The clinical potential of adipogenesis and obesity-related microRNAs.

Obesity is a growing health problem commonly associated with numerous metabolic disorders including type 2 diabetes, hypertension, cardiovascular disease, and some forms of cancer. The burden of obesity and associated cardiometabolic diseases are believed to arise through complex interplay between genetics and epigenetics predisposition, nutrition, environment, and lifestyle. However, the molecular basis and the repertoire of obesity-affecting factors are still unknown. Emerging evidence is connecting microRNAs (miRNAs) dysregulation with adipogenesis and obesity. Alteration in miRNAs expression could result in changes in the pattern of genes controlling a range of biological processes including inflammation, lipid metabolism, insulin resistance and adipogenesis. Hence, understanding exact roles of miRNAs as well as the degree of their contribution to the regulation of adipogenesis and fat cell development in obesity would provide new therapeutic targets for the development of novel and effective anti-obesity drugs. The objective of the current review is to: (i) discuss some of the latest development on relevant miRNAs dysregulation mainly in human adipogenesis and obesity, (ii) emphasize the role of circulating miRNAs as new promising therapeutics and attractive potential biomarkers for treating obesity and associated risk factor diseases, (iii) describe how dietary factors may influence obesity through modulation of miRNAs expression, (iv) highlight some of the actual limitations to the promise of miRNAs as novel therapeutics as well as to their translation for the benefit of patients, and finally (v) provide recommendations for future research on miRNA-based therapeutics that could lead to a breakthrough in the treatment of obesity and its associated pathologies.

Cardiovascular pharmacogenetics: a promise for genomically-guided therapy and personalized medicine.

Cardiovascular disease (CVD) is the leading cause of death worldwide. The basic causes of CVD are not fully understood yet. Substantial evidence suggests that genetic predisposition plays a vital role in the physiopathology of this complex disease. Hence, identification of genetic contributors to CVD will likely add diagnostic accuracy and better prediction of an individual's risk. With high-throughput genetics and genomics technology and newer genome-wide study approaches, a number of genetic variations across the human genome were uncovered. Evidence suggests that genetic defects could influence CVD development and inter-individual responses to widely used cardiovascular drugs like clopidogrel, aspirin, warfarin, and statins, and therefore, they may be integrated into clinical practice. If clinically validated, better understanding of these genetic variations may provide new opportunities for personalized diagnostic, pharmacogenetic-based drug selection and best treatment in personalized medicine. However, numerous gaps remain unsolved due to the lack of underlying pathological mechanisms for how genetic predisposition could contribute to CVD. This review provides an overview of the extraordinary scientific progress in our understanding of genetic and genomic basis of CVD as well as the development of relevant genetic biomarkers for this disease. Some of the actual limitations to the promise of these markers and their translation for the benefit of patients will be discussed.

Identifying early events of gene expression in breast cancer with systems biology phylogenetics.

Advanced omics technologies such as deep sequencing and spectral karyotyping are revealing more of cancer heterogeneity at the genetic, genomic, gene expression, epigenetic, proteomic, and metabolomic levels. With this increasing body of emerging data, the task of data analysis becomes critical for mining and modeling to better understand the relevant underlying biological processes. However, the multiple levels of heterogeneity evident within and among populations, healthy and diseased, complicate the mining and interpretation of biological data, especially when dealing with hundreds to tens of thousands of variables. Heterogeneity occurs in many diseases, such as cancers, autism, macular degeneration, and others. In cancer, heterogeneity has hampered the search for validated biomarkers for early detection, and it has complicated the task of finding clonal (driver) and nonclonal (nonexpanded or passenger) aberrations. We show that subtyping of cancer (classification of specimens) should be an a priori step to the identification of early events of cancers. Studying early events in oncogenesis can be done on histologically normal tissues from diseased individuals (HNTDI), since they most likely have been exposed to the same mutagenic insults that caused the cancer in their neighboring tissues. Polarity assessment of HNTDI data variables by using healthy specimens as outgroup(s), followed by the application of parsimony phylogenetic analysis, produces a hierarchical classification of specimens that reveals the early events of the disease ontogeny within its subtypes as shared derived changes (abnormal changes) or synapomorphies in phylogenetic terminology.

[Toxoplasmosis in Sfax, Tunisia]. / Etat actuel de la toxoplasmose dans la région de Sfax, Tunisie.

Abstract The frequency of toxoplasmosis depends on life-style and environment. Our objective was to study different epidemiological, clinical and biological aspects of toxoplasmosis in the Sfax area (Tunisia). This retrospective study has been performed on seria of 40,566 pregnant women in the Parasitology-Mycology Laboratory of Habib-Bourguiba Sfax hospital-Tunisia for 13 years from 1994 to 2006; 1,691 patients presenting with lymphadenopathy; 191 immunocompromised patients (78 HIV infected patients and 113 transplanted patients) and 21 patients presenting clinical signs of ocular toxoplasmosis. In pregnant women, the seroprevalence was 39.3% (15,952/40,567). Among 24,089 seronegative women, only 6,890 (28.6%) had been followed up during their pregnancy. An active toxoplasmosis possibly acquired during pregnancy was detected in 1.3% of cases. Sixteen congenital toxoplasmosis were detected. Toxoplasmosis was confirmed in 13.7% of the 169 patients with lymphadenitis. For HIV positive patients, 11.7% had cerebral toxoplasmosis. It revealed the HIV infection in four cases. Among transplant recipients, one case of active toxoplasmosis was diagnosed in a renal transplant recipient who received transplant from a seronegative donor. Twenty-one patients presenting toxoplasmic retinochoroiditis were treated by subconjonctival injections of clindamycin and systemic corticotherapy at a dose of 1 mg/kg per day. This clinical toxoplasmosis diversity explains the need for bioclinical confrontation to establish diagnosis.

8-[(1E)-1-(2-Aminophenyl-iminio)eth-yl]-2-oxo-2H-chromen-7-olate.

The title Schiff base, C(17)H(14)N(2)O(3), exists as an NH tautomer with the H atom of the phenol group transferred to the imine N atom. The iminium H atom is involved in a strong intra-molecular N(+)-H⋯O(-) hydrogen bond to the phenolate O atom, forming an S(6) motif. In the crystal structure, N-H⋯O hydrogen bonds form a C(9) chain parallel to [100] and a C(11) chain parallel to [010], while C-H⋯O hydrogen bonds form a C(11) chain parallel to [010]. The combination of N-H⋯O and C-H⋯O hydrogen bonds generates R(4) (3)(30) rings parallel to the ab plane.

[Schizophrenia and violence, incidence and risk factors: a Tunisian sample]. / Schizophrénie et violence: incidence et facteurs de risque à propos d'une population tunisienne.

INTRODUCTION: Schizophrenia appears to be the mental pathology the most associated with violence. The aim of this study is to show the incidence and the different risk factors of violence among schizophrenics. MATERIAL AND METHOD: We have compared a group of 30 violent schizophrenic inpatients with another group of 30 nonviolent schizophrenic inpatients hospitalised during the same period. These two groups have been matched according to age and gender. The comparison concerned: sociodemographic parameters, family and personal psychiatric history, legal antecedents, social insertion, clinic, Clinical Global Impressions (CGI), Global Impairment Scale (GIS) and Positive And Negative Syndrome Scale (PANSS) scores for admissions, familial support and insight, compliance to treatment, administered treatments, and awareness degree. RESULTS: Violent schizophrenics represent 18.07% of all hospitalisations and 26.08% of schizophrenic patients. When compared to violent schizophrenic patients, nonviolent schizophrenic patients have a better socioeconomic level (77% versus 43%), better professional adaptation (67% versus 10%) and familial support (60% versus 10%), better insight (87% versus 23%) and therapeutic control (70% versus 17%). Differences are significant. We found significantly more personal antecedents of inflicted violence within violent schizophrenics (50% versus 13%), more addictive behavior (53% versus 13%), and more paranoid and indifferentiated forms (87% versus 47%) than in nonviolent schizophrenics. The average of CGI scores was significantly higher within violent schizophrenics (5.27+/-0.8 versus 3.77+/-0.5). Conversely, the average of EGF scores was lowest (37.6+/-6.5 versus 47.8+/-5.6). The comparison of PANSS scores revealed that violent schizophrenic subjects are characterised by the existence of more positive signs and more general symptoms (34.4+/-4.7 versus 20.2+/-4.5; 55.1+/-11.4 versus 46.1+/-6.9). Violent schizophrenics are characterised by higher neuroleptic doses (2375+/-738 mg/d versus 1610+/-434 mg/d). Differences here are also significant. DISCUSSION: Addictive behaviour seems to considerably increase the risk of turning to violence. Thus in our study, 53% of violent patients showed an addictive behaviour. These results have also been reported by other authors. It is obvious that alcohol and drug abuse double the risk of violence among schizophrenic subjects. Psychotic decompensation and rich symptomatology increase the violent potential among the schizophrenics. In our study, the PANSS scores were higher among violent subjects. Nonviolent schizophrenic subjects have a lesser symptomatology of psychiatric disorders and a better outcome as shown by the CGI and EGF scores. In our study, the group of violent subjects needed higher neuroleptic doses and were noncompliant. Compliance permits the acquisition, and then maintains, the stability of the mental status and plays an essential role in decreasing dangerousness. In fact, violent schizophrenics exhibit low insight, implying diminished awareness of the legal implications of their acts, and are little aware of their illness and its dangerousness. In our study, we noted better familial support among nonviolent subjects. According to the literature, violent schizophrenics are characterised by a particularly hostile and rejecting familial environment. CONCLUSION: Awareness of these factors will allow us to provide improved prevention of violence within schizophrenic subjects.

Injection safety at primary health care level in south-western Saudi Arabia.

In a study of injection safety in Abha health district, Saudi Arabia, data were collected from 47 physicians and 85 nurses at 24 primary health care centres, using an observation checklist and an interview questionnaire. All centres used individually packed disposable syringes and puncture-proof containers to collect used needles. Needlestick injury in the previous year was reported by 14.9% of physicians and 16.5% of nurses (0.21 and 0.38 injuries/person/year respectively). Logistic regression analysis identified recapping the needle after use (physicians and nurses) and bending the needle before disposal (physicians) as significant risk factors for injury.

Insecticidal effects of extracts of seven plant species on larval development, alpha-amylase activity and offspring production of Tribolium castaneum (Herbst) (Insecta: Coleoptera: Tenebrionidae).

Bioinsecticidal effects of methanol extracts from seven plant species on Tribolium castaneum were investigated. Centaurium erythraea, Peganum harmala, Ajuga iva, Aristolochia baetica, Pteridium aquilinum and Raphanus raphanistrum extracts inhibit growth of larvae. C. erythraea was the most toxic with 63% mortality 10 days after treatment, followed by P. harmala with 58%. C. erythraea and P. aquilinum reduce the emergence rate respectively of 66% and 19%. The duration of larval period was shortened by Launaea arborescens, P. aquilinum and A. iva extracts, whereas R. raphanistrum and P. harmala extracts extend the larval period when compared to the control. Extracts of C. erythraea, P. harmala, A. iva and A. baetica inhibited F1 progeny production. Larvae possess three alpha-amylase isoforms as determined by SDS-PAGE. Larvae fed on treated diet had lower alpha-amylase activity than larvae feed on untreated diet. C. erythraea and P. harmala are the most potent extracts. These plant extracts could be useful to reduce seed damage caused by this pest species.

[Unipolar versus bipolar depression: clues toward predicting bipolarity disorder]. / Dépression unipolaire versus dépression bipolaire: facteurs prédictifs d'une évolution bipolaire.

Bipolar and unipolar disorders share a common depressive clinical manifestation. It is important to distinguish between these two forms of depression for several reasons. First, prescribing antidepressors in monotherapy indubitably worsens the prognosis of bipolarity disorders. Second, postponing the prescription of a mood stabilizer reduces the efficacy of the treatment and multiplies the suicidal risks by two. The object of this study is to reveal the factors that distinguish between unipolar and bipolar depression. This is a retrospective study on patients' files. It includes 186 patients divided according to DSM IV criteria into two groups: patients with bipolar disorder type I or II with a recent depressive episode (123 patients) and patients with recurrent depressive disorder (63 patients). A medical record card was filled-in for every patient. It included socio-demographic data, information about the disorder, family antecedents, CGI score (global clinical impressions), physical comorbidity, substance abuse and personality disorder. In order to sort out the categorization variables, the two groups were compared using chi2 test or Fischer's test. With regard to the quantitative variables, the two groups were compared using Krostal Wallis's test or Ancova. Our study has revealed that bipolar disorder differs significantly from unipolar disorder in the following respects: bipolar disorder is prevalent among men (sex-ratio 2) while unipolar disorder is prevailing among women (sex-ratio 0.8); patients with bipolar disorder are younger than patients with unipolar disorder (38.1 +/- 5 years vs. 49.7 +/- years); the age at the onset of bipolar disorder is earlier than that of unipolar disorder (20.8 +/- 2 years vs. 38.7 +/- 5 years); family antecedents are more important in bipolar patients than in unipolar patients (51.1% vs. 33%). More importantly, bipolar disorder differs from unipolar disorder in the following aspects: The number of suicidal attempts (25.3% vs. 23.6%); the degree of substance abuse (15.4% vs. 14.5%); the level of somatic comorbidity (20.3% vs. 17.4%); the amount of anxiety manifestations (5.6% vs. 4.8%); the extent of personality disorder (30.8% vs. 23.8%); the degree of socio-professional impairment (bachelorhood and unemployment). On the other hand, we noted that unipolar patients differ from bipolar patients in terms of the frequency of hospitalizations (3.5 vs. 3.1) and the length of stays in hospitals (25.8 vs. 20.7 days) with significant differences of 0.003 and 0.0000001 respectively. Moreover, the CGI scores of unipolar patients are higher than those of bipolar patients. However, the difference is not significant. Consequently, an early distinction between bipolar and unipolar disorder is of utmost importance for the treatment of these two illnesses.

Structure, function and regulation of the mitochondrial peripheral-type benzodiazepine receptor.

Steroid biosynthesis begins with the transfer of cholesterol from intracellular stores into mitochondria. Through in vitro and in vivo studies using various steroidogenic cell models and with the help of pharmacological, biochemical, morphological and molecular approaches we demonstrated that the peripheral-type benzodiazepine receptor (PBR) is an 18 kDa mitochondrial protein that interacts with other proteins in the outer mitochondrial membrane to form a multimeric complex. PBR is required for the binding, uptake and release, upon ligand activation, of the substrate cholesterol. Thus, cholesterol becomes available to the inner mitochondrial membrane P450scc where steroid biosynthesis begins. The presence of mitochondrial PBR is also critical in maintaining outer mitochondrial membrane stability and in preventing apoptosis. Considering these functions of PBR and the fact that PBR is a ubiquitous protein, it is suggested that this drug receptor may serve as a target to control various mitochondrial and cell functions and to protect against experimentally or pathologically induced mitochondrial and cell toxicity.

The quality of clinical notes at a Psychiatric Hospital in Saudi Arabia.

OBJECTIVE: This study aims to display the quality of the clinical notes at Abha Psychiatric Hospital out-patient clinics. METHODS: In this chart review study, a total of 380 charts were randomly collected, and reviewed for the clinical items that should be included in the psychiatric clinical notes. Each chart note quality was indicated as good, fair or poor based on means and standard deviations. RESULTS: The quality of clinical notes was good in 16.3%, fair in 71.1% and poor in 12.6% of the total records. The most frequent clearly present items were medications (92.1%), and personal data (91.1%); while the most frequent clearly absent items were psychotherapy (90.8%), and functioning (80.8%). CONCLUSION: The quality of psychiatric clinical notes needs to be improved. Practical recommendations regarding this were stressed.

Drug-induced inhibition of the peripheral-type benzodiazepine receptor expression and cell proliferation in human breast cancer cells.

The peripheral-type benzodiazepine receptor (PBR) expression and localization correlate with human breast cancer cell proliferation and aggressive phenotype expression. The standardized extract of Ginkgo biloba leaves (EGb 761) and isolated ginkgolide B (GKB) were shown to decrease PBR mRNA expression in adrenal cells. We examined the effect of EGb 761 and GKB on PBR expression and cell proliferation in human breast cancer cells. EGb 761 and GKB decreased in a time- and dose-dependent manner PBR expression and cell proliferation in the highly aggressive, rich in PBR, human breast cancer cell line MDA-231 whereas they did not affect the proliferation of the non-aggressive human breast cancer cell line MCF-7, which contains very low PBR levels. This effect was reversible and not due to the antioxidant properties of the compounds tested. Using a human cDNA expression array we determined that EGb 761 treatment altered, in addition to PBR, the expression of 36 gene products involved in various pathways regulating cell proliferation. These in vitro data were further validated in an in vivo model where EGb 761 and GKB significantly inhibited the nuclear PBR expression and growth of MDA-231 cell xenografts in nude mice. Taken together, these data suggest that the manipulation of PBR expression could be used to control tumor growth and that EGb 761 and GKB, under the conditions used, exert cytostatic properties.

In vitro studies on the role of the peripheral-type benzodiazepine receptor in steroidogenesis.

In vitro studies using isolated cells, mitochondria and submitochondrial fractions demonstrated that in steroid synthesizing cells, the peripheral-type benzodiazepine receptor (PBR) is an outer mitochondrial membrane protein, preferentially located in the outer/inner membrane contact sites, involved in the regulation of cholesterol transport from the outer to the inner mitochondrial membrane, the rate-determining step in steroid biosynthesis. Mitochondrial PBR ligand binding characteristics and topography are sensitive to hormone treatment suggesting a role of PBR in the regulation of hormone-mediated steroidogenesis. Targeted disruption of the PBR gene in Leydig cells in vitro resulted in the arrest of cholesterol transport into mitochondria and steroid formation; transfection of the mutant cells with a PBR cDNA rescued steroidogenesis demonstrating an obligatory role for PBR in cholesterol transport. Molecular modeling of PBR suggested that it might function as a channel for cholesterol. This hypothesis was tested in a bacterial system devoid of PBR and cholesterol. Cholesterol uptake and transport by these cells was induced upon PBR expression. Amino acid deletion followed by site-directed mutagenesis studies and expression of mutant PBRs demonstrated the presence in the cytoplasmic carboxy-terminus of the receptor of a cholesterol recognition/interaction amino acid consensus sequence. This amino acid sequence may help for recruiting the cholesterol coming from intracellular sites to the mitochondria.

Pharmacological studies of two antidiabetic plants: Globularia alypum and Zygophyllum gaetulum.

Investigations were carried out to evaluate the hypoglycaemic activity of the infusions of Globularia alypum and Zygophyllum gaetulum. Oral and intraperitoneal administration of the plants (0.7 g/kg) produced a significant hypoglycaemic effect in normal as well as in hyperglycaemic rats. The infusions increased significantly plasma insulin levels in normal rats. It is suggested that the hypoglycaemic activity of these plants may be mediated through enhancement of peripheral metabolism of glucose and an increase in insulin release.

In vivo studies on the role of the peripheral benzodiazepine receptor (PBR) in steroidogenesis.

In various steroidogenic cell models, mitochondrial preparations and submitochondrial fractions, the expression of the mitochondrial 18 kDa peripheral-type benzodiazepine receptor (PBR) protein confers the ability to take up and release, upon ligand activation, cholesterol. Thus, cholesterol becomes available to P450scc on the inner mitochondrial membrane. These in vitro studies were validated by in vivo experiments. Treatment of rats with ginkgolide B (GKB), specifically reduced the ligand binding capacity, protein, and mRNA expression of the adrenocortical PBR and circulating glucocorticoid levels. Treatment with GKB also resulted in inhibition of PBR protein synthesis and corticosterone production by isolated adrenocortical cells in response to ACTH. The ontogeny of both PBR binding capacity and protein directly paralleled that of ACTH-inducible steroidogenesis in rat adrenal cells and in rats injected with ACTH. In addition, the previously described suppression of luteal progesterone synthesis in the pregnant rat by continuous in vivo administration of a gonadotropin-releasing hormone agonist may be due to decreased luteal PBR ligand binding and mRNA. These results suggest that (i) PBR is an absolute prerequisite for adrenocortical and luteal steroidogenesis, (ii) regulation of adrenal PBR expression may be used as a tool to control circulating glucocorticoid levels and (iii) the stress hypo-responsive period of neonatal rats may result from decreased adrenal cortical PBR expression.

Contribution to the study of the chemical composition of Verticillium albo-atrum secretions in liquid media.

Verticillium albo-atrum culture filtrates contain numerous metabolites such as enzymes and toxins. Their purification was carried out by high phase liquid chromatography (HPLC). In this study, we set out to look for other lighter metabolites that could be identified by gas chromatography coupled to mass spectrometry (GC-MS). The analyses revealed the presence of many volatile chemical compounds, some of which are of great economic interest such as methyl paraben, propyl paraben tethane and xylene. Verticillium albo-atrum, which is a pathogen of many market-garden and fodder crops, is able to produce substances that can be used in both the agro-food and cosmetic industries.

Ex vivo regulation of adrenal cortical cell steroid and protein synthesis, in response to adrenocorticotropic hormone stimulation, by the Ginkgo biloba extract EGb 761 and isolated ginkgolide B.

We previously demonstrated that repeated treatment of rats with the standardized extract of Ginkgo biloba leaves, EGb 761, and its bioactive component ginkgolide B (GKB), specifically reduces the ligand binding, and protein and messenger RNA expression of the adrenal mitochondrial peripheral benzodiazepine receptor (PBR), a key element in the regulation of cholesterol transport, resulting in decreased circulating corticosterone levels. Adrenocortical cells were isolated from rats treated with EGb 761 or GKB and cultured for 2 and 12 days. The effect of ACTH on normal and metabolically labeled cells was examined. Corticosterone levels were measured by RIA, and protein synthesis was analyzed by two-dimensional gel electrophoresis. Ex vivo treatment with EGb 761 and GKB resulted, respectively, in 50% and 80% reductions of ACTH-stimulated corticosterone production by adrenocortical cells cultured for 2 days compared with that by cells isolated from saline-treated rats. Two-dimensional gel electrophoresis analysis revealed that in cells from both control and drug-treated animals, ACTH induced the synthesis, at the same level, of a 29-kDa and pI 6.4-6.7 protein identified as the adrenal steroidogenic acute regulatory protein (StAR). In addition, treatment with EGb 761 and GKB specifically altered the synthesis of seven proteins, including inhibition of synthesis of a 17-kDa, identified as PBR. After 12 days in culture, ACTH-stimulated adrenocortical cell steroid synthesis was maintained, and it was identical among the cells isolated from animals treated with GKB or saline. Under the same conditions, the expression of PBR was recovered, whereas no effect of ACTH on the 29-kDa and 6.4-6.7 pI protein (StAR) or other protein synthesis could be seen. A comparative analysis of the effects of GKB and EGb 761 on adrenocortical steroidogenesis and protein synthesis identified, in addition to the 17-kDa PBR, target proteins of 32 kDa (pI 6.7) and 40 kDa (pI 5.7-6.0) as potential mediators of the effect of EGb 761 and GKB on ACTH-stimulated glucocorticoid synthesis. In conclusion, these results 1) validate and extend our previous in vivo findings on the effect of EGb 761 and GKB on ACTH-stimulated adrenocortical steroidogenesis, 2) demonstrate the specificity and reversibility of EGb 761 and GKB treatment, 3) question the role of the 29-kDa, 6.4-6.7 pI protein (mature StAR) as the sole mediator of ACTH-stimulated steroid production, and 4) demonstrate the obligatory role of PBR in hormone-regulated steroidogenesis.

Peripheral benzodiazepine receptor in cholesterol transport and steroidogenesis.

Steroidogenesis begins with the metabolism of cholesterol to pregnenolone by the inner mitochondrial membrane cytochrome P450 side-chain cleavage (P450scc) enzyme. The rate of steroid formation, however, depends on the rate of cholesterol transport from intracellular stores to the inner mitochondrial membrane and loading of P450scc with cholesterol. In previous in vitro studies, we demonstrated that a key element in the regulation of cholesterol transport is the mitochondrial peripheral-type benzodiazepine receptor (PBR). We also showed that the polypeptide diazepam binding inhibitor (DBI), an endogenous PBR ligand, stimulates cholesterol transport and promotes loading of cholesterol to P450scc in vitro, and that its presence is vital for hCG-induced steroidogenesis by Leydig cells. Based on these data and the observations that i) the mitochondrial PBR binding and topography are regulated by hormones; ii) the 18-kDa PBR protein is functionally coupled to the mitochondrial contact site voltage-dependent anion channel protein; iii) the 18-kDa PBR protein is a channel for cholesterol, as shown by molecular modeling and in vitro reconstitution studies; iv) targeted disruption of the PBR gene in steroidogenic cells dramatically reduces the ability of the cells to transport cholesterol in the mitochondria and produce steroids; v) endocrine disruptors, with known anisteroidogenic effect, inhibit PBR ligand binding; and vi) in vivo reduction of adrenal PBR expression results in reduced circulating glucocorticoid levels, we conclude that PBR is an indispensable element of the steroidogenic machinery.

Targeted disruption of the peripheral-type benzodiazepine receptor gene inhibits steroidogenesis in the R2C Leydig tumor cell line.

To evaluate the role of the mitochondrial peripheral-type benzodiazepine receptor (PBR) in steroidogenesis, we developed a molecular approach based on the disruption of the PBR gene, by homologous recombination, in the constitutive steroid producing R2C rat Leydig tumor cell line. Inactivation of one allele of the PBR gene resulted in the suppression of PBR mRNA and ligand binding expression. Immunoblot and electron microscopic immunogold labeling analyses confirmed the absence of the 18-kDa PBR protein in the selected clone. Although mitochondria from the PBR-negative cells contained high levels of the constitutively expressed 30-kDa steroidogenic activity regulator protein, these cells produced minimal amounts of steroids compared with normal cells (5%). Moreover, mitochondria from PBR-negative cells failed to produce pregnenolone when supplied with exogenous cholesterol. Addition of the hydrosoluble cholesterol derivative, 22R-hydroxycholesterol, increased steroid production by the PBR-negative R2C cells, indicating that the cholesterol transport mechanism was impaired. Stable transfection of the PBR-negative R2C Leydig cells with a vector containing the PBR cDNA resulted in the recovery of the steroidogenic function of the cells. These data demonstrate that PBR is an indispensable element of the steroidogenic machinery, where it mediates the delivery of the substrate cholesterol to the inner mitochondrial side chain cleavage cytochrome P-450.