Biomedical subjects

O Okladnova

Publications and source records attributed to O Okladnova.

18 recordsLinked to original sources

PubMed2002

Increased hippocampal DNA oxidation in serotonin transporter deficient mice.

The serotonin transporter (5HTT) is the molecule responsible for the high-affinity reuptake of 5HT from the synaptic cleft. Mice lacking the 5HTT exhibit highly elevated extracellular concentrations of 5HT. We assessed whether the glutathione detoxification system is altered in 5HTT-deficient mice. While levels of reduced and oxidized glutathione were unchanged, glutathione metabolising enzymes showed a differential pattern of modulation. Glutathione peroxidase was reduced in frontal cortex, brainstem, and cerebellum of 5HTT-deficient mice, though not to a statistically significant extent, while a putative isoform of the detoxifying enzyme glutathione-S-transferase pi was decreased in a number of brain regions, especially in brainstem. At the level of the DNA, we found an increase of oxidative DNA adducts in the hippocampus of 5HTT-deficient mice. Given the importance of the hippocampus in learning and memory, this may be the most important neurochemical consequence of the absence of the 5HTT.

Animals↗

PubMed2001

Association analysis of the functional monoamine oxidase A gene promoter polymorphism in psychiatric disorders.

Functional characterization studies revealed that transcriptional activity of the human monoamine oxidase A (MAOA) gene is modulated by a polymorphic repetitive sequence located approximately 1.2 kb upstream of the ATG codon. To investigate the possible influence of the allelic variants of the MAOA gene-linked polymorphic region (MAOA-LPR) on the genetic predisposition to psychiatric disorders, we have performed a case-control association study. 174 patients with affective disorders and 258 patients with schizophrenia according to DSM-IV, as well as 229 population controls were tested. Statistical analysis showed no significant differences in allele or genotype frequencies between control and patient groups. Our results suggest that there is no association between MAOA-LPR genotype and susceptibility to recurrent major depression, bipolar disorder, and schizophrenia in our population.

Adult↗

PubMed2000

Serotonin transporter function is modulated by brain-derived neurotrophic factor (BDNF) but not nerve growth factor (NGF).

The serotonin transporter (5-HTT) regulates serotonergic neurotransmission by determining the magnitude and duration of serotonergic responses. We have recently described a polymorphism in the 5-HTT gene promoter (5-HTTLPR) which influences the function of the 5-HTT and is associated with several psychiatric disorders. Immortalized B lymphocytes express the 5-HTT, and a B lymphocyte line has been shown to express the receptor for brain-derived neurotrophic factor, trkB. Since brain-derived neurotrophic factor (BDNF) is a specific growth and differentiation factor for serotonergic neurons, we assessed whether BDNF is able to modulate 5-HTT function in B lymphoblasts. Nerve growth factor (NGF), another neurotrophin which acts via the trkA receptor, was also studied. Eight immortalized B lymphoblast lines were generated and genotyped for the 5-HTTLPR. After treatment with BDNF or NGF, 5-HT uptake and proliferation of the cell lines were assessed. Two of the B cell lines showed a dose-dependent reduction of 5-HT uptake after exposure to BDNF. Both of these cell lines were homozygous for the long allele of the 5-HTTLPR. NGF did not influence 5-HT uptake or cellular proliferation in any of the cell lines. Thus, BDNF but not NGF may influence 5-HT uptake in some B lymphocytes. The fact that regulation of the 5-HTT was observed preferentially in cells of the long/long genotype indicates that presence of a short allele confers reduced regulatory capacity on the 5-HTT. In conclusion, B lymphoblasts represent a practical model for functional regulation of the 5-HTT by neurotrophins in serotonergic neurons.

Alleles↗

PubMed2000

Polymorphic MAO-A and 5-HT-transporter genes: analysis of interactions in panic disorder.

