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S Dubois

Publications and source records attributed to S Dubois.

At least 37 records · Page 2Linked to original sources
PubMed2001

Characterization of an IL-2 mimetic with therapeutic potential.

Human interleukin-2 (IL-2) interacts with two types of functional receptors (IL-2R alpha betagamma and IL-2R betagamma) and acts on a broad range of target cells involved in inflammatory reactions and immune responses. IL-2 is also used in different clinical trials aimed at improving the treatment of some cancers and the recovery of CD4 lymphocytes by HIV patients. The therapeutic index of IL-2 is limited by various side effects dominated by the vascular leak syndrome. We have shown that a chemically synthesised fragment of the IL-2 sequence can fold into a helical tetramer likely mimicking the quatemary structure of an hemopoietin. Indeed, peptide p1-30 (containing amino acids 1 to 30, including the sequence corresponding to the entire alpha helix A of IL-2) spontaneously folds into an alpha-helical homotetramer and stimulates the growth of T-cell lines expressing human IL-2R beta, whereas shorter versions of the peptide lack helical structure and are inactive. At the cellular level, p1-30 induces lymphokine-activated killer (LAK) cells and preferentially activates CD8 low lymphocytes and natural killer cells, which constitutively express IL-2R beta. A significant IFN-gamma production is also detected following p1-30 stimulation. A mutant form of p1-30 (Asp20-->Lys) which is likely unable to induce vascular leak syndrome remains capable to generate LAK cells like the original p1-30 peptide. Altogether our data suggest that p1-30 has therapeutic potential.

CD8-Positive T-Lymphocytes↗
PubMed2000

IL-2-induced activation-induced cell death is inhibited in IL-15 transgenic mice.

A transgenic (Tg) mouse expressing human IL-15 was generated to define the role of IL-15 in the normal immune response. Overexpression of IL-15 resulted in an increase of NK, CD44(hi)CD8 memory T cells, and gammadelta T cells. Additionally, we observed the emergence of a novel type of NK-T cells with CD8alphaalpha' expression. Due to the expansion and activation of NK cells, the IL-15Tg mouse showed enhanced innate immunity. In adaptive T cell immunity, the roles of IL-15 contrasted with those of IL-2. IL-15 inhibited IL-2-induced T cell death, which plays a role in the maintenance of peripheral self-tolerance. IL-15 thus seems to contribute to enhanced immune memory by selectively propagating memory T cells and by blocking T cell death mediated by IL-2.

Animals↗
PubMed2000

Systematic implementation of an advance directive program in nursing homes: a randomized controlled trial.

CONTEXT: Although advance directives are commonly used in the community, little is known about the effects of their systematic implementation. OBJECTIVES: To examine the effect of systematically implementing an advance directive in nursing homes on patient and family satisfaction with involvement in decision making and on health care costs. DESIGN: Randomized controlled trial conducted June 1, 1994, to August 31, 1998. SETTING AND PARTICIPANTS: A total of 1292 residents in 6 Ontario nursing homes with more than 100 residents each. INTERVENTION: The Let Me Decide advance directive program included educating staff in local hospitals and nursing homes, residents, and families about advance directives and offering competent residents or next-of-kin of mentally incompetent residents an advance directive that provided a range of health care choices for life-threatening illness, cardiac arrest, and nutrition. The 6 nursing homes were pair-matched on key characteristics, and 1 home per pair was randomized to take part in the program. Control nursing homes continued with prior policies concerning advance directives. MAIN OUTCOME MEASURES: Residents' and families' satisfaction with health care and health care services utilization over 18 months, compared between intervention and control nursing homes. RESULTS: Of 527 participating residents in intervention nursing homes, 49% of competent residents and 78% of families of incompetent residents completed advance directives. Satisfaction was not significantly different in intervention and control nursing homes. The mean difference (scale, 1-7) between intervention and control homes was -0.16 (95 % confidence interval [CI], -0.41 to 0.10) for competent residents and 0.07 (95% CI, -0.08 to 0.23) for families of incompetent residents. Intervention nursing homes reported fewer hospitalizations per resident (mean, 0.27 vs 0.48; P = .001) and less resource use (average total cost per patient, Can $3490 vs Can $5239; P = .01) than control nursing homes. Proportion of deaths in intervention (24%) and control (28%) nursing homes were similar (P = .20). CONCLUSION: Our data suggest that systematic implementation of a program to increase use of advance directives reduces health care services utilization without affecting satisfaction or mortality.

Advance Directives↗
PubMed2000

The first alpha helix of interleukin (IL)-2 folds as a homotetramer, acts as an agonist of the IL-2 receptor beta chain, and induces lymphokine-activated killer cells.

Interleukin (IL)-2 interacts with two types of functional receptors (IL-2Ralphabetagamma and IL-2Rbetagamma) and acts on a broad range of target cells involved in inflammatory reactions and immune responses. For the first time, we show that a chemically synthesized fragment of the IL-2 sequence can fold into a molecule mimicking the quaternary structure of a hemopoietin. Indeed, peptide p1-30 (containing amino acids 1-30, covering the entire alpha helix A of IL-2) spontaneously folds into an alpha-helical homotetramer and stimulates the growth of T cell lines expressing human IL-2Rbeta, whereas shorter versions of the peptide lack helical structure and are inactive. We also demonstrate that this neocytokine interacts with a previously undescribed dimeric form of IL-2Rbeta. In agreement with its binding to IL-2Rbeta, p1-30 activates Shc and p56(lck) but unlike IL-2, fails to activate Janus kinase (Jak)1, Jak3, and signal transducer and activator of transcription 5 (STAT5). Unexpectedly, we also show that p1-30 activates Tyk2, thus suggesting that IL-2Rbeta may bind to different Jaks depending on its oligomerization. At the cellular level, p1-30 induces lymphokine-activated killer (LAK) cells and preferentially activates CD8(low) lymphocytes and natural killer cells, which constitutively express IL-2Rbeta. A significant interferon gamma production is also detected after p1-30 stimulation. A mutant form of p1-30 (Asp20-->Lys), which is likely unable to induce vascular leak syndrome, remains capable of generating LAK cells, like the original p1-30 peptide. Altogether, our data suggest that p1-30 has therapeutic potential.

Amino Acid Sequence↗
PubMed2000

Chromosomal numerical aberrations are frequent in oesophageal and gastric adenocarcinomas: a study using in-situ hybridization.

AIMS: (i) To investigate and compare the numerical aberrations of chromosomes 7, 8, 11, 17 and Y in a series of 60 adenocarcinomas of the oesophagus, gastric cardia and gastric antrum; and (ii) to specify the sequence of chromosomal aberrations occurring during the neoplastic progression of Barrett's oesophagus. METHODS AND RESULTS: Chromosomal in-situ hybridization was performed on deparaffinized tissue sections from 20 Barrett's adenocarcinomas, 20 adenocarcinomas of the cardia and 20 adenocarcinomas of the antrum, with centromeric alpha satellite DNA probes specific for chromosomes 7, 8, 11, 17 and Y, labelled with digoxigenin. Signals were detected by immunoperoxidase staining. The copy number for each chromosome was counted in 200 tumour cells nuclei and 100 lymphocytes as controls. In parallel, the DNA content of the nuclear suspensions was measured by flow cytometry. Numerical abnormalities of the five chromosomes (loss of the Y chromosome, monosomy, trisomy, and tetrasomy) were frequently observed in the three groups of adenocarcinomas (from 40% to 65% of the cases). Sixty per cent to 75% of oesophagus and gastric adenocarcinomas were DNA-aneuploid. Chromosomal aberrations progressively increased with advancing degrees of dysplasia in Barrett's mucosa, with an increasing frequency of trisomy and loss of the Y chromosome from non-dysplastic Barrett's mucosa to invasive adenocarcinoma, and with monosomy and tetrasomy present only in invasive cancers. CONCLUSION: Our study confirms the high frequency of chromosomal numerical aberrations in oesophageal and gastric adenocarcinomas, without differences between adenocarcinomas of the gastric cardia and antrum. We have shown that these alterations occur early during the neoplastic transformation of Barrett's mucosa.

