Expression of alpha-synuclein is increased in the hippocampus of rats with high levels of innate anxiety.

A genomic region neighboring the alpha-synuclein gene, on rat chromosome 4, has been associated with anxiety- and alcohol-related behaviors in different rat strains. In this study, we have investigated potential molecular and physiological links between alpha-synuclein and the behavioral differences observed between Lewis (LEW) and Spontaneously Hypertensive (SHR) inbred rats, a genetic model of anxiety. As expected, LEW rats appeared more fearful than SHR rats in three anxiety models: open field, elevated plus maze and light/dark box. Moreover, LEW rats displayed a higher preference for alcohol and consumed higher quantities of alcohol than SHR rats. alpha-Synuclein mRNA and protein concentrations were higher in the hippocampus, but not the hypothalamus of LEW rats. This result inversely correlated with differences in dopamine turnover in the hippocampus of LEW and SHR rats, supporting the hypothesis that alpha-synuclein is important in the downregulation of dopamine neurotransmission. A novel single nucleotide polymorphism was identified in the 3'-untranslated region (3'-UTR) of the alpha-synuclein cDNA between these two rat strains. Plasmid constructs based on the LEW 3'-UTR sequence displayed increased expression of a reporter gene in transiently transfected PC12 cells, in accordance with in-vivo findings, suggesting that this nucleotide exchange might participate in the differential expression of alpha-synuclein between LEW and SHR rats. These results are consistent with a novel role for alpha-synuclein in modulating rat anxiety-like behaviors, possibly through dopaminergic mechanisms. Since the behavioral and genetic differences between these two strains are the product of independent evolutionary histories, the possibility that polymorphisms in the alpha-synuclein gene may be associated with vulnerability to anxiety-related disorders in humans requires further investigation.

Collagen content, but not the ratios of collagen type III/I mRNAs, differs among hypertensive, alcoholic, and idiopathic dilated cardiomyopathy

Cardiac interstitial fibrosis may contribute to ventricular dysfunction and the prognosis of patients with dilated cardiomyopathy. The objective of the present study was to determine if total myocardial collagen content and collagen type III/I (III/I ratio) mRNAs differ in hypertensive, alcoholic, and idiopathic dilated cardiomyopathy subjects. Echocardiography and exercise cardiopulmonary testing were performed in patients with idiopathic (N = 22), hypertensive (N = 12), and alcoholic (N = 11) dilated cardiomyopathy. Morphometric analysis of collagen was performed in fragments obtained by endomyocardial biopsy with picrosirius red staining. The collagen III/I ratio was determined by reverse transcription polymerase chain reaction. Samples of controls (N = 10) were obtained from autopsy. Echocardiographic variables and maximal oxygen uptake were not different among dilated cardiomyopathy groups. Collagen was higher in all dilated cardiomyopathy groups (idiopathic, hypertensive and alcoholic, 7.36 ± 1.09 percent) versus controls (1.12 ± 0.18 percent), P < 0.05. Collagen was lower in idiopathic dilated cardiomyopathy (4.97 ± 0.83 percent) than hypertensive (8.50 ± 1.11 percent) and alcoholic (10.77 ± 2.09 percent) samples (P < 0.005 for both). The collagen III/I ratio in all samples from dilated cardiomyopathy patients was higher compared to that in controls (0.29 ± 0.04, P < 0.05) but was the same in the samples from idiopathic (0.77 ± 0.07), hypertensive (0.75 ± 0.07), and alcoholic (0.81 ± 0.16) dilated cardiomyopathy groups. Because of the different physical properties of the types of collagen, the higher III/I ratio may contribute to progressive ventricular dilation and dysfunction in dilated cardiomyopathy patients.

Collagen content, but not the ratios of collagen type III/I mRNAs, differs among hypertensive, alcoholic, and idiopathic dilated cardiomyopathy.

Cardiac interstitial fibrosis may contribute to ventricular dysfunction and the prognosis of patients with dilated cardiomyopathy. The objective of the present study was to determine if total myocardial collagen content and collagen type III/I (III/I ratio) mRNAs differ in hypertensive, alcoholic, and idiopathic dilated cardiomyopathy subjects. Echocardiography and exercise cardiopulmonary testing were performed in patients with idiopathic (N = 22), hypertensive (N = 12), and alcoholic (N = 11) dilated cardiomyopathy. Morphometric analysis of collagen was performed in fragments obtained by endomyocardial biopsy with picrosirius red staining. The collagen III/I ratio was determined by reverse transcription polymerase chain reaction. Samples of controls (N = 10) were obtained from autopsy. Echocardiographic variables and maximal oxygen uptake were not different among dilated cardiomyopathy groups. Collagen was higher in all dilated cardiomyopathy groups (idiopathic, hypertensive and alcoholic, 7.36 +/- 1.09%) versus controls (1.12 +/- 0.18%), P < 0.05. Collagen was lower in idiopathic dilated cardiomyopathy (4.97 +/- 0.83%) than hypertensive (8.50 +/- 1.11%) and alcoholic (10.77 +/- 2.09%) samples (P < 0.005 for both). The collagen III/I ratio in all samples from dilated cardiomyopathy patients was higher compared to that in controls (0.29 +/- 0.04, P < 0.05) but was the same in the samples from idiopathic (0.77 +/- 0.07), hypertensive (0.75 +/- 0.07), and alcoholic (0.81 +/- 0.16) dilated cardiomyopathy groups. Because of the different physical properties of the types of collagen, the higher III/I ratio may contribute to progressive ventricular dilation and dysfunction in dilated cardiomyopathy patients.

Anabolic steroids induce cardiac renin-angiotensin system and impair the beneficial effects of aerobic training in rats.

We evaluated the effects of swimming and anabolic steroids (AS) on ventricular function, collagen synthesis, and the local renin-angiotensin system in rats. Male Wistar rats were randomized into control (C), steroid (S; nandrolone decanoate; 5 mg/kg sc, 2x/wk), steroid + losartan (SL; 20 mg.kg(-1).day(-1)), trained (T), trained + steroid (T+S), and trained + steroid + losartan (T+SL; n = 14/group) groups. Swimming was performed 5 times/wk for 10 wk. Serum testosterone increased in S and T+S. Resting heart rate was lower in T and T+S. Percent change in left ventricular (LV) weight-to-body weight ratio increased in S, T, and T+S. LV systolic pressure declined in S and T+S. LV contractility increased in T (P < 0.05). LV relaxation increased in T (P < 0.05). It was significantly lower in T+S compared with C. Collagen volumetric fraction (CVF) and hydroxyproline were higher in S and T+S than in C and T (P < 0.05), and the CVF and LV hypertrophy were prevented by losartan treatment. LV-ANG I-converting enzyme activity increased (28%) in the S group (33%), and type III collagen synthesis increased (56%) in T+S but not in T group. A positive correlation existed between LV-ANG I-converting enzyme activity and collagen type III expression (r(2) = 0.88; P < 0.05, for all groups). The ANG II and angiotensin type 1a receptor expression increased in the S and T+S groups but not in T group. Supraphysiological doses of AS exacerbated the cardiac hypertrophy in exercise-trained rats. Exercise training associated with AS induces maladaptive remodeling and further deterioration in cardiac performance. Exercise training associated with AS causes loss of the beneficial effects in LV function induced by exercising. These results suggest that aerobic exercise plus AS increases cardiac collagen content associated with activation of the local renin-angiotensin system.

Social isolation and expression of serotonergic neurotransmission-related genes in several brain areas of male mice.

Early-life events influence brain development and evoke long-lasting behavioral consequences. Postweaning social isolation in rodents induces emotional and neurochemical alterations similar to those observed among some human psychopathologies. Central serotonergic neurotransmission is intimately involved in the observed adjustments, but the impact of social deprivation on serotonergic gene expression is unknown. We investigated the effects of prolonged early social isolation on emotion-related behaviors and 5-hydroxytryptamine (5-HT)-related gene transcription in mice. After weaning, male C57BL/6J mice were reared singly or in groups of four for 6 weeks. Gene expression of 5-HT(1A), 5-HT(1B), 5-HT(2A), 5-HT(2C), 5-HT(3A), 5-HT(6) and 5-HT(7) receptors and of 5-HT transporter and tryptophan hydroxylase-2 was determined by quantitative real-time polymerase chain reaction in distinct brain areas. Single-housed mice were hyperactive in a novel environment and showed signs of aggressive behavior. Housing condition did not alter weight gain or body temperature. Isolation markedly reduced transcription of all postsynaptic 5-HT receptors in the prefrontal cortex and reduced 5-HT(1B), 5-HT(2A) and 5-HT(2C) in both hypothalamus and midbrain. In contrast, the only alteration in the hippocampus was 5-HT(6) overexpression. Neither 5-HT transporter nor synthetic enzyme gene transcription differed between housing conditions. In conclusion, early social isolation in mice induces robust changes in postsynaptic 5-HT receptors gene transcription, motor hyperactivity and behavioral disinhibition. The overall pattern of decreased gene expression in the prefrontal cortex highlights its high vulnerability to environment. Furthermore, this is the first study to present a general representation of 5-HT-related gene expression in specific brain areas after social isolation and identifies novel candidates that may be critical for underlying molecular mechanisms.

