Interaction between SLC6A4 promoter variants and childhood trauma on the age at onset of bipolar disorders.

Age at onset (AAO) of bipolar disorders (BD) could be influenced both by a repeat length polymorphism (5HTTLPR) in the promoter region of the serotonin transporter gene (SLC6A4) and exposure to childhood trauma. We assessed 308 euthymic patients with BD for the AAO of their first mood episode and childhood trauma. Patients were genotyped for the 5HTTLPR (long/short variant) and the rs25531. Genotypes were classified on functional significance (LL, LS, SS). A sample of 126 Brazilian euthymic patients with BD was used for replication. In the French sample, the correlation between AAO and trauma score was observed only among 'SS' homozygotes (p = 0.002) but not among 'L' allele carriers. A history of at least one trauma decreased the AAO only in 'SS' homozygotes (p = 0.001). These results remained significant after correction using FDR. Regression models suggested an interaction between emotional neglect and 'SS' genotype on the AAO (p = 0.009) and no further interaction with other trauma subtypes. Partial replication was obtained in the Brazilian sample, showing an interaction between emotional abuse and 'LS' genotype on the AAO (p = 0.02). In conclusion, an effect of childhood trauma on AAO of BD was observed only in patients who carry a specific stress responsiveness-related SLC6A4 promoter genotype.

The absence of VGLUT3 predisposes to cocaine abuse by increasing dopamine and glutamate signaling in the nucleus accumbens.

Tonically active cholinergic interneurons (TANs) from the nucleus accumbens (NAc) are centrally involved in reward behavior. TANs express a vesicular glutamate transporter referred to as VGLUT3 and thus use both acetylcholine and glutamate as neurotransmitters. The respective roles of each transmitter in the regulation of reward and addiction are still unknown. In this study, we showed that disruption of the gene that encodes VGLUT3 (Slc17a8) markedly increased cocaine self-administration in mice. Concomitantly, the amount of dopamine (DA) release was strongly augmented in the NAc of VGLUT3(-/-) mice because of a lack of signaling by metabotropic glutamate receptors. Furthermore, dendritic spines and glutamatergic synaptic transmission on medium spiny neurons were increased in the NAc of VGLUT3(-/-) mice. Increased DA and glutamate signaling in the NAc are hallmarks of addiction. Our study shows that TANs use glutamate to reduce DA release and decrease reinforcing properties of cocaine in mice. Interestingly, we also observed an increased frequency of rare variations in SLC17A8 in a cohort of severe drug abusers compared with controls. Our findings identify VGLUT3 as an unexpected regulator of drug abuse.

Clinical evidence-based cutoff limits for GH stimulation tests in children with a backup of results with reference to mass spectrometry.

CONTEXT: Cutoff limits of GH stimulation tests to diagnose GH deficiency (GHD) in children and adolescents are not sufficiently validated by clinical studies due to discrepancies in the performance of GH immunoassays and lack of available study populations. OBJECTIVE: We aimed to establish new cutoff limits for GH stimulation tests based on clinical evidence and compared these immunoassay-based values with an antibody-independent mass spectrometric method. DESIGN AND SETTING: In a retrospective study, GH cutoff limits for eight different immunoassays and isotope dilution mass spectrometry (ID-MS) were calculated from hGH peak concentrations of short-statured children with and without GHD. PATIENTS: We compared the serum GH peak concentrations at GH stimulation test of 52 short-statured children and adolescents, who have normal GH secretion at initial workup and normal growth in the follow-up, with the serum GH peak concentrations of 44 GHD patients in the same age range, in order to optimize the cutoff limit calculation. RESULTS: Discriminant analysis of re-measured GH led to a new cutoff limit of 7.09 µg/l using the iSYS assay (IDS) and the limits for the other seven hGH assays varied between 4.32 and 7.77 µg/l. For ID-MS, cutoffs of 5.48 µg/l (22k GH) and 7.43 µg/l (total GH) were ascertained. CONCLUSION: The establishment of method-specific clinical evidence-based GH cutoff limits is of importance to ensure adequate clinical diagnosis and treatment of children and adolescents with GHD. ID-MS may become an important tool for providing both reliable and sustainable SI traceability of GH measurements in the future.

An ASMT variant associated with bipolar disorder influences sleep and circadian rhythms: a pilot study.

Patients with bipolar disorder (BD) experience persistent circadian rhythm and sleep abnormalities during periods of remission, and biological studies have shown that these patients have abnormal melatonin secretion profiles or reactivity to light. We previously reported the association with BD of a common polymorphism (rs4446909) of the promoter of the acetylserotonin O-methyltransferase (ASMT) gene, encoding one of the two enzymes involved in melatonin biosynthesis. This variant was associated with weaker transcription and lower levels of ASMT activity in lymphoblastoid cell lines. Actigraphy, based on the use of a mobile portable device for the analysis of sleep/wake cycles in natural conditions, may be useful for studies of carriers of the at-risk allele. We studied the association between the ASMT rs4446909 variant and sleep/activity, as assessed with the Pittsburgh Sleep Quality Index (PSQI) and by actigraphy, in 53 subjects (25 patients with BD in remission and 28 healthy controls). The two groups were similar for age, sex ratio, current mood symptoms, body mass index and risk of sleep apnea syndrome. In the total sample, the GG at-risk genotype was associated with longer sleep duration (P = 0.03), greater activity in active periods of sleep (P = 0.015) and greater interday stability (P = 0.003). These associations remained significant when disease status was included in the model. Only the association with interday stability remained significant after correction for multiple testing. This pilot study thus shows that a BD-associated functional variant involved in the melatonin synthesis pathway influences sleep and circadian rhythms in bipolar patients in remission and controls.

Molecular characteristics of Human Endogenous Retrovirus type-W in schizophrenia and bipolar disorder.

Epidemiological and genome-wide association studies of severe psychiatric disorders such as schizophrenia (SZ) and bipolar disorder (BD), suggest complex interactions between multiple genetic elements and environmental factors. The involvement of genetic elements such as Human Endogenous Retroviruses type 'W' family (HERV-W) has consistently been associated with SZ. HERV-W envelope gene (env) is activated by environmental factors and encodes a protein displaying inflammation and neurotoxicity. The present study addressed the molecular characteristics of HERV-W env in SZ and BD. Hundred and thirty-six patients, 91 with BD, 45 with SZ and 73 healthy controls (HC) were included. HERV-W env transcription was found to be elevated in BD (P<10-4) and in SZ (P=0.012) as compared with HC, but with higher values in BD than in SZ group (P<0.01). The corresponding DNA copy number was paradoxically lower in the genome of patients with BD (P=0.0016) or SZ (P<0.0003) than in HC. Differences in nucleotide sequence of HERV-W env were found between patients with SZ and BD as compared with HC, as well as between SZ and BD. The molecular characteristics of HERV-W env also differ from what was observed in Multiple Sclerosis (MS) and may represent distinct features of the genome of patients with BD and SZ. The seroprevalence for Toxoplasma gondii yielded low but significant association with HERV-W transcriptional level in a subgroup of BD and SZ, suggesting a potential role in particular patients. A global hypothesis of mechanisms inducing such major psychoses is discussed, placing HERV-W at the crossroads between environmental, genetic and immunological factors. Thus, particular infections would act as activators of HERV-W elements in earliest life, resulting in the production of an HERV-W envelope protein, which then stimulates pro-inflammatory and neurotoxic cascades. This hypothesis needs to be further explored as it may yield major changes in our understanding and treatment of severe psychotic disorders.

Reference measurement procedure for Delta9-tetrahydrocannabinol in serum.

An isotope dilution gas chromatography/mass spectrometry (ID-GC/MS) reference measurement procedure for Delta9-tetrahydrocannabinol (THC) in serum was developed and validated. The method complies with the concept of a ratio primary reference measurement procedure. The uncertainty was determined for two concentrations of THC in serum (1 ng/mL and 2.4 ng/mL). The calculation procedure is based on the Guide to the Expression of Uncertainty in Measurement (GUM). The relative expanded uncertainty was found to be less than 2% for both concentration levels, corresponding to a 95% confidence interval. For the reference method, it was shown that the measurement of THC within the concentration range covered by the current threshold values is very accurate. The method has the potential to provide traceability for the methods used in practical forensics.

Application of the combination of isotope ratio monitoring with isotope dilution mass spectrometry to the determination of glucose in serum.

Isotope ratio monitoring combined with n((13)C)/n((12)C) isotope dilution mass spectrometry (IRM/IDMS) provides results of low uncertainty of the order of 0.1% if it is applied to the analysis of simple mixtures as found in organic chemistry, even if only low (13)C spike additives to the sample are used. If the method is applied to the analysis of systems that require large-scale sample preparation prior to the measurement, such as the determination of glucose in serum, the results obtained exhibit a higher uncertainty that is comparable to that of the conventional gas chromatography/isotope dilution mass spectrometry (GC/IDMS) method. The reason for this observation is that the small contribution that the IRM/IDMS method makes to the uncertainty budget of the result is superimposed on a large contribution due to the sample preparation. It appears therefore that the IRM/IDMS method has no advantage over the conventional GC/IDMS method. However, if a series of measurements is carried out, and if a suitable experimental design is chosen, the IRM/IDMS method can provide valuable additional information. The influence of sample preparation on each individual result can be quantified as its deviation from the average value of all results of the series. From these data conclusions can be drawn for an improvement in sample preparation.

Role of keratinocyte growth factor in the control of surfactant synthesis by fetal lung mesenchyme.

Fetal lung maturation is regulated by mesenchymal-epithelial cell communication, which plays a major role in the control of surfactant synthesis by alveolar type II cells. We have recently shown that keratinocyte growth factor (KGF), also called fibroblast growth factor-7, enhances the maturation of fetal alveolar epithelial type II cells. Here, we investigated, among the factors produced by lung mesenchyme, the part attributable to KGF in the control of surfactant synthesis. Using a KGF-neutralizing antibody, we assessed surfactant phospholipid synthesis by measuring choline incorporation into disaturated phosphatidylcholine of isolated fetal type II cells. We found that KGF accounts for about half of the stimulating activity present in fetal lung fibroblast-conditioned medium (FCM). By contrast, the use of an epidermal growth factor-neutralizing antibody did not alter the FCM-stimulating activity. To further delineate KGF properties as a mesenchymal mediator, we wondered about its possibility to relay glucocorticoid-stimulating activity on the synthesis of the phospholipid moiety of surfactant in fetal lung fibroblasts. A 24-h exposure to dexamethasone led us to detect a 50% increase in the level of KGF messenger RNA (mRNA) in isolated fetal lung fibroblasts. Moreover, anti-KGF antibody totally abolished the further increase of FCM-stimulating activity induced by dexamethasone. Thus, KGF seems to be a major player in mediating glucocorticoid stimulation of fetal lung maturation.

Memory effects in combining isotope ratio monitoring with isotope dilution mass spectrometry.

Combining isotope ratio monitoring with isotope dilution techniques provides very accurate results in the quantitative analysis of volatile organic chemical compounds by gas chromatography/mass spectrometry (GC/MS). However, this method requires that spikes highly enriched in (13)C be used. This may lead to memory effects which will be investigated in more detail. They occur when the component of the mixture to be investigated exhibits an isotope ratio which is different from that of the component eluted earlier from the column during the chromatographic separation process. A residue of this component, which is shown in the gas chromatogram as tailing, falsifies the result of the isotope ratio measurement. This also leads to false amount-of-substance measurement results. Memory effects can be avoided by using spikes of low (13)C content, by adjusting the composition of the reference solution to that of the sample, or by ensuring effective sample preparation, thus separating disturbing mixture components prior to the measurement. Copyright 1999 John Wiley & Sons, Ltd.

Differential roles of upstream stimulatory factors 1 and 2 in the transcriptional response of liver genes to glucose.

USF1 and USF2 are ubiquitous transcription factors of the basic helix-loop-helix leucine zipper family. They form homo- and heterodimers and recognize a CACGTG motif termed E box. In the liver, USF binding activity is mainly accounted for by the USF1/USF2 heterodimer, which binds in vitro the glucose/carbohydrate response elements (GlRE/ChoRE) of glucose-responsive genes. To assign a physiological role of USFs in vivo, we have undertaken the disruption of USF1 and USF2 genes in mice. We present here the generation of USF1-deficient mice. In the liver of these mice, we demonstrate that USF2 remaining dimers can compensate for glucose responsiveness, even though the level of total USF binding activity is reduced by half as compared with wild type mice. The residual USF1 binding activity was similarly reduced in the previously reported USF2 -/- mice in which an impaired glucose responsiveness was observed (Vallet, V. S., Henrion, A. A., Bucchini, D., Casado, M. , Raymondjean, M., Kahn, A., and Vaulont, S. (1997) J. Biol. Chem. 272, 21944-21949). Taken together, these results clearly suggest differential transactivating efficiencies of USF1 and USF2 in promoting the glucose response. Furthermore, they support the view that USF2 is the functional transactivator of the glucose-responsive complex.

Glucose-dependent liver gene expression in upstream stimulatory factor 2 -/- mice.

Upstream stimulatory factors (USF) 1 and 2 belong to the Myc family of transcription factors characterized by a basic/helix loop helix/leucine zipper domain responsible for dimerization and DNA binding. These ubiquitous factors form homo- and heterodimers and recognize in vitro a CACGTG core sequence termed E box. Through binding to E boxes of target genes, USF factors have been demonstrated to activate gene transcription and to enhance expression of some genes in response to various stimuli. In particular, in the liver USF1 and USF2 have been shown to bind in vitro glucose/carbohydrate response elements of glycolytic and lipogenic genes and have been proposed, from ex vivo experiments, to be involved in their transcriptional activation by glucose. However, the direct involvement of these factors in gene expression and nutrient gene regulation in vivo has not yet been demonstrated. Therefore, to gain insight into the specific role of USF1 and USF2 in vivo, and in particular to determine whether the USF products are required for the response of genes to glucose, we have created, by homologous recombination, USF2 -/- mice. In this paper, we provide the first evidence that USF2 proteins are required in vivo for a normal transcriptional response of L-type pyruvate kinase and Spot 14 genes to glucose in the liver.