Adenocarcinoma↗
PubMed2000

Hepatocellular carcinoma occurring in nonfibrotic liver: epidemiologic and histopathologic analysis of 80 French cases.

Hepatocellular carcinoma (HCC) occurring in nonfibrotic liver represents a rare, ill-defined subgroup of HCC without cirrhosis in which mechanisms of hepatocarcinogenesis remain unclear. The aim of our study was to assess epidemiological factors and detailed histopathologic changes in the nontumoral liver of patients developing such tumors. Of 330 HCCs resected in our institution between 1985 and 1998, we retrospectively analyzed 80 cases (53 men, 27 women; mean age, 51 +/- 16 years) in which the nontumoral liver showed no (n = 28) or minimal (n = 52) portal fibrosis without any septal fibrosis. In the group with no portal fibrosis there was no male predominance, and patients were significantly younger (44 +/- 19 years vs. 54 +/- 14 years) than those with minimal portal fibrosis. Sixty-seven tumors were typical HCCs, 8 were of fibrolamellar type, and 5 were hepatocholangiocarcinomas. Mean tumor size was 10 +/- 5 cm. Risk factors for HCC development were found in 30 patients: hepatitis B (n = 17) or C (n = 2) virus infections, alcohol consumption (n = 11), and hemochromatosis (n = 1). In the nontumoral liver, periportal and lobular necrosis, mild portal inflammation, steatosis, and iron overload were present in 15%, 57%, 52%, and 54% of cases, respectively. Liver cell changes were noted in 6%. This study emphasizes the need for strict criteria to classify HCC without cirrhosis. HCC in nonfibrotic liver is a distinct subgroup in which nontumoral liver shows nonspecific minimal changes without regeneration or premalignant lesion. Etiologic factors are often unidentified, although presence of HBV infection in 21% suggests a direct oncogenic role of this virus.

Adult↗
PubMed2000

Subunit structure of the high and low affinity human interleukin-15 receptors.

Radio-iodinated cytokines and monoclonal antibodies directed at the IL-2R beta- and gamma-chains were used to analyze the structure of the cell-surface IL-15 and IL-2 receptors expressed by the human lymphoma cell clone YT-2C2. YT-2C2 cells are IL-2R alpha negative and express IL-2R gamma (15,000 molecules/cell) in excess of IL-2R beta (11,000 molecules/cell). Accordingly, they display a number of beta/gamma complexes of intermediate affinity for IL-2 and IL-15 which is equivalent to the number of beta-chains. Both cytokines compete for binding to this beta/gamma complex. There are about 800 high affinity IL-15 receptors, suggesting the presence of a similar number of IL-15R alpha-chains. Within the common intermediate affinity beta/gamma-complex, the anti-beta-chain A41 mAb defines an epitope which is similarly engaged in IL-2 and IL-15 binding, whereas the anti-beta-chain 284 mAb defines an epitope which does not display similar interaction with either cytokines. Thus, although IL-2 and IL-15 compete for binding to this beta/gamma-complex, they do not use similar binding areas. Cross-linking and immunoprecipitation experiments have shown that the high affinity IL-15 receptors comprises IL-2R beta/gamma, in association with IL-15R alpha and that the three chains can be efficiently cross-linked to IL-15 and co-immunoprecipitated. Contrary to the intermediate affinity situation, high affinity IL-15 binding and subunit cross-linking were not affected by excess amounts of IL-2, A41 or 284 mAb, suggesting that when engaged in the IL-15 high affinity complex, the beta- and gamma-chains adopt different conformations, at least with respect to IL-15 binding. Finally, we provide evidence for the participation of a novel 35 kDa component within the high affinity structure. This component is immunoprecipitated with anti-IL-2R gamma mAb but not with anti-IL-2R beta mAb and might correspond to a truncated form of IL-2R gamma-chain.