The contribution of 700,000 ORF sequence tags to the definition of the human transcriptome.

Open reading frame expressed sequences tags (ORESTES) differ from conventional ESTs by providing sequence data from the central protein coding portion of transcripts. We generated a total of 696,745 ORESTES sequences from 24 human tissues and used a subset of the data that correspond to a set of 15,095 full-length mRNAs as a means of assessing the efficiency of the strategy and its potential contribution to the definition of the human transcriptome. We estimate that ORESTES sampled over 80% of all highly and moderately expressed, and between 40% and 50% of rarely expressed, human genes. In our most thoroughly sequenced tissue, the breast, the 130,000 ORESTES generated are derived from transcripts from an estimated 70% of all genes expressed in that tissue, with an equally efficient representation of both highly and poorly expressed genes. In this respect, we find that the capacity of the ORESTES strategy both for gene discovery and shotgun transcript sequence generation significantly exceeds that of conventional ESTs. The distribution of ORESTES is such that many human transcripts are now represented by a scaffold of partial sequences distributed along the length of each gene product. The experimental joining of the scaffold components, by reverse transcription-PCR, represents a direct route to transcript finishing that may represent a useful alternative to full-length cDNA cloning.

Glucocorticoid regulation of angiotensin-converting enzyme in primary culture of adult cardiac fibroblasts.

The effect of dexamethasone on angiotensin-converting enzyme (ACE) in primary culture of adult cardiac fibroblasts was analyzed in this study. ACE is central to cardiac remodeling in conditions such as myocardial infarction (MI). Some studies indicate that glucocorticoids are often increased post-MI, whereas other studies suggest that glucocorticoids stimulate ACE activity in various cell types. Most cardiac cells are fibroblasts, which have an important function in cardiac remodeling. Therefore, we studied the effects of glucocorticoids on ACE activity and mRNA levels in primary cultures of adult rat cardiac fibroblasts. Steady-state ACE activity was very low, but it increased sixfold with dexamethasone (1 microM for 48 h) treatment. ACE activation occurred within 12 h and peaked at 96 h, after treatment. RNase-protection assays revealed an associated threefold increase (P < 0.05) in ACE mRNA. Dexamethasone's stimulatory effect was abolished by an RNA synthesis inhibitor (actinomycin D, 5 microg/ml) but was potentiated by a protein synthesis inhibitor (cycloheximide, 5 microg/ml). The glucocorticoid-mediated response appears to be specific, because mineralocorticoid treatment did not alter ACE activity. These findings indicate that both transcriptional and posttranscriptional mechanisms are involved in glucocorticoid regulation of ACE expression in rat cardiac fibroblasts.

Identification of human chromosome 22 transcribed sequences with ORF expressed sequence tags.

Transcribed sequences in the human genome can be identified with confidence only by alignment with sequences derived from cDNAs synthesized from naturally occurring mRNAs. We constructed a set of 250,000 cDNAs that represent partial expressed gene sequences and that are biased toward the central coding regions of the resulting transcripts. They are termed ORF expressed sequence tags (ORESTES). The 250,000 ORESTES were assembled into 81,429 contigs. Of these, 1, 181 (1.45%) were found to match sequences in chromosome 22 with at least one ORESTES contig for 162 (65.6%) of the 247 known genes, for 67 (44.6%) of the 150 related genes, and for 45 of the 148 (30.4%) EST-predicted genes on this chromosome. Using a set of stringent criteria to validate our sequences, we identified a further 219 previously unannotated transcribed sequences on chromosome 22. Of these, 171 were in fact also defined by EST or full length cDNA sequences available in GenBank but not utilized in the initial annotation of the first human chromosome sequence. Thus despite representing less than 15% of all expressed human sequences in the public databases at the time of the present analysis, ORESTES sequences defined 48 transcribed sequences on chromosome 22 not defined by other sequences. All of the transcribed sequences defined by ORESTES coincided with DNA regions predicted as encoding exons by genscan. (http://genes.mit.edu/GENSCAN.html).