Mouse USF1 gene cloning: comparative organization within the c-myc gene family.

Upstream stimulatory factors (USF/MLTF) belong to the c-myc family of transcription factors. Through binding to target DNA as dimers, the ubiquitous USF proteins regulate a variety of genes. USF proteins are encoded by two genes, USF1 and USF2. Protein sequences of USF1 and 2 are highly homologous across species, suggesting functional conservation. To determine whether the genomic organization was conserved between USF1 and USF2, we isolated the murine USF1 gene and characterized its genomic structure. Both genes are similarly organized in 10 exons spanning over 10 kbp. By the 5'-rapid amplification of cDNA ends and S1 nuclease mapping methods, exon 1 was defined and the transcription initiation sites were mapped. The sequence of 8 kb of the gene, including 1.75 kb of 5'-flanking DNA, was determined. The promoter region is GC rich and lacks a typical TATA or CCAAT element. Strikingly, a comparison of the murine and human untranslated sequences reveals regions that exhibit greater than 73% sequence identity. A genomic alignment of the dimerization and DNA binding domains is presented for five genes of the c-myc family, suggesting a hypothetical common ancestor gene.

Bcl-2 protects from lethal hepatic apoptosis induced by an anti-Fas antibody in mice.

Fas is an apoptosis-signalling cell surface antigen that has been shown to trigger cell death upon specific ligand or antibody binding. Treatment of mice with an anti-Fas antibody causes fulminant hepatic failure due to massive apoptosis. To test a putative protective effect of the anti-apoptotic Bcl-2 protein, transgenic mice were generated to express the human bcl-2 gene product in hepatocytes. Early onset of massive hepatic apoptosis leading to death was observed in all nontransgenic mice treated with an anti-Fas antibody. By contrast, hepatic apoptosis was delayed and dramatically reduced in transgenic animals, yielding a 93% survival rate. These results demonstrate that Bcl-2 is able to protect from in vivo Fas-mediated cytotoxicity, and could be of significance for preventing fulminant hepatic failure due to viral hepatitis in humans.

Immunochemical characterization and transacting properties of upstream stimulatory factor isoforms.

The ubiquitous upstream stimulatory factor (USF) transcription factors encoded by two distinct genes (USF1 and USF2) exist under the form of various dimers able to bind E-boxes. We report the molecular cloning and functional characterization of USF2 isoforms, corresponding to a 44-kDa subunit, USF2a, and a new 38-kDa subunit, USF2b, generated by differential splicing. Using specific anti-USF antibodies, we define the different binding complexes in various nuclear extracts. In vivo, the USF1/USF2a heterodimer represents over 66% of the USF binding activity whereas the USF1 and USF2a homodimers represent less than 10%, which strongly suggests an in vivo preferential association in heterodimers. In particular, an USF1/USF2b heterodimer accounted for almost 15% of the USF species in some cells. The preferential heterodimerization of USF subunits was reproduced ex vivo, while the in vitro association of cotranslated subunits, or recombinant USF proteins, appeared to be random. In transiently transfected HeLa or hepatoma cells, USF2a and USF1 homodimers transactivated a minimal promoter with similar efficiency, whereas USF2b, which lacks an internal 67-amino acid domain, was a poor transactivator. Additionally, USF2b was an efficient as USF1 and USF2a homodimers in transactivating the liver-specific pyruvate kinase gene promoter.

Structure, sequence, and chromosomal location of the gene for USF2 transcription factors in mouse.

The ubiquitously expressed upstream stimulatory factor (USF) involved in the transcription of a wide variety of cellular genes is defined as dimers of c-myc-related proteins, composed of a basic helix-loop-helix/leucine zipper region. The USF family consists of different members that split into two groups: MLTF or USF1 and USF2 or FIP. We present here evidence that USF1 and USF2 are distinct closely related genes in human, rat, and mouse. Based on the recent cloning of rat and human new cDNAs, we have isolated genomic clones encompassing the murine USF2 gene, which consists of at least 10 exons spanning a minimum of 10 kb of genomic DNA. Unexpectedly, the organization of USF2 appears very split up by introns (0.08 to over 6 kb in size), compared to the myc gene structure. The entire gene (but the larger intron) and 1.6 kb of the 5' flanking region were sequenced. This 5' flanking region is GC-rich, contains several putative transcription binding sites, and has no apparent TATA box. Gene mapping of murine USF2 and USF1 has been determined by in situ hybridization, indicating the localization of USF2 on chromosome 7 and of USF1 on chromosomes 1 and 11.