Antibodies, Monoclonal↗
PubMed2000

The effect of dietary fibre on energy utilisation and partitioning of heat production over pregnancy in sows.

A low (L) and high-fibre (H) diet were fed to six multiparous sows during gestation in a 2 x 2 repeated Latin square design. A single meal per day was given that provided 37.2 MJ digestible energy/d. The kinetics of heat production (HP) and its partitioning (fasting HP, activity HP, and thermic effect of feeding (TEF)) were determined. The TEF was partitioned between a dynamic component (TEFst) and a constant component (TEFlt). Digestibility of energy and nutrients was lower for the diet H. In spite of the lower metabolisable energy (ME) intake (33.9 v. 35.4 MJ/d for diets H and L respectively), HP was higher for diet H (30.5 v. 28.9 MJ/d) resulting in a lower energy retention. The estimated fasting HP was 270 kJ/kg body weight 0.75 per d at day 0 of gestation and increased with advancement of pregnancy. The TEFlt was not significantly different from zero for diet L, but represented 4.1% of ME intake for diet H. The TEFst was not affected by the diet but diet H delayed the postprandial peak of HP. Total TEF was higher for diet H than for diet L (11.7 v. 8.2% of ME intake). The longer duration of eating with diet H was compensated for by less physical activity between meals, so that activity HP was equivalent for both diets. The activity HP represented 20% of ME intake but was variable between sows. The ME requirements for maintenance averaged 440 kJ/kg body weight 0.75 per d. Feeding high-fibre diets increases HP, delays the postprandial peak of HP and maintains the basal HP at a higher level.

Animals↗
PubMed1999

Natural splicing of exon 2 of human interleukin-15 receptor alpha-chain mRNA results in a shortened form with a distinct pattern of expression.

We report the existence of eight different interleukin-15 receptor alpha-chain (IL-15Ralpha) transcripts resulting from exon-splicing mechanisms within the IL-15Ralpha gene. Two main classes of transcripts can be distinguished that do or do not (Delta2 isoforms) contain the exon 2-coding sequence. Both classes were expressed in numerous cell lines and tissues (including peripheral blood lymphocytes) at comparable levels and could be transcribed in COS-7 cells, and the proteins were expressed at the cell surface. Both receptor forms displayed numerous glycosylation states, reflecting differential usage of a single N-glycosylation site as well as extensive O-glycosylations. Whereas IL-15Ralpha bound IL-15 with high affinity, Delta2IL-15Ralpha was unable to bind IL-15, thus revealing the indispensable role of the exon 2-encoded domain in cytokine binding. A large proportion of IL-15Ralpha was expressed at the nuclear membrane with some intranuclear localization, supporting a potential direct action of the IL-15.IL-15Ralpha complex at the nuclear level. In sharp contrast, Delta2IL-15Ralpha was found only in the non-nuclear membrane compartments, indicating that the exon 2-encoded domain (which is shown to contain a potential nuclear localization signal) plays an important role in receptor post-translational routing. Together, our data indicate that exon 2 splicing of human IL-15Ralpha is a natural process that might play regulatory roles at different levels.

Animals↗
PubMed1999

PET study of the human foveal fixation system.

