a well-characterized vanilloid receptor antagonist. Capsazepine was also observed to reverse cumulative responses to 20-HETE and capsaicin, a TRPV1 agonist. In addition, capsazepine pretreatment largely modified the sustained inotropic responses to 20-HETE, suggesting that 20-HETE cross-reacted with TRPV1 receptors with a low affinity (microM) or that its specific receptor was inhibited by the vanilloid antagonist. Data obtained using RHC-80267, ONO-RS-082, and eicosatetraynoic acid, respective inhibitors of diacylglycerol-lipase, phospholipase A2, and CYP-450 omega-hydroxylase, reveal that intracellular arachidonic acid production and its 20-HETE metabolite may be responsible for the activation of nonselective cationic channels and tonic responses.

5,8,11,14-Eicosatetraynoic Acid↗

PubMed2003

Effect of sildenafil on cyclic nucleotide phosphodiesterase activity, vascular tone and calcium signaling in rat pulmonary artery.

(1) Sildenafil (viagra) is a potent PDE5 inhibitor and thus a relaxant drug in corpus carvernosum smooth muscle. In the present work, we evidenced the presence of PDE5 isozyme and investigated the effect of sildenafil on the specific cyclic nucleotide phosphodiesterase (PDE) activity, smooth muscle tone and calcium signaling in the rat main pulmonary artery (MPA). (2) The PDE activity was measured in cytosolic and microsomal fractions. Total cAMP and cGMP-PDE activities were mainly present in the cytosolic fraction. Sildenafil (0.1 micro M) reduced by 72% cGMP-PDE activity, whereas zaprinast (10 micro M), a relatively selective PDE5 inhibitor, reduced this activity by 63%. Sildenafil (0.1 micro M) also inhibited significantly (22%) the cAMP-PDE activity. (3) Western blot analysis revealed the expression of PDE5 mainly in the cytosolic fraction of MPA. Sildenafil concentration-dependently inhibited (IC(50)=3.4 nM) the activity of MPA PDE5 partially purified by HPLC. (4) Sildenafil (0.1 nM-50 micro M) concentration-dependently relaxed MPA rings precontracted with phenylephrine (0.5 micro M). The potency of sildenafil (IC(50)=11 nM) was similar to that of a nitric oxide donor, sodium nitroprusside, but higher than that of zaprinast (IC(50)=600 nM). The vasorelaxant effect of sildenafil was not altered by endothelium removal or in the presence of KT 5823 (1 micro M) and H89 (1 micro M), potent inhibitors of PKG and PKA, respectively. (5) In isolated MPA myocytes, which had been loaded with the calcium fluorophore indo-1, sildenafil (10-100 nM) antagonized ATP- and endothelin-1-induced calcium oscillations but had no effect on the transient caffeine-induced [Ca(2+)](i) response. (6) This study demonstrates the presence of a functional and highly sildenafil-sensitive PDE5 isozyme in rat MPA. Inhibition of this isozyme mainly accounts for the potent pulmonary vasodilator action of sildenafil, which involves alteration in the inositol triphosphate-mediated calcium signaling pathway.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2003

CDKN2A, CDKN2B and p14ARF are frequently and differentially methylated in ependymal tumours.

Ependymal tumours are histologically and clinically varied lesions. Numerical abnormalities of chromosome 9 are frequently associated with these tumours. Nevertheless, the three important tumour suppressor genes located in this chromosome, CDKN2A, CDKN2B and p14 ARF, have not been reported to be commonly altered in them. We studied promoter methylation of these genes, an important mechanism associated with gene silencing in a series of 152 ependymal tumours of WHO grades I to III. Methylation status of the CDKN2A, CDKN2B and p14 ARF promoters was assessed by methylation-specific polymerase chain reaction and the genetic results were correlated to clinicopathological features. We observed promoter methylation for CDKN2A in 21% (26/123) of tumours, for CDKN2B in 32% (23/71) and p14 ARF in 21% (23/108). For all three genes, posterior fossa ependymomas were less frequently methylated in paediatric patients than in adults. For CDKN2B, extracranial tumours were more frequently methylated than intracranial ones. For CDKN2B and p14 ARF, methylation was more frequent in low-grade tumours; the reverse was observed for CDKN2A. CDKN2A, CDKN2B and p14 ARF promoters were methylated in 21-32% of the tumours. Frequencies of methylation varied according to clinicopathological features. This suggests a role for these genes in ependymoma tumorigenesis.

Adolescent↗

PubMed2003

Tumour necrosis and microvascular proliferation are associated with 9p deletion and CDKN2A alterations in 1p/19q-deleted oligodendrogliomas.

A subset of oligodendrogliomas and oligoastrocytomas has been associated with 1p/19q deletion. Subsequently, this genetic alteration was linked to chemosensitivity and classic histology of oligodendrogliomas. Tumoural progression includes deletions of 9p, 10q and alterations of CDKN2A. However, these (epi)genetic changes have not been associated with specific histological features. In a series of 45 gliomas including oligodendrogliomas, oligoastrocytomas and astrocytomas, deletions of chromosomal regions implied in these tumours (1p, 9p, 10, 17p13, 19q and 22) were looked for by microsatellite analysis. Tumours that were deleted for 1p and 19q were selected. Subsequently, presence of deletions in the other studied regions, (epi)genetic changes in p14ARF, CDKN2A and CDKN2B, as well as histological features, were associated to these tumours. 1p/19q deletion was observed in 22 tumours. Twenty-one of them presented regions of classic histology of oligodendroglioma. A deletion of 9p was found in eight of them, always in association with tumour necrosis and/or microvascular proliferation. In addition, (epi)genetic alterations of CDKN2A were observed in 71% of these tumours. Presence of regions of classic histology of oligodendroglioma in a tumour sample is predictive of 1p/19q deletions. Necrosis and/or microvascular proliferation are signs of an additional 9p deletion. Finally, as CDKN2A (epi)genetic alterations were found in 71% of the 1p/19q/9p-deleted oligodendrogliomas, CDKN2A may have a role in oligodendroglioma-associated microvascular proliferation.

Central Nervous System Neoplasms↗

PubMed2003

[Surgical treatment of genital prolapse with a new lateral prosthetic hysteropexia technique combining vaginal and laparoscopic methods].

We describe a new surgical treatment of pelvic organ prolapse. The anterior and medium compartments are treated by a transversal hysteropexy by means of an anterior prosthesis fixed by vaginal route and by laparoscopy. The principle is based on the operative technique described by Kapandji but the prosthesis is fixed to the fascia of the external oblique muscle in a subperitoneal path. The posterior compartment is treated by a vaginal route exclusively. A posterior prosthesis is placed in the rectovaginal space and fixed to the elevator muscles. Further studies are necessary to evaluate this new operative technique.

Colposcopy↗

PubMed2002

Characterisation of cyclic nucleotide phosphodiesterase isoforms in the media layer of the main pulmonary artery.

Cyclic nucleotides are involved in the control of pulmonary vascular tone. In the present study, we measured the cyclic nucleotide specific phosphodiesterase (PDE) activity in the media of bovine isolated main pulmonary artery (MPA). Total cAMP- and cGMP-PDE activities were measured in microsomal and cytosolic fractions. Both cyclic nucleotides were hydrolysed in these subcellular fractions at consistently higher rate in the cytosolic than in the microsomal fraction. Using different classes of PDE modulator, at least four PDE isoforms (PDE1, 3, 4 and 5) were identified in these fractions. PDE3 (cilostamide-sensitive), PDE4 (rolipram-sensitive) and PDE5 (zaprinast- and DMPPO-sensitive) isoforms appeared as the main isozymes implicated in the cAMP and cGMP hydrolytic activities. Calcium-camodulin stimulated PDE activity (PDE1) was mainly present in the cytosolic fraction. PDE2, although present, had a lower hydrolytic activity since addition of its specific inhibitor, erythro-9-(2-hydroxy-3nonyl)adenine (EHNA), to a combination of inhibitors of PDE3, 4 and 5 produced no further significant reduction in the enzymatic activity. Resolution of PDE activities from the cytosolic fraction using anion exchange chromatography confirmed this finding. Functional experiments performed in endothelium-denuded rings of rat MPA revealed that all specific PDE inhibitors used relaxed precontracted vascular smooth muscle preparations in a concentration-dependent manner. The rank order of potency was cilostamide >zaprinast>rolipram>>EHNA. The present study demonstrates the presence in the smooth muscle cells-containing layer of MPA of PDE1, 3, 4 and 5 isoforms and suggests that PDE3, 4 and 5 are the main enzymes involved in the control of vascular tone.

1-Methyl-3-isobutylxanthine↗

PubMed2001

The gene structure of the Drosophila melanogaster homolog of the human proto-oncogene fos.