Recurrent panic attacks, anticipatory anxiety and phobic avoidance characterise panic disorder. The influence of genetic factors on liability to the disease has been the object of several linkage and association studies and appears to relate to an oligo- or polygenic rather than a monogenic mode of inheritance. Recently, an excess of high activity monoamine oxidase A (MAO-A) gene promoter alleles was found in female patients with panic disorder. An analysis of possible synergistic effects of the MAO-A gene promoter variant and the short serotonin transporter (5-HTT) gene promoter variant in panic disorder was performed in a German and an Italian sample (combined panic disorder n = 144, combined controls n = 175). There was no significant difference in odds ratios, suggesting that the observed increase of genetic liability by the long MAO-A gene promoter allele is not modified by the 5-HTT gene promoter polymorphism.

Adult↗

PubMed1999

Functional PAX-6 gene-linked polymorphic region: potential association with paranoid schizophrenia.

BACKGROUND: Early differentiation of the nervous system and adult CNS neuroplasticity is modulated by PAX-6. We have shown previously that a highly polymorphic, functional AC/AG repeat in the 5' regulatory region of the gene showed significantly increased promoter activity, if containing > or = 29 repeats, and that the heterozygous genotype (< or = 28/> or = 29) revealed increased mRNA PAX-6 levels in human brain tissue compared to the homozygous short variant. METHODS: In a case-control study of 655 unrelated individuals, allele frequencies and genotype distributions of the functional PAX-6 promoter polymorphism were investigated comprising patients with DSM-IV schizophrenia, patients with affective disorders, and population controls. RESULTS: No allelic or genotypic association of the PAX-6 promoter polymorphism to affective disorder or to schizophrenia as one disease entity was observed. After subtyping schizophrenia into paranoid and nonparanoid forms, potential evidence was found for a genotypic association of the high-activity variant with the paranoid subtype of schizophrenia (p = .02). The estimated odds ratio was 1.7 (95% CI .98 to 2.95) for those heterozygous and 1.4 (95% CI .82 to 2.42) for those heterozygous or homozygous for the high-activity variant compared to the homozygous low-activity variant. CONCLUSIONS: Our finding indicates that early developmental genes may be involved in the etiopathogenesis of schizophrenia subtypes via variable transcriptional regulation in the developing and adult human brain.

Adult↗

PubMed1999

Association of a regulatory polymorphism in the promoter region of the monoamine oxidase A gene with antisocial alcoholism.

We analyzed a novel functional 30-bp repeat polymorphism in the promoter region of the X-chromosomal monoamine oxidase A gene (MAOA) to test whether length variation of the repeat polymorphism contributes to variation in the individual vulnerability to antisocial behavior and liability to alcohol dependence. The repeat number (3-5) of the MAOA polymorphism was assessed in 488 male subjects of German descent, a sample comprising 185 psychiatrically screened control subjects and 303 alcohol-dependent subjects including 59 alcoholics with antisocial personality disorder. The frequency of the low-activity 3-repeat allele was significantly increased in 59 antisocial alcoholics compared to 185 control subjects (51 vs. 35%; P = 0.031) and to 244 alcoholics without antisocial personality disorder (51 vs. 32%; P = 0.008), respectively. We found no significant difference in the frequency of the 3-repeat allele between 244 alcoholics without an antisocial personality disorder and the control subjects. Our findings suggest that the low-activity 3-repeat allele of the MAOA promoter polymorphism confers increased susceptibility to antisocial behavior rather than alcohol dependence per se in alcohol-dependent males.

Adult↗

PubMed1999

Functional characterization of the human PAX3 gene regulatory region.

Spatiotemporal expression of the PAX3 gene is tightly regulated during development. We have isolated and sequenced the 5'-flanking regulatory region of human PAX3. Primer extension and ribonuclease protection mapping revealed that transcription is initiated from a single start site downstream of a TATA-like motif in human brain and peripheral tissues. Functional dissection of the gene's 5'-flanking region, which had been fused to a luciferase reporter gene and transiently expressed in rhabdomyosarcoma (RD) and cos-7 cells, indicated that the upstream region of PAX3 contains multiple positive and negative cis-acting regulatory elements. While the basal promoter is likely to be driven by two CCAAT boxes located at nucleotide positions -90 and -135, a cluster of regulatory elements acting as a strong repressor was detected between nucleotides -1200 and -650. Comparison of human and murine sequences revealed more than 90% identity in this segment. A polymorphic (CA)n repeat sequence and a G/C substitution are located 337 bp and 328 bp upstream of the transcription start site, respectively. PCR-based systematic screening for length variations in 225 unrelated individuals of a Caucasian population showed a bimodal distribution of multiple alleles containing between 13 and 30 repeat units. Although the (CA)25 variant of this PAX3 gene-linked polymorphic region (PAX3LPR) conferred lower transcriptional efficiency on the PAX3 promoter, a regulatory impact of the PAX3LPR on PAX3 expression related to brain plasticity and function remains to be demonstrated.

Alleles↗

PubMed1999

Mosaicism for a serotonin transporter gene promoter-associated deletion: decreased recombination in depression.

Transcriptional activity of the human serotonin transporter gene (5HTT) is modulated by complex interaction of multiple genomic and cellular factors. Variability of a polymorphic repetitive element (5HTTLPR) is associated with anxiety, depression, and aggression-related traits and may influence the risk to develop affective spectrum disorders. 5HTTLPR variants display a unique DNA secondary structure that has the potential to regulate the transcriptional activity of the associated 5HTT promoter. The structure of the 5HTTLPR is also likely to precipitate a 381-bp somatic deletion in the 5HTT's promoter region [del(17)(q11.2)] that is observed in 20-60% of genomic DNA isolated from mononuclear blood cells and postmortem brain. The localization of the deletion breakpoints adjacent to identical putative signal sequences (CAGCC) suggests a V(D)J recombinase-like rearrangement event. In comparison with healthy controls, del(17)(q11.2)/wildtype sequence ratios showed a decrease of the deleted variant in recurrent unipolar depression. Our results also suggest that mosaicism of del(17)(q11.2) is likely to be regulated by tissue-specific as well as 5HTTLPR-dependent mechanisms. The findings confirm that the pericentric region of human chromosome 17 is highly unstable and furnishs additional evidence for intricate complexity of 5HTT regulation under physiological condition and in disease.

Adult↗

PubMed1999

Association analysis of a regulatory promoter polymorphism of the PAX-6 gene with idiopathic generalized epilepsy.

The PAX-6 gene is a member of the paired-box-containing (PAX) gene family, encoding a transcriptional activator, that plays an important role in the development of the central nervous system. The present association study tested the hypothesis that length variation of a novel regulatory dinucleotide repeat polymorphism in the promoter region of the PAX-6 gene (PAX-6 gene-linked polymorphic region, PAX-6LPR) confers susceptibility to the epileptogenesis of common subtypes of idiopathic generalized epilepsy (IGE). The repeat length of the regulatory dinucleotide repeat polymorphism was assessed in 354 German control subjects and 125 German IGE patients, comprising 70 patients with juvenile myoclonic epilepsy (JME) and 55 patients with an idiopathic absence epilepsy (IAE). The allelic distribution of the PAX-6LPR did not deviate significantly between the controls and the IGE patients (Wilcoxon Rank-Sum test: P > 0.76), or both subgroups of either JME patients (P > 0.78) or IAE patients (P > 0.87). Our results do not provide evidence that length variation of the polymorphic dinucleotide sequence in the PAX-6LPR contributes a frequent and relevant effect to the pathogenesis of common subtypes of IGE.

Alleles↗

PubMed1999

Excess of high activity monoamine oxidase A gene promoter alleles in female patients with panic disorder.

A genetic contribution to the pathogenesis of panic disorder has been demonstrated by clinical genetic studies. Molecular genetic studies have focused on candidate genes suggested by the molecular mechanisms implied in the action of drugs utilized for therapy or in challenge tests. One class of drugs effective in the treatment of panic disorder is represented by monoamine oxidase A inhibitors. Therefore, the monoamine oxidase A gene on chromosome X is a prime candidate gene. In the present study we investigated a novel repeat polymorphism in the promoter of the monoamine oxidase A gene for association with panic disorder in two independent samples (German sample, n = 80; Italian sample, n = 129). Two alleles (3 and 4 repeats) were most common and constituted >97% of the observed alleles. Functional characterization in a luciferase assay demonstrated that the longer alleles (3a, 4 and 5) were more active than allele 3. Among females of both the German and the Italian samples of panic disorder patients (combined, n = 209) the longer alleles (3a, 4 and 5) were significantly more frequent than among females of the corresponding control samples (combined, n = 190, chi2 = 10.27, df = 1, P = 0.001). Together with the observation that inhibition of monoamine oxidase A is clinically effective in the treatment of panic disorder these findings suggest that increased monoamine oxidase A activity is a risk factor for panic disorder in female patients.

Alleles↗

PubMed1998

Short CAG repeats within the hSKCa3 gene associated with schizophrenia: results of a family-based study.

In a family-based association study we investigated transmission of a multiallelic CAG repeat in a novel neuronal potassium channel gene, hSKCa3, in 59 parent/ offspring trios. In contrast to recent reports of an association of moderately large repeats with schizophrenia in case-control studies, our findings indicate that short CAG repeats (< or=19 repeats) are transmitted at an increased frequency to schizophrenic offspring (p=0.014), particularly among familial cases (p=0.007). No evidence for a parent-of-origin effect was found. Multiallelic TDT procedure showed no association of individual CAG repeats to schizophrenia. Further studies using family-based designs should clarify whether hSKCa3 is a susceptibility factor to schizophrenia or co-segregates with a major disease gene in tight linkage.

Adolescent↗

PubMed1998

Regulation of PAX-6 gene transcription: alternate promoter usage in human brain.

We have isolated and characterized the 5'-flanking regulatory region of the human PAX-6 gene. Mapping of transcription initiation sites revealed the existence of an additional non-coding 5' exon, exon 1A. Functional analyses indicated that PAX-6 transcription is regulated by two distinct promoters, A and B, resulting in alternative transcription of exon 1A or 1B and joint transcription of exons 2 to 13. While a single initiation site was identified for exon 1A, transcription of exon 1B appears to be initiated from more than one site downstream of the promoter B-associated TATA motif. Multiple potential binding sites for transcription factors were found in the regions of promoter A and B. Although a 1.1-kb fragment of promoter A and a 1.5 kb fragment of promoter B, which had been fused to a reporter gene and transiently expressed in cell lines, displayed constitutive promoter activity, transcription of PAX-6 driven by promoter B was considerably higher than by promoter A in various regions of human postmortem brain. Transcript PAX-6B was primarily expressed in cerebellar cortex, whereas relatively low concentrations were detected in other brain areas. Functional dissection by serial deletions revealed several clusters of both activating elements and cell-selective silencers within the regulatory regions upstream of exon 1A and 1B. Coexpression of the promoter B constructs with a vector expressing PAX-6 modulated promoter B activity, thus indicating autoregulation by PAX-6 transcription. In conclusion, our findings suggest that PAX-6 transcription is regulated by alternate usage of promoter A and B, and that in adult human brain expression of PAX-6 is primarily controlled by promoter B. Alternate promoter usage and differential PAX-6 transcription are likely to play a critical role in brain development and neuroplasticity.

3T3 Cells↗

PubMed1998

A promoter-associated polymorphic repeat modulates PAX-6 expression in human brain.

The PAX-6 gene plays a critical role in neurodevelopment and brain plasticity. While transcription of human PAX-6 is regulated by alternate usage of two distinct promoters termed A and B, expression in adult human brain is primarily controlled by promoter B. We now report that a novel polymorphic dinucleotide repeat sequence with the structure (AC)m(AG)n is located approximately 1 kb upstream of the transcription initiation site associated with promoter B. PCR-based systematic screening for length variations in a caucasian population showed a skewed distribution of multiple alleles containing between 24 and 36 repeat units. In 217 unrelated individuals, the frequency of alleles in the range between 25 and 29 repeats was 90%, with the 26 repeat allele alone accounting for 50%; the heterozygosity rate was 65%. Variants of this PAX-6 gene-linked polymorphic region (PAX-6LPR) had different transcriptional efficiencies when fused to a luciferase reporter gene and transfected into Cos-7 cells. Promoter activity of variants with >/=29 repeats was 4- to 9-fold higher than that of the 26 repeat allele. The influence of the PAX-6LPR on PAX-6 expression was confirmed in postmortem cerebellum from individuals with different genotypes. mRNA levels were 2-fold higher in genotypes with long alleles compared to those with short alleles. Allelic variation in PAX-6 expression may be a determinant of interindividual differences in brain plasticity and function.

Alleles↗

PubMed1998

Different ocular abnormalities in individuals of a three-generation family caused by a new nonsense mutation in the PST domain of the PAX6 gene. Mutations in brief no. 189. Online.

PAX6 is a candidate gene for familial aniridia. We have carried out a mutational analysis of the PAX6 gene in a three-generation family from Germany, containing 5 individuals affected with ocular abnormalities. In all affected individuals, a heterozygous mutation was detected in the PAX6 gene, exchanging tyrosine 369 by a stop codon. The mutation is located in the 3' moiety of the PST domain, at the C terminus of the PAX6 protein. In the affected family members, the same heterozygous mutation leads to distinct phenotypes of varying severity. Most notably, no aniridia was observed in one of the family members carrying the mutation, although other ocular abnormalities (underdeveloped iris and cataracts) were present. We discuss the possibility that small C terminal truncations of the PAX6 protein might lead to less severe or more divergent phenotypes than trancations at internal positions.

Aniridia↗

PubMed1998

Cloning and functional characterization of the human norepinephrine transporter gene promoter.

The norepinephrine transporter (NET) plays a critical role in brain norepinephrine homeostasis and is a target for antidepressants and drugs of abuse. We have analyzed the 5'-flanking regulatory region of the human NET gene (SLC6A2). Primer extension and 5' RACE revealed a single transcription start site, alternative splicing of exon 1 due to alternate splice donor usage, and a variable splice acceptor of intron 1. A TA-rich motif 35 bp upstream of the transcription start and several potential binding sites for transcription factors including a cAMP response element (CRE)-like motif are present in the 5'-flanking region. A 4.0-kb fragment, which had been fused to the luciferase reporter gene and transiently expressed in a NET+ cell line, displayed both constitutive and inducible promoter activity. Functional analysis by serial deletions revealed several clusters of cell-selective enhancer elements. Our findings indicate that (1) the NET gene promoter is active in NET-expressing cells and the information contained within approximately 4 kb of the 5'-flanking sequence is required to confer its cell-selective expression, (2) the expression of NET is regulated by a combination of positive cis-acting elements operating through a basal promoter defined by a TA-rich motif, and (3) the promoter responds to cAMP-dependent induction. Fusion of the human NET gene promoter to selected genes will facilitate their cell-selective expression in gene transfer strategies.

Animals↗

PubMed1998

Enhancement of serotonin transporter function by tumor necrosis factor alpha but not by interleukin-6.

Serotonin (5-HT) is a prime candidate for studies of the interaction between the nervous and immune systems, since it is both an important neurotransmitter and released at high concentrations at sites of inflammation. Serotonergic neurotransmission is regulated by the 5-HT transporter (5-HTT), which determines the magnitude and duration of serotonergic responses. Since tumor necrosis factor alpha (TNF-alpha) and interleukin-6 are two inflammatory mediators that are central to the initiation of inflammation, we studied the impact of these cytokines on the 5-HTT. As model system we used a cell line which constitutively expresses the 5-HTT, namely the choriocarcinoma cell line JAR. We found that TNF-alpha enhances 5-HT uptake, with a doubling of the maximal velocity of uptake. Interleukin-6, on the other hand, had no effect. We thus show for the first time that the cytokine TNF-alpha modulates 5-HTT function. Furthermore, we propose a molecular mechanism for this effect. Since both 5-HT and TNF-alpha are elevated at sites of inflammation, TNF-alpha may act to renormalize 5-HT levels by way of its effect on the 5-HTT. This is especially important for the central nervous system, where the TNF-alpha effect shown here can aid in preventing disturbances of serotonergic neurotransmission.

Carrier Proteins↗

PubMed1997

Determination of functional domains of the human transcription factor PAX8 responsible for its nuclear localization and transactivating potential.

The conserved structure of the transcription factors of the Pax gene family may reflect functional conservation. We have demonstrated that the human Pax8 transcription factor is organized in several functional domains and contains two regions responsible for its nuclear localization, in addition to an activating region at the carboxy terminus of the protein and an inhibitory region encoded by the exon 9 present only in a splice variant PAX8a. Regions of PAX8 determining the nuclear localization of the PAX8A/lacZ fusions contain short amino acid sequences similar to several described nuclear localization sites (NLS). These NLS were identified in the paired domain and between the octapeptide and the residual homeodomain, respectively. The activating domain is encoded by the exons 10 and 11 and its function is modulated by the adjacent domains encoded by the exons 9 and 12. The domain encoded by exon 9 significantly inhibits the function of the activating domain. Pax8 is expressed in thyroid cells and its product binds promoters of the thyroglobulin and thyroperoxidase genes through its paired domain. Thyroid cell growth and differentiation depend on thyrotropin which, by stimulating cAMP synthesis, activates the cAMP-dependent protein kinase A (PKA). We have investigated a link between thyrotropin stimulation and gene activation by Pax8. Stimulation of cAMP synthesis augments Pax8-specific transcription in thyroid cells, indicating that PKA is involved in Pax8 activation. Cotransfection of GAL4/PAX8 fusions and the catalytic subunit of PKA in A126, a PKA-deficient derivative of the PC12 pheochromocytoma cell line, synergistically activates the GAL4-specific reporter, suggesting the activating domain of PAX8 is dependent upon the catalytic subunit of the PKA. We propose that this dependence is due to a hypothetical adaptor which forms a target for PKA and interacts with the activating domain of PAX8. We show that PAX8 isolated from the thyroid cell line FTRL5 is a phosphoprotein in which phosphorylation is not dependant on cAMP pathway activation. Our results suggest that Pax8 is part of the cAMP signaling pathway and mediates thyrotropin-dependent gene activation in thyroid cells. Investigation of the PAX8 expression in a panel of Wilms' tumors shows a striking correlation between the expression of PAX8 and another transcription factor, WT1, indicating that these two genes may interact in vivo.

Animals↗

PubMed1997

The genomic organization of the murine Pax 8 gene and characterization of its basal promoter.

Lambda phage clones containing the murine Pax 8 gene were isolated from a C57BL/6 kidney genomic mouse library using mouse cDNA fragments as probes. A clone encompassing about 16 kb of the 5' untranslated region of the murine Pax 8 gene was isolated from a mouse embryonic stem cell (D3) library. The murine Pax 8 gene has a size of approximately 26 kb and contains the coding sequence for mRNA in 12 exons. The major and several minor transcription initiation sites were identified. Position +1 is located 488 nucleotides upstream of the ATG initiation codon and 24 bases downstream of a TATA-like sequence, ATAAAA. The translation initiation and termination sites are located in exons 2 and 12, respectively. Further analysis of 570 bases of the 5' flanking sequence revealed AP2, SP1, PEA3, zeste, NF-kappaB, and CCAAT consensus binding sites. Ribonuclease protection assays with a probe spanning the first two exons of mouse Pax 8 cDNA on total RNA samples isolated from different tissues of newborn mice show that the murine Pax 8 gene is predominantly expressed in kidney tissue. Low levels of Pax 8 gene expression were also found in the liver, spleen, lung, brain, and heart. The same transcription initiation sites are utilized in different tissues of newborn mice and embryo at Day 10.5 postconception. A FISH assay shows that the murine Pax 8 gene is located on chromosome 2, map position B.

Animals↗