Positron emission tomography (PET) was used to investigate the functional anatomy of the foveal fixation system in 10 subjects scanned under three different conditions: at rest (REST), during the fixation of a central point (FIX), and while fixating the same foveal target during the presentation of peripheral visual distractors (DIS). Compared with the REST condition, both FIX and DIS tasks activated a common set of cortical areas. First, in addition to the involvement of the occipital visual cortex, both the frontal eye field (FEF) and the intraparietal sulcus (IPS) were bilaterally activated. Right frontal activation was also found in the dorsolateral prefrontal cortex, the inferior part of the precentral gyrus, and the inferior frontal gyrus. These results suggest that both FEF and IPS may constitute the main cortical regions subserving bilaterally the foveal fixation system in humans. The remaining right frontal activations may be considered as part of the anterior attentional network, supporting a role for the right frontal lobe in the allocation of the attentional mechanisms. Compared with the FIX condition, the DIS task also revealed the perceptual and cognitive processes related to the presence of peripheral visual distractors during foveal fixation. In addition to a bilateral activation of the V5/MT motion-sensitive area, a right FEF-IPS network was activated which may correspond to the engagement of the visuospatial attention. Finally, normalized regional cerebral blood flow (NrCBF) decreases were also observed during both DIS and FIX condition performance. Such NrCBF decreases were centered in the superior and middle temporal gyri, the prefrontal cortex, and the precuneus and the posterior retrosplenial part of the cingulate gyrus.

Adult↗
PubMed1999

Neuronal mechanisms of perceptual learning: changes in human brain activity with training in orientation discrimination.

Using 15O-water 3D positron emission tomography, regional cerebral blood flow was measured twice in six human subjects: before and after extensive training in orientation discrimination. In each session subjects performed two orientation discrimination tasks, during which they discriminated the orientation of a grating at either the trained or untrained reference orientation, and a control task, during which they detected a randomly textured pattern. By comparing the discrimination to the detection tasks, we observed a main effect of task bilaterally in the posterior occipital cortex, extending into the left posterior fusiform gyrus and the right inferior occipital gyrus, bilaterally in the intraparietal sulcus, as well as in the cerebellum, thalamus, and brainstem. When we compared the activation pattern before and after the training period, all the changes observed were activity decreases. The nonspecific changes, which were not related to the orientation used during the training, were situated in the cerebellum and bilaterally in the extrastriate visual cortex. The orientation-specific changes, on the other hand, were restricted to the striate and extrastriate visual cortex, more precisely the right calcarine sulcus, the left lingual gyrus, the left middle occipital, and the right inferior occipital gyrus. These findings confirm our hypothesis concerning the existence of learning related changes at early levels of visual processing in human adults and suggest that mechanisms resulting in neuronal activity decreases might be involved in the present kind of learning.

Adult↗
PubMed1999

Effect of familiarity on the processing of human faces.

Most brain imaging studies on face perception have investigated the processing of unknown faces and addressed mainly the question of specific face processing in the human brain. The goal of this study was to highlight the effects of familiarity on the visual processing of faces. Using [15O]water 3D Positron Emission Tomography, regional cerebral blood flow distribution was measured in 11 human subjects performing an identical task (gender categorization) on both unknown and known faces. Subjects also performed two control tasks (a face recognition task and a visual pattern discrimination task). They were scanned after a training phase using videotapes during which they had been familiarized with and learned to recognize a set of faces. Two major results were obtained. On the one hand, we found bilateral activations of the fusiform gyri in the three face conditions, including the so-called fusiform-face area, a region in the right fusiform gyrus specifically devoted to face processing. This common activation suggests that different cognitive tasks performed on known and unknown faces require the involvement of this fusiform region. On the other hand, specific regional cerebral blood flow changes were related to the processing of known and unknown faces. The left amygdala, a structure involved in implicit learning of visual representations, was activated by the categorization task on unknown faces. The same task on known faces induced a relative decrease of activity in early visual areas. These differences between the two categorization tasks reveal that the human brain processes known and unknown faces differently.

Adult↗
PubMed1999

[A comparison between group-average and individual evoked potential analysis].

PURPOSE: Event-related potentials (ERPs) studies in human subjects have shown inter-individual response variations, probably linked to anatomical and functional brain disparities. The present study was conducted to compare the results obtained by a standard grand-average method and a single subject analysis of VEPs to faces. MATERIAL AND METHOD: Fifty-eight channel ERPs (analysis time: 1,024 ms) were recorded in 13 normal volunteers during gender or familiarity judgements on unknown and known faces, as well as on a control task using meaningless patterns. Data were then submitted to individual and group averages. RESULTS AND CONCLUSION: Three activities were identified by both procedures: a P1/N1 complex, a vertex positive potential (P2 or VPP) associated with a temporal negativity, and a N2 negativity. These peaks displayed a marked inter-individual topographical variability. Regarding the outcome of statistical analyses, a certain number of differences were found: on P1, in which individual analyses revealed a strong effect of experimental conditions, while the grand-average method did not; on VPP, in which grand-average analyses suggested an interaction between experimental conditions, face familiarity and cerebral lateralization, while individual analyses did not; and on N2, in which grand-average data showed a clear lateralization effect, while individual analyses did not. A P3 component (Pz, 250 ms) was also defined in grand-average data, but could not be clearly described in individual data. Statistical analyses on this P3 component were thus only performed on group data and revealed a right lateralization and an interaction between face familiarity and experimental conditions. These findings confirmed the existence of a marked topographical variability of ERPs to face and, therefore, question the validity of grand-average studies. Moreover, these results suggest a better efficiency of individual analyses for studying short and middle-latency peaks, while grand-averages appear to be better suited for studying late components.

Adult↗
PubMed1999

Task modulation of brain activity related to familiar and unfamiliar face processing: an ERP study.

In order to investigate stimulus-related and task-related electrophysiological activity relevant for face processing, event-related potentials (ERPs) from 58 electrodes at standard EEG sites were recorded while subjects performed a simple visual discrimination (control) task, in addition to various face processing tasks: recognition of previously learned faces and gender decision on familiar and unfamiliar faces. Three electrophysiological components or dipolar complex were recorded in all subjects: an occipital early component (P1, around 110 ms); a vertex positive potential (VPP; around 158 ms) which appeared to be specific to faces; and a negative central component, N2 (around 230 ms). Parametric analysis and source localization were applied to these components by means of a single-subject analysis methodology. No effect of familiarity was observed on any of these early component. While the VPP appears to be independent of the kind of processing performed, face task modulations of the early P1 and the N2 were observed, with a higher amplitude for the recognition than for the gender discrimination task. An attentional modulation of early visual areas is proposed for the first effect (P1 modulation), while the N2 seems to be related to general visual memory processing. This study strongly suggests that the VPP reflects an early visual stage of face processing in the fusiform gyrus that is strictly stimulus-related and independent of familiarity. It also shows that source localization algorithms may give reliable solutions on single subject averages for early visual components despite high inter-subject variability of the surface characteristics of ERPs.

Adult↗
PubMed1999

Metabolic utilization of energy and maintenance requirements in growing pigs: effects of sex and genotype.

An experiment was conducted in which the metabolic utilization of energy was measured in individually penned pigs from seven groups that differed in genotype and(or) sex and ranged in body weight between 20 and 107 kg. The animals were fed a diet containing, on a DM basis, 14.7 MJ ME and at least 21% CP. Heat production was measured in an open-circuit calorimeter, and energy, nitrogen, and fat balances were determined at regular intervals over the growing period; a total of 177 measurements were performed. Body composition of the animals was measured by serial slaughter, and these data were used for estimating the body composition of an animal at a given weight through allometric regression. A factorial analysis procedure was used to estimate the utilization of ME by regressing the ME intake on the observed protein and lipid deposition rates. The intercept of this equation is the maintenance energy requirement (MEm) and was represented either as a function of body weight with group-specific parameters (MEm = a(i) BWb) or as a function of the muscle and visceral mass with an additional additive group effect (MEm = aM muscle(b) + a(v) viscera(b) + G(i)). With BW as dependent variable, the exponent b was close to .60 and differed significantly from .75. The regression coefficient (a(i)) averaged 1.02 MJ ME/kg.60 but it was different for most groups, indicating that different groups of animals have different maintenance requirements. Fixing the exponent to .75 consistently underestimated the maintenance requirement. When the exponent b was not fixed to .75 but estimated, the partial efficiencies for protein and lipid deposition were .62 and .84, respectively. Body muscle and visceral mass could explain a large part of the variation in MEm. Viscera contributed three times more to MEm (per kilogram of mass raised to the .70 power) than did muscle. Even though the muscle mass exceeds to a large extent the visceral mass in animals, the contribution of muscle to MEm was lower than that of viscera for most groups.

Animals↗
PubMed1998

Cotranscription and intergenic splicing of human galactose-1-phosphate uridylyltransferase and interleukin-11 receptor alpha-chain genes generate a fusion mRNA in normal cells. Implication for the production of multidomain proteins during evolution.

In the past 10 years, much attention has been focused on transcription preinitiation complex formation as a target for regulating gene expression, and other targets such as transcription termination complex assemblage have been less intensively investigated. We established the existence of poly(A) site choice and fusion splicing of two adjacent genes, galactose-1-phosphate uridylyltransferase (GALT) and interleukin-11 receptor alpha-chain (IL-11Ralpha), in normal human cells. This 16-kilobase (kb) transcription unit contains two promoters (the first one is constitutive, and the second one, 8 kb downstream, is highly regulated) and two cleavage/polyadenylation signals separated by 12 kb. The promoter from the GALT gene yields two mRNAs, a 1.4-kb mRNA encoding GALT and a 3-kb fusion mRNA when the first poly(A) site is spliced out and the second poly(A) is used. The 3-kb mRNA codes for a fusion protein of unknown function, containing part of the GALT protein and the entire IL-11Ralpha protein. The GALT promoter/IL-11Ralpha poly(A) transcript results from leaky termination and alternative splicing. This feature of RNA polymerase (pol) II transcription, which contrasts with efficient RNA pol I and pol III termination, may be involved, together with chromosome rearrangements, in the generation of fusion proteins with multiple domains and would have major evolutionary implications in terms of natural processes to generate novel proteins with common motifs. Our results, together with accumulation of genomic informations, will stimulate new considerations and experiments in gene expression studies.

Animals↗
PubMed1998

The CYP2B2 phenobarbital response unit contains an accessory factor element and a putative glucocorticoid response element essential for conferring maximal phenobarbital responsiveness.

Hepatic cytochrome P450s play a critical role in the metabolism of hydrophobic xenobiotics. One of the major unsolved problems in xenobiotic metabolism is the molecular mechanism whereby phenobarbital induces hepatic enzymes, particularly CYP2B1 and CYP2B2 in rat liver. By using primary rat hepatocytes for transfection analyses, we previously identified in the CYP2B2 5'-flank a 163-base pair Sau3AI fragment that confers phenobarbital inducibility on a cat reporter gene and that has the properties of a transcriptional enhancer. Transfection experiments with sub-regions of the Sau3AI fragment now indicate that a central core together with an upstream or downstream accessory element within the fragment can confer phenobarbital responsiveness. One such accessory element, AF1, was identified and localized. DNase I footprinting analysis revealed the presence of a footprint overlapping this AF1 element. It also identified three other major protected regions, two of which are putative recognition sites for known transcription factors. Site-directed mutagenesis indicated that a putative glucocorticoid response element as well as a nuclear factor 1 site and an associated nuclear receptor hexamer half-site are essential for conferring maximal phenobarbital inducibility. Taken together, the results indicate that phenobarbital induction of CYP2B2 requires interactions among multiple regulatory proteins and cis-acting elements constituting a phenobarbital response unit.

Animals↗