The Drosophila melanogaster homolog of the human proto-oncogene fos is Dfos. It is the only fos homolog in the Drosophila genome. Fos functions as a subunit of the heterodimeric transcription factor AP-1. There are two models of the Dfos gene. The first comes from a cDNA sequence of Dfos (Perkins et al., Genes Dev. 4 (1990) 822). The second is from the gene sequence published by the Drosophila genome project (Adams et al., Science 287 (2000) 2185), and there are notable contradictions between the two models. The promoter and the 5' end of the transcript sequence were not identified in either model. In this paper, we present the gene structure of Dfos and identify the promoter. This promoter has an initiator and a downstream promoter element sequence, but lacks a TATA box. Through comparison of the mRNA and genomic DNA sequences, three introns varying in length from 66 bp to 17.57 kb were found and verified by RT-PCR. The Dfos gene is 21.2 kb in length, giving a transcript of 3438 bp, coding for a predicted protein of 595 amino acids. The 3' untranslated region is confirmed to be 1092 bp in length.

3' Untranslated Regions↗

PubMed2001

EETs relax airway smooth muscle via an EpDHF effect: BK(Ca) channel activation and hyperpolarization.

Epoxyeicosatrienoic acids (EETs) are produced from arachidonic acid via the cytochrome P-450 epoxygenase pathway. EETs are able to modulate smooth muscle tone by increasing K(+) conductance, hence generating hyperpolarization of the tissues. However, the molecular mechanisms by which EETs induce smooth muscle relaxation are not fully understood. In the present study, the effects of EETs on airway smooth muscle (ASM) were investigated using three electrophysiological techniques. 8,9-EET and 14,15-EET induced concentration-dependent relaxations of the ASM precontracted with a muscarinc agonist (carbamylcholine chloride), and these relaxations were partly inhibited by 10 nM iberiotoxin (IbTX), a specific large-conductance Ca(2+)-activated K(+) (BK(Ca)) channel blocker. Moreover, 3 microM 8,9- or 14,15-EET induced hyperpolarizations of -12 +/- 3.5 and -16 +/- 3 mV, with EC(50) values of 0.13 and 0.14 microM, respectively, which were either reversed or blocked on addition of 10 nM IbTX. These results indicate that BK(Ca) channels are involved in hyperpolarization and participate in the relaxation of ASM. In addition, complementary experiments demonstrated that 8,9- and 14,15-EET activate reconstituted BK(Ca) channels at low free Ca(2+) concentrations without affecting their unitary conductance. These increases in channel activity were IbTX sensitive and correlated well with the IbTX-sensitive hyperpolarization and relaxation of ASM. Together these results support the view that, in ASM, the EETs act through an epithelium-derived hyperpolarizing factorlike effect.

8,11,14-Eicosatrienoic Acid↗

PubMed2001

Functional properties of the native type 3 ryanodine receptor Ca2+-release channel from canine diaphragm.

mRNA and protein analyses have previously shown that the diaphragm expresses two ryanodine receptor isoforms: RyR1 and RyR3. RyR1 is the main Ca2+-releasing pathway in this muscle type. We now report the conducting, gating, and immunological properties of the native and purified forms of the less abundant RyR3 channel. The conductance of this native Ca2+-release channel was 330 pS in 50 mM/250 mM trans/cis CsCH3SO3. It was activated by Ca2+ concentrations of 1-1000 microM, and did not inactivate at mM concentrations of Ca2+. Both isoforms were purified by either a sucrose density gradient or immunoprecipitation as > 450 kDa proteins on SDS-PAGE. Western blot analysis confirmed the presence of RyR1 and RyR3, which displayed conductances of 740 +/- 30 and 800 +/- 25 pS, respectively, in 250 mM KCl. We thus provide evidence that one form of the diaphragm SR Ca2+-release channels may be classified as RyR3, with gating properties different from those of the well-characterized RyR1 and RyR2 isoforms.

Animals↗

PubMed2000

Characterization and immunohistochemical localization of nucleoside triphosphate diphosphohydrolase (NTPDase) in pig adrenal glands (presence of a non-sedimentable isoform).

Considering that adrenal glands possess a variety of purinoceptors associated with various cell types and that some of these cells (chromaffin cells) secrete large amounts of adenine nucleotides, it was of interest to localize nucleoside triphosphate diphosphohydrolase (NTPDase) in these glands and to define the biochemical characteristics of this ectonucleotidase. Immunolocalization produced a moderate reaction in capsula and medulla, with no signal in zona glomerulosa and zona reticularis. In contrast, a very strong reaction was found in zona fasciculata. Biochemical analysis of particulate fractions isolated from whole glands revealed high levels of ATPase and ADPase activities. This appeared to be attributable to the NTPDase since the level of ADPase was as high as ATPase. Both ATPase and ADPase activities were similarly inhibited by sodium azide. Additionally electrophoretograms with these two substrates showed comparable patterns. Western blots with 'Ringo', an antibody that recognizes the different isoforms of mammalian NTPDases, showed the presence of isoforms of NTPDases at 54 and 78 kDa, respectively. Interestingly, the 54 kDa isoform remains in the supernatant of a chromaffin granule lysate after ultracentrifugation. Up until now little interest has been given to the relationship between adrenal medulla and cortex. Presence of purinoceptors and ectonucleotidases in both these regions together with the effects of ATP in vivo and in vitro in different species indicate that purines play a significant role in adrenal glands.

Acid Anhydride Hydrolases↗

PubMed2000

Patch-clamp study of liver nuclear ionic channels reconstituted into giant proteoliposomes.

Nuclear ionic channels (NICs) represent ubiquitous structures of living cells, although little is known about their functional properties and encoding genes. To characterize NICs, liver nuclear membrane vesicles were reconstituted into either planar lipid bilayers or proteoliposomes. Reconstitution of nuclear envelope (NE) vesicles into planar lipid bilayer proceeded with low efficiency. NE vesicle reconstitution into proteoliposomes led to NIC observations by the patch-clamp technique. Large conductance, voltage-gated, K(+)-permeant and Cl(-)-permeant NICs were characterized. An 80-105-pS K(+)-permeant NIC with conducting sub-state was also recorded. Our data establish that NICs can be characterized upon reconstitution into giant proteoliposomes and retain biophysical properties consistent with those described for native NICs.

Animals↗

PubMed2000

Characterization of the sarcoplasmic reticulum k(+) and Ca(2+)-release channel-ryanodine receptor-in human atrial cells.

Since the role of sarcoplasmic reticulum (SR) in the E-C coupling of mammalian atrial cells has long been a subject of debate, biochemical, electrophysiological and immunological assays were performed in order to define and compare the properties of the Ca(2+)-release channel-ryanodine receptor (RyR)-from atrial and ventricular tissues. Cardiac SR preparations from human, canine and ovine tissues were compared using [(3)H]ryanodine binding, channel reconstitution into planar lipid bilayers and Western blot analysis involving RyR antibodies. [(3)H]ryanodine binding assays revealed a K(d)value of; 2.5 n M for all investigated cardiac tissues. Bound [(3)H]ryanodine was Ca(2+)-dependent with similar EC(50)values of 0.43, 0.49 and 0.79 microM for human atrium, canine ventricle and ovine atrium, respectively. However the density of binding sites was 4.5 times lower in atrial than in ventricular tissues. Beyond the presence of selective K(+)channels (gamma=188 pS) recorded in the SR enriched fraction of human atrium, the activity of a large conducting (gamma=671 pS) cationic channel was also observed. The latter displayed typical characteristics of Ca(2+)-release channels which were activated by 10 microM free [Ca(2+)] and 2 m M ATP. Western blot analysis revealed the presence of the RyR2 isoform in atrial and ventricular samples whereas no immunoreactivity was detected with specific RyR1 and RyR3 antibodies. Our results, obtained at the molecular level, are consistent with the presence of functional SR in human atrial cells. The human atrial Ca(2+)-release channel displays binding and regulating properties typical of the RyR2 isoform.

Aged↗

PubMed1999

Characterization of cyclic nucleotide phosphodiesterase isoforms associated to isolated cardiac nuclei.

The identity and location of nuclear cyclic nucleotide phosphodiesterases (PDE) has yet to be ascertained. Intact cardiac nuclei and subnuclear fractions from ovine hearts were isolated to determine cAMP-specific PDE activity which was 3-fold greater than that of cGMP PDE, the latter being insensitive to Ca-calmodulin and zaprinast. Specific hydrolytic activities of the cardiac nuclear envelopes (NE) were similar to those measured in the corresponding intact nuclei, thus suggesting that most PDE activity is associated with the nuclear membrane. Moreover, the main hydrolytic activities in cardiac nuclei were attributed to PDE4 (56%) and PDE3 (44%). The pharmacological sensitivity of each isoform in terms of IC(50), K(m) and K(i) values was typical of previously characterized cardiac PDE 3 and 4 isoforms. PDE2 (cGMP-stimulated PDE) represented a minor component (8-9%) of total hydrolytic activity. Solubilization of nuclear envelopes and HPLC separation also yielded rolipram-sensitive PDE activities. Upon 1% Triton X-100 extractions, the presence of PDE3 and PDE4 was revealed in a low speed, nucleopore complex-enriched, P1 pellet. In addition, Western blot analysis demonstrated the presence of PDE4B and PDE4D subtypes in the nuclei as well as enrichment in NE. However, in the same preparations, the presence of PDE4A could not be ascertained. Altogether, these results suggest an intrinsic and predominant association of these nuclear PDEs with the NE and much likely with nucleopore complexes.

1-Methyl-3-isobutylxanthine↗

PubMed1999

Identification and immunolocalization of two isoforms of ATP-diphosphohydrolase (ATPDase) in the pig immune system.

The occurrence of a variety of purine receptors in the immune system indicates that extracellular purines play important functional roles. Extracellular purine concentrations are, in great part, determined by ectonucleotidases, namely, the ATP diphosphohydrolase, also identified as CD39, a lymphocyte cell surface marker. The latter enzyme converts triphospho- and diphosphonucleosides to nucleoside monophosphates. In this study, high levels of ATPase and ADPase activities have been found in homogenates of the different pig lymphoid organs. Specific activities decreased in the following order: spleen > bone marrow > thymus > lymph glands. The parallel decrease in ATPase and ADPase activities, in the presence of sodium azide, indicated that an ATP diphosphohydrolase (ATPDase) was responsible for these activities. Particulate fractions, prepared from the different lymphoid organs by ultracentrifugation on a sucrose cushion, showed about a 10-fold enrichment of ATPDase activity. Identity of ATPDase was confirmed by electrophoretograms of the particulate fractions and Western immunoblots, with an antibody that recognizes ATPDases from different sources. Two isoforms of ATPDase were found (I and II), corresponding to molecular masses of 78,000 and 54,000, respectively, as estimated by SDS-PAGE. Immunohistochemical localization was carried out on these different organs: In spleen, reaction was found in both white and red pulps. A particularly intense reaction was put in evidence in nervous fibers of this organ. Immunolocalization also showed positive reactions with tonsilar lymphoid structures, diffuse lymphoid tissues, and nodules associated with stomach, duodenum, jejunum, and ileum. In addition, our observations establish the presence of ATPDase in lymphocytes and macrophages of the pig immune system.

Adenosine Triphosphatases↗

PubMed1999

[Preparation of cell therapy products: contribution of closed systems].

A phase I clinical trial is being currently performed in our institution, aiming at evaluating the feasibility and toxicity related to the administration of Herpes Simplex-thymidine kinase gene-expressing human primary T lymphocytes following allogeneic hematopoietic stem cell transplantation. The need for safe and standardized preparation conditions for gene-modified cells is crucial. We describe the closed culture system used in the current trial for ex vivo retroviral-mediated gene transfer and transduced cell selection. Cell handling is performed in closed systems using sampling and transfer pack bags, culture bags and a sterile connection device which avoids opening the culture system. This closed system allows safe and reproducible ex vivo preparation of gene-modified primary T-lymphocytes for clinical use.

Cell Culture Techniques↗

PubMed1999

Comparative involvement of cyclic nucleotide phosphodiesterases and adenylyl cyclase on adrenocorticotropin-induced increase of cyclic adenosine monophosphate in rat and human glomerulosa cells.

The present study investigated the role and identity of cyclic nucleotide phosphodiesterases (PDEs) in the regulation of basal and ACTH-stimulated levels of intracellular cAMP in human and rat adrenal glomerulosa cells. Comparative dose-response curves indicated that maximal hormone-stimulated cAMP accumulation was 11- and 24-fold higher in human and rat cells, compared with cAMP production obtained in corresponding membranes, respectively. Similarly to 3-isobutyl-1-methyl-xanthine, 25 microM erythro-9-[2-hydroxy-3-nonyl]adenine (EHNA, a specific PDE2 inhibitor), caused a large increase in ACTH-stimulated cAMP accumulation; by contrast, it did not change cAMP production in membranes. Moreover, in membrane fractions, addition of 10 microM cGMP inhibited ACTH-induced cAMP production, an effect completely reversed by addition of 25 microM EHNA. These results indicate that PDE2 activity is involved in the regulation of cAMP accumulation induced by ACTH, and suggest that ACTH inhibits this activity. Indeed, time-course studies indicated that ACTH induced a rapid decrease in cGMP production, resulting in PDE2 inhibition, which in turn, contributed [with adenylyl cyclase (AC) activation] to an accumulation in cAMP for 15 min. Thereafter, cAMP content decreased, because of cAMP-stimulated PDE2, as confirmed by measurement of PDE activity that was activated by ACTH, but only after a 10-min incubation. Hence, we demonstrate that the ACTH-induced increase in intracellular cAMP is the result of a balance between activation of AC and direct modulation of PDE2 activity, an effect mediated by cGMP content. Although similar results were observed in both models, PDE2 involvement is more important in rat than in human adrenal glomerulosa cells, whereas AC is more stimulated in human than in rat glomerulosa cells.

1-Methyl-3-isobutylxanthine↗