Transcriptome alterations of prefrontal cortical parvalbumin neurons in schizophrenia.

Schizophrenia (SZ) is associated with dysfunction of the dorsolateral prefrontal cortex (DLPFC). This dysfunction is manifest as cognitive deficits that appear to arise from disturbances in gamma frequency oscillations. These oscillations are generated in DLPFC layer 3 (L3) via reciprocal connections between pyramidal cells (PCs) and parvalbumin (PV)-containing interneurons. The density of cortical PV neurons is not altered in SZ, but expression levels of several transcripts involved in PV cell function, including PV, are lower in the disease. However, the transcriptome of PV cells has not been comprehensively assessed in a large cohort of subjects with SZ. In this study, we combined an immunohistochemical approach, laser microdissection, and microarray profiling to analyze the transcriptome of DLPFC L3 PV cells in 36 matched pairs of SZ and unaffected comparison subjects. Over 800 transcripts in PV neurons were identified as differentially expressed in SZ subjects; most of these alterations have not previously been reported. The altered transcripts were enriched for pathways involved in mitochondrial function and tight junction signaling. Comparison with the transcriptome of L3 PCs from the same subjects revealed both shared and distinct disease-related effects on gene expression between cell types. Furthermore, network structures of gene pathways differed across cell types and subject groups. These findings provide new insights into cell type-specific molecular alterations in SZ which may point toward novel strategies for identifying therapeutic targets.

Oncogenic activity of amplified miniature chromosome maintenance 8 in human malignancies.

Miniature chromosome maintenance (MCM) proteins play critical roles in DNA replication licensing, initiation and elongation. MCM8, one of the MCM proteins playing a critical role in DNA repairing and recombination, was found to have overexpression and increased DNA copy number in a variety of human malignancies. The gain of MCM8 is associated with aggressive clinical features of several human cancers. Increased expression of MCM8 in prostate cancer is associated with cancer recurrence. Forced expression of MCM8 in RWPE1 cells, the immortalized but non-transformed prostate epithelial cell line, exhibited fast cell growth and transformation, while knock down of MCM8 in PC3, DU145 and LNCaP cells induced cell growth arrest, and decreased tumour volumes and mortality of severe combined immunodeficiency mice xenografted with PC3 and DU145 cells. MCM8 bound cyclin D1 and activated Rb protein phosphorylation by cyclin-dependent kinase 4 in vitro and in vivo. The cyclin D1/MCM8 interaction is required for Rb phosphorylation and S-phase entry in cancer cells. As a result, our study showed that copy number increase and overexpression of MCM8 may play critical roles in human cancer development.

Effects of Kras activation and Pten deletion alone or in combination on MUC1 biology and epithelial-to-mesenchymal transition in ovarian cancer.

Mucin1 (MUC1) is an epithelial glycoprotein overexpressed in ovarian cancer and actively involved in tumor cell migration and metastasis. Using novel in vitro and in vivo MUC1-expressing conditional (Cre-loxP) ovarian tumor models, we focus here on MUC1 biology and the roles of Kras activation and Pten deletion during cell transformation and epithelial-to-mesenchymal transition (EMT). We generated several novel murine ovarian cancer cell lines derived from the ovarian surface epithelia (OSE) of mice with conditional mutations in Kras, Pten or both. In addition, we also generated several tumor-derived new cell lines that reproduce the original tumor phenotype in vivo and mirror late stage metastatic disease. Our results demonstrate that de novo activation of oncogenic Kras does not trigger increased proliferation, cellular transformation or EMT, and prevents MUC1 upregulation. In contrast, Pten deletion accelerates cell proliferation, triggers cellular transformation in vitro and in vivo, and stimulates MUC1 expression. Ovarian tumor-derived cell lines MKP-Liver and MKP-Lung cells reproduce in vivo EMT and represent the first immune competent mouse model for distant hematogenous spread. Whole genome microarray expression analysis using tumor and OSE-derived cell lines reveal a 121 gene signature associated with EMT and metastasis. When applied to n=542 cases from The Cancer Genome Atlas (TCGA) ovarian cancer dataset, the gene signature identifies a patient subset with decreased survival (P=0.04). Using an extensive collection of novel murine cell lines we have identified distinct roles for Kras and Pten on MUC1 and EMT in vivo and in vitro. The data has implications for future design of combination therapies targeting Kras mutations, Pten deletions and MUC1 vaccines.

Distinctive transcriptome alterations of prefrontal pyramidal neurons in schizophrenia and schizoaffective disorder.

Schizophrenia is associated with alterations in working memory that reflect dysfunction of dorsolateral prefrontal cortex (DLPFC) circuitry. Working memory depends on the activity of excitatory pyramidal cells in DLPFC layer 3 and, to a lesser extent, in layer 5. Although many studies have profiled gene expression in DLPFC gray matter in schizophrenia, little is known about cell-type-specific transcript expression in these two populations of pyramidal cells. We hypothesized that interrogating gene expression, specifically in DLPFC layer 3 or 5 pyramidal cells, would reveal new and/or more robust schizophrenia-associated differences that would provide new insights into the nature of pyramidal cell dysfunction in the illness. We also sought to determine the impact of other variables, such as a diagnosis of schizoaffective disorder or medication use at the time of death, on the patterns of gene expression in pyramidal neurons. Individual pyramidal cells in DLPFC layers 3 or 5 were captured by laser microdissection from 36 subjects with schizophrenia or schizoaffective disorder and matched normal comparison subjects. The mRNA from cell collections was subjected to transcriptome profiling by microarray followed by quantitative PCR validation. Expression of genes involved in mitochondrial (MT) or ubiquitin-proteasome system (UPS) functions were markedly downregulated in the patient group (P-values for MT-related and UPS-related pathways were <10(-7) and <10(-5), respectively). MT-related gene alterations were more prominent in layer 3 pyramidal cells, whereas UPS-related gene alterations were more prominent in layer 5 pyramidal cells. Many of these alterations were not present, or found to a lesser degree, in samples of DLPFC gray matter from the same subjects, suggesting that they are pyramidal cell specific. Furthermore, these findings principally reflected alterations in the schizophrenia subjects were not present or present to a lesser degree in the schizoaffective disorder subjects (diagnosis of schizoaffective disorder was the most significant covariate, P<10(-6)) and were not attributable to factors frequently comorbid with schizophrenia. In summary, our findings reveal expression deficits in MT- and UPS-related genes specific to layer 3 and/or layer 5 pyramidal cells in the DLPFC of schizophrenia subjects. These cell type-specific transcriptome signatures are not characteristic of schizoaffective disorder, providing a potential molecular-cellular basis of differences in clinical phenotypes.

Plasma biosignature and brain pathology related to persistent cognitive impairment in late-life depression.

Cognitive impairment is highly prevalent among individuals with late-life depression (LLD) and tends to persist even after successful treatment. The biological mechanisms underlying cognitive impairment in LLD are complex and likely involve abnormalities in multiple pathways, or 'cascades,' reflected in specific biomarkers. Our aim was to evaluate peripheral (blood-based) evidence for biological pathways associated with cognitive impairment in older adults with LLD. To this end, we used a data-driven comprehensive proteomic analysis (multiplex immunoassay including 242 proteins), along with measures of structural brain abnormalities (gray matter atrophy and white matter hyperintensity volume via magnetic resonance imaging), and brain amyloid-ß (Aß) deposition (PiB-positron emission tomography). We analyzed data from 80 older adults with remitted major depression (36 with mild cognitive impairment (LLD+MCI) and 44 with normal cognitive (LLD+NC)) function. LLD+MCI was associated with differential expression of 24 proteins (P<0.05 and q-value <0.30) related mainly to the regulation of immune-inflammatory activity, intracellular signaling, cell survival and protein and lipid homeostasis. Individuals with LLD+MCI also showed greater white matter hyperintensity burden compared with LLD+NC (P=0.015). We observed no differences in gray matter volume or brain Aß deposition between groups. Machine learning analysis showed that a group of three proteins (Apo AI, IL-12 and stem cell factor) yielded accuracy of 81.3%, sensitivity of 75% and specificity of 86.4% in discriminating participants with MCI from those with NC function (with an averaged cross-validation accuracy of 76.3%, sensitivity of 69.4% and specificity of 81.8% with nested cross-validation considering the model selection bias). Cognitive impairment in LLD seems to be related to greater cerebrovascular disease along with abnormalities in immune-inflammatory control, cell survival, intracellular signaling, protein and lipid homeostasis, and clotting processes. These results suggest that individuals with LLD and cognitive impairment may be more vulnerable to accelerated brain aging and shed light on possible mediators of their elevated risk for progression to dementia.

Beyond modules and hubs: the potential of gene coexpression networks for investigating molecular mechanisms of complex brain disorders.

In a research environment dominated by reductionist approaches to brain disease mechanisms, gene network analysis provides a complementary framework in which to tackle the complex dysregulations that occur in neuropsychiatric and other neurological disorders. Gene-gene expression correlations are a common source of molecular networks because they can be extracted from high-dimensional disease data and encapsulate the activity of multiple regulatory systems. However, the analysis of gene coexpression patterns is often treated as a mechanistic black box, in which looming 'hub genes' direct cellular networks, and where other features are obscured. By examining the biophysical bases of coexpression and gene regulatory changes that occur in disease, recent studies suggest it is possible to use coexpression networks as a multi-omic screening procedure to generate novel hypotheses for disease mechanisms. Because technical processing steps can affect the outcome and interpretation of coexpression networks, we examine the assumptions and alternatives to common patterns of coexpression analysis and discuss additional topics such as acceptable datasets for coexpression analysis, the robust identification of modules, disease-related prioritization of genes and molecular systems and network meta-analysis. To accelerate coexpression research beyond modules and hubs, we highlight some emerging directions for coexpression network research that are especially relevant to complex brain disease, including the centrality-lethality relationship, integration with machine learning approaches and network pharmacology.

Expression of GALNT2 in human extravillous trophoblasts and its suppressive role in trophoblast invasion.

Extravillus trophoblast (EVT) invasion plays a critical role in placental development. Integrins bind to extracellular matrix (ECM) proteins to mediate EVT cell adhesion, migration, and invasion. Changes in O-glycans on ß1-integrin have been found to regulate cancer cell behavior. We hypothesize that O-glycosyltransferases can regulate EVT invasion through modulating the glycosylation and function of ß1-integrin. Here, we found that the GALNT1 and GALNT2 mRNA were highly expressed in HTR8/SVneo and first trimester EVT cells. Immunohistochemstry and immunofluorescence staining showed that GALNT2 was expressed in subpopulations of EVT cells in deciduas, but not in syncytiotrophoblasts and cytotrophoblasts of placental villi. The percentage of GALNT2-positive EVT cells increased with gestational ages. Overexpression of GALNT2 in HTR8/SVneo cells significantly enhanced cell-collagen IV adhesion, but suppressed cell migration and invasion. Notably, we found that GALNT2 increased the expression of Tn antigen (GalNAc-Ser/Thr) on ß1-integrin as revealed by Vicia Villosa agglutinin (VVA) binding. Furthermore, GALNT2 suppressed the phosphorylation of focal adhesion kinase (FAK), a crucial downstream signaling molecule of ß1-integrin. Our findings suggest that GALNT2 is a critical initiating enzyme of O-glycosylation for regulating EVT invasion.

Molecular evidence for BDNF- and GABA-related dysfunctions in the amygdala of female subjects with major depression.

Women are twice as likely as men to develop major depressive disorder (MDD) and are more prone to recurring episodes. Hence, we tested the hypothesis that the illness may associate with robust molecular changes in female subjects, and investigated large-scale gene expression in the post-mortem brain of MDD subjects paired with matched controls (n=21 pairs). We focused on the lateral/basolateral/basomedian complex of the amygdala as a neural hub of mood regulation affected in MDD. Among the most robust findings were downregulated transcripts for genes coding for γ-aminobutyric acid (GABA) interneuron-related peptides, including somatostatin (SST), tachykinin, neuropeptide Y (NPY) and cortistatin, in a pattern reminiscent to that previously reported in mice with low brain-derived neurotrophic factor (BDNF). Changes were confirmed by quantitative PCR and not explained by demographic, technical or known clinical parameters. BDNF itself was significantly downregulated at the RNA and protein levels in MDD subjects. Investigating putative mechanisms, we show that this core MDD-related gene profile (including SST, NPY, TAC1, RGS4 and CORT) is recapitulated by complementary patterns in mice with constitutive (BDNF-heterozygous) or activity-dependent (exon IV knockout) decreases in BDNF function, with a common effect on SST and NPY. Together, these results provide both direct (low RNA/protein) and indirect (low BDNF-dependent gene pattern) evidence for reduced BDNF function in the amygdala of female subjects with MDD. Supporting studies in mutant mice models suggest a complex mechanism of low constitutive and activity-dependent BDNF function in MDD, particularly affecting SST/NPY-related GABA neurons, thus linking the neurotrophic and GABA hypotheses of depression.

Methylcobalamin, but not methylprednisolone or pleiotrophin, accelerates the recovery of rat biceps after ulnar to musculocutaneous nerve transfer.

Using ulnar nerve as donor and musculocutaneous nerve as recipient we recently demonstrated that end-to-end neurorrhaphy in young adult male Wistar rats resulted in good recovery following protracted survival. Here we explored whether anti-inflammatory drug- methylprednisolone, regeneration/myelination-enhancing agent- methylcobalamin and neurite growth-enhancing and angiogenic factor- pleiotrophin accelerated its recovery. Methylprednisolone suppressed the perineuronal microglial reaction and periaxonal ED-1 expression while pleiotrophin increased the blood vessel density and nerve fiber densities in the reconnected nerve as expected. Neither methylprednisolone nor methylcobalamin altered the expression of growth associated protein 43 in the neurons examined suggesting that they did not interfere with axonal regeneration attempt. Surprisingly methylcobalamin enhanced the recovery of compound muscle action potentials and motor end plate innervation and the performance on sticker removal grooming test and augmented the diameters and myelin thicknesses of regenerated axons dramatically while enhancing S-100 expression in Schwann cells; remarkable recovery was achieved 1 month following neurorrhaphy. Simultaneous methylcobalamin and pleiotrophin treatment resulted in quick and persistent supernumerary reinnervation but failed to enhance the recovery over that of the former alone. Methylprednisolone transiently suppressed the enumeration of regrowing axons. In conclusion, methylcobalamin may be preferred over methylprednisolone to facilitate the recovery of peripheral nerves following end-to-end neurorrhaphy. The long-term effect of this treatment however remains to be clarified.

Inhibition of prostate cancer growth and metastasis using small interference RNA specific for minichromosome complex maintenance component 7.

Minichromosome complex maintenance component 7 (MCM7) is a critical component of DNA replication licensing. Amplification and overexpression of MCM7 leads to high rate of prostate cancer metastasis. Recent studies indicate that MCM7 genome encodes a putative 'super-oncogene' cluster including MCM7 oncogene and a miRNA cluster that knocks down the expression of several critical tumor-suppressor genes. In this study, we constructed a vector that constitutively expresses small interference RNA (siRNA) specific for MCM7. Introduction of this vector into prostate cancer cell lines PC3 or Du145 decreases the expression of MCM7 by 80%. The vector inhibits DNA synthesis and generates growth arrest of these cancer cells. Severe combined immunodeficient mice were xenografted PC3 or Du145 tumors, and subsequently treated with this vector through tail vein injection with polyethylenimine. The animals had dramatically smaller tumor volume, less metastasis and better survival rate in comparison with the controls. As a result, intervention of MCM7 expression using siRNA approach may hold the promise for treating androgen refractory prostate cancer.

The immediate large-scale dendritic plasticity of cortical pyramidal neurons subjected to acute epidural compression.

Head trauma and acute disorders often instantly compress the cerebral cortex and lead to functional abnormalities. Here we used rat epidural bead implantation model and investigated the immediate changes following acute compression. The dendritic arbors of affected cortical pyramidal neurons were filled with intracellular dye and reconstructed 3-dimensionally for analysis. Compression was found to shorten the apical, but not basal, dendrites of underlying layer III and V cortical pyramidal neurons and reduced dendritic spines on the entire dendritic arbor immediately. Dendrogram analysis showed that in addition to distal, proximal apical dendrites also quickly reconfigured. We then focused on apical dendritic trunks and explored how proximal dendrites were rapidly altered. Compression instantly twisted the microtubules and deformed the membrane contour of dendritic trunks likely a result of the elastic nature of dendrites as immediate decompression restored it and stabilization of microtubules failed to block it. Subsequent adaptive remodeling restored plasmalemma and microtubules to normal appearance in 3 days likely via active mechanisms as taxol blocked the restoration of microtubules and in addition partly affected plasmalemmal reorganization which presumably engaged recycling of excess membrane. In short, the structural dynamics and the associated mechanisms that we revealed demonstrate how compression quickly altered the morphology of cortical output neurons and hence cortical functions consequently.

Efficacy of an intravenous proton pump inhibitor after endoscopic therapy with epinephrine injection for peptic ulcer bleeding in patients with uraemia: a case-control study.

BACKGROUND: Patients with peptic ulcer bleeding and uraemia are prone to re-bleeding. AIM: To compare the efficacy of an intravenous proton pump inhibitor in treating peptic ulcer bleeding in patients with uraemia and those without uraemia. METHODS: High-risk peptic ulcer bleeding patients received endoscopic therapy with epinephrine (adrenaline) injection plus intravenous omeprazole (40 mg bolus followed by 40 mg infusion every 12 h) for 3 days. Re-bleeding, volume of blood transfusion, hospital stay, need for surgery, and mortality were analysed. RESULTS: The uraemic group had similar 7-day re-bleeding rate (6/42, 14.29% vs. 6/46, 13.04%, P = 0.865) to that of non-uraemic patients, but more re-bleeding episodes beyond 7 days (4/42, 9.52% vs. 0/46, 0%, P = 0.032, OR [95% CI] = 1.105 [1.002-1.219]) and all-cause mortality (4/42 vs. 0/46 P = 0.032, OR [95% CI] = 1.105 [1.002-1.219]). The uraemic group also had more units of blood transfusion after endoscopic therapy (mean +/- s.d. 4.33 +/- 3.35 units vs. 2.15 +/- 1.65 units, P < 0.001), longer hospital stay (mean +/- s.d. 8.55 +/- 8.12 days vs. 4.11 +/- 1.60 days, P < 0.001) and complications during hospitalization (9/42 vs. 0/46, P = 0.001, OR [95% CI] = 1.273 [1.087-1.490]). CONCLUSION: Endoscopic therapy with epinephrine injection plus an intravenous proton pump inhibitor can offer protection against early re-bleeding in uraemic patients with peptic ulcer bleeding, but has a limited role beyond 7 days.

Fatigue reversibly reduced cortical and hippocampal dendritic spines concurrent with compromise of motor endurance and spatial memory.

Fatigue could be induced following forced exercise, sickness, heat stroke or sleep disturbance and impaired brain-related functions such as concentration, attention and memory. Here we investigated whether fatigue altered the dendrites of central neurons. Central fatigue was induced by housing rats in cage with 1.5-cm deep water for 1-5 days. Three days of sleep deprivation seriously compromised rats' performance in weight-loaded forced swimming and spatial learning tests, and 5 days of treatment worsened it further. Combinations of intracellular dye injection and three-dimensional analysis revealed that dendritic spines on retrