High levels of DNA methyltransferase 1 (DNMT1), hypermethylation, and downregulation of GAD(67) and reelin have been described in GABAergic interneurons of patients with schizophrenia (SZ) and bipolar (BP) disorders. However, overexpression of DNMT1 is lethal, making it difficult to assess the direct effect of high levels of DNMT1 on neuronal development in vivo. We therefore used Dnmt1(tet/tet) mouse ES cells that overexpress DNMT1 as an in vitro model to investigate the impact of high levels of DNMT1 on neuronal differentiation. Although there is down-regulation of DNMT1 during early stages of differentiation in wild type and Dnmt1(tet/tet) ES cell lines, neurons derived from Dnmt1(tet/tet) cells showed abnormal dendritic arborization and branching. The Dnmt1(tet/tet) neuronal cells also showed elevated levels of functional N-methyl d-aspartate receptor (NMDAR), a feature also reported in some neurological and neurodegenerative disorders. Considering the roles of reelin and GAD(67) in neuronal networking and excitatory/inhibitory balance, respectively, we studied methylation of these genes' promoters in Dnmt1(tet/tet) ES cells and neurons. Both reelin and GAD(67) promoters were not hypermethylated in the Dnmt1(tet/tet) ES cells and neurons, suggesting that overexpression of DNMT1 may not directly result in methylation-mediated repression of these two genes. Taken together, our results suggest that overexpression of DNMT1 in ES cells results in an epigenetic change prior to the onset of differentiation. This epigenetic change in turn results in abnormal neuronal differentiation and upregulation of functional NMDA receptor.

Deletion analysis of mouse DNMT1, the primary maintenance methyltransferase in mammals, showed that most of the N-terminal regulatory domain (amino acid residues 412-1112) is required for its enzymatic activity. Although analysis of deletion mutants helps to identify regions of a protein sequence required for a particular activity, amino acid deletions can have drastic effects on protein structure and/or stability. Alternative approaches represented by rational design and directed evolution are resource demanding, and require high-throughput selection or screening systems. We developed Regional Frame-shift Mutagenesis (RFM) as a new approach to identify portions required for the methyltransferase activity of DNMT1 within the N-terminal 89-905 amino acids. In this method, a short stretch of amino acids in the wild-type protein is converted to a different amino acid sequence. The resultant mutant protein retains the same amino acid length as the wild type, thereby reducing physical constrains on normal folding of the mutant protein. Using RFM, we identified three small regions in the amino-terminal one-third of the protein that are essential for DNMT1 function. Two of these regions (amino acids 124-160 and 341-368) border a large disordered region that regulates maintenance methylation activity. This organization of DNMT1's amino terminus suggests that the borders define the position of the disordered region within the DNMT1 protein, which in turn allows for its proper function.

PURPOSE: The relationship between obstructive sleep apnea (OSA) and atherosclerosis-related inflammation has been poorly investigated, particularly focusing on functional responses of immune cells playing a key role in atherogenesis and in comparison with control groups with similar cardiovascular risk factors which are known to be themselves associated with inflammation. We sought to determine cellular tumor necrosis factor-alpha (TNF-alpha) production from peripheral blood mononuclear cells (PBMCs) and interleukin (IL)-8 release from neutrophils (PMNs) in patients studied for suspected OSA. METHODS: Thirty-six consecutive patients who underwent a nocturnal complete cardiorespiratory evaluation for suspected OSA were initially evaluated. Serum, PBMCs, and PMNs were isolated (at baseline and after 12 weeks) from patients with apnea-ipopnea index (AHI)>20 (OSA group, n = 16) and from control patients with AHI <5 (nonOSA group, n = 11). All patients continued the same pharmacological therapy for 12 weeks; the OSA group was additionally treated with nocturnal continuous positive-airway-pressure ventilation (cPAP). RESULTS: The two groups had similar clinical characteristics (prevalence of hypertension, dyslipidemia, diabetes, and cardio-metabolic therapies) except for obesity. Resting and stimulated TNF-alpha production from PBMCs and IL-8 release from PMNs were similar in the two groups. Serum cytokines resulted within the normal range. In the OSA group, cPAP was not associated with changes in cellular responses. CONCLUSIONS: In patients showing similar prevalence of major cardiovascular risk factors and cardio-metabolic therapies, differing for the presence or absence of OSA, cytokine productions from PBMC and PMN were similar and were not modified during cPAP therapy. Studies designed to investigate OSA-associated inflammation should carefully match the control group subjects.

Long-term treatment of Parkinson disease (PD) is frequently associated with l-3,4-dihydroxyphenylalanine (l-DOPA)-induced dyskinesias (LIDs). l-DOPA-induced dyskinesias are likely due to changes in the signal transduction pathways, at the striatal level, related to pulsatile stimulation of dopamine receptors. We investigated whether markers of this phenomenon can also be detected peripherally. We analyzed mRNA expression for D5 (D1-like) and D3 (D2-like) receptors and levels of second messengers, such as cAMP and free intracellular Ca ([Ca]i), in peripheral blood lymphocytes of PD patients with (LID+) or without LIDs (LID-). Patients with PD showed depressed [Ca]i rise in response to mitogen-induced activation. The defect was more pronounced in LID+(-33% with respect to healthy controls) than in LID- patients (-20%). Peripheral blood lymphocyte levels of cAMP were decreased in both LID+(3.8 +/- 2.9 pmol/10 cells) and LID- patients (4.2 +/- 2.4 pmol/10 cells), with respect to controls (6 +/- 2.6 pmol/10 cells). No differences were found in dopamine receptor mRNA expression. Our results demonstrate that second messenger levels are altered in the peripheral blood lymphocytes of PD patients treated with dopaminergic agents and that patients with LIDs show further alterations in the regulation of [Ca]i homeostasis. This may represent a distinctive trait of patients prone to develop dyskinetic movements.

CD4(+)CD25(+) regulatory T lymphocytes (Treg) are specialized T cells playing a key role in the control of immune homeostasis. Here we show that human Treg constitutively express tyrosine hydroxylase (TH, EC 1.14.16.2), the rate-limiting enzyme in the synthesis of catecholamines, and contain substantial amounts of dopamine, norepinephrine and epinephrine, which are released upon treatment with reserpine. Catecholamine release results in reduced production of interleukin-10 and transforming growth factor-beta by Treg, and in down regulation of Treg-dependent inhibition of effector T lymphocyte (Teff) proliferation, which occurs without affecting the production of tumor necrosis factor-alpha or interferon-gamma. Treg and Teff express on the cell membrane both D1-like and D2-like dopaminergic receptors to a similar extent (12-29% of the cells). Catecholamine-dependent down regulation of Treg is however selectively reversed by pharmacological blockade of dopaminergic D1-like receptors, which in Treg only (and not in Teff) are expressed also at the level of mRNA and are functionally coupled to intracellular production of cAMP. These findings indicate that in human Treg endogenous catecholamines subserve an autocrine/paracrine loop involving dopaminergic pathways and resulting in down regulation of Treg function.

Intracellular free calcium concentrations (Ca++i) were studied in polymorphonuclear leukocytes (PMNs) from 13 athyreotic patients who had been previously treated by total thyroidectomy and radioiodine therapy for differentiated thyroid carcinoma, and from age- and sex-matched euthyroid healthy controls. Patients were studied twice, when hypothyroid (visit 1) and after restoration of euthyroidism by L-T4 TSH-suppressive therapy (visit 2). PMNs from patients at visit 1 had significantly lower resting (Ca++)i levels compared to both visit 2 and controls. Values at visit 2 did not differ from those of the controls. Stimulus-induced (Ca++)i rise was also significantly blunted at visit 1 and normalized at visit 2, possibly through a differential contribution of distinct intracellular Ca++ stores, as suggested by the response pattern to the chemotactic agent, N-formyl-Met-Leu-Phe (fMLP), to the selective SERCA pump inhibitor, thapsigargine, and to the mitochondrial uncoupler, carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone (FCCP). In vitro treatment of PMNs from healthy subjects with high TSH concentrations impaired intracellular Ca++ store function. Both resting (Ca++)i levels and fMLP-induced (Ca++)i rise increased in the presence either of low-concentration TSH or of T4, but effects of TSH and T4 were not additive. T3, rT3, and TRIAC had no effect. In conclusion, this study provides evidence for a direct relationship between thyroid status and (Ca++)i homeostasis in human PMNs, mainly related to direct actions of TSH and T4 on these cells.

This paper presents the methodology and the main results of a study that has as its principal aim, the experimental identification the application and the quantification of the sustainable indicator 'Percentage of the population exposed to acoustic pollution levels' in the 'Hospital' area of Turin. The investigation of this indicator was prompted by the province of Turin's previous categorisation as 'Lead City' in the project 'Towards a support description at local the level--European Community Indicators'. Alongside, the process of experimenting, applying and quantifying the sustainable indicator--and as a logical fit with what was foreseen by the Italian normative regulation on protection against environmental acoustic pollution--the study supplies the base elements to analyse the environmental acoustic climate conditions in an important area of the city and defines the interaction tools that are the environmental indicators for the town council's future Acoustic Restoration Plan (PRA).

Twenty-seven patients with hay fever had a carbachol inhalation challenge both out of season and during the pollen season. Eight patients with allergic asthma were used as a control group. Only three patients (11.1%) demonstrated a value of a provocative dose causing a 20% fall in FEV1 in the asthmatic range out of pollen season, but during pollen exposure, the number of positive responses significantly increased to 13 (48.1%). We observed differences regarding mean age, age of onset of symptoms, sex, and family history between patients with positive responses and patients who failed to react to inhalation challenge. It appears reasonable that an aspecific bronchial provocation test, performed during the pollen season, can detect with greater sensitivity patients with hay fever at risk of developing asthma in the future, and it also appears reasonable that these patients should be treated differently from subjects with "pure" allergic rhinitis. We expect the ongoing follow-up to clarify the prognostic value to be attributed to these findings.

The pharmacokinetic profiles of cimetropium bromide, after either intravenous injection of 10 mg or oral ingestion of 200 mg, were determined in eight healthy volunteers. After intravenous administration, the plasma levels and urinary excretion indicated that the drug is distributed and eliminated at a rapid rate (terminal half-life, 50 +/- 8 min) and that urinary excretion is not the exclusive route of elimination (46 +/- 2%) of the administered dose). After oral administration, a low percentage of the drug is absorbed (1-4% of the administered dose), however, the amount is sufficient for therapeutic effect. The absorption is discontinuous, with two distinct phases, and ends abruptly during the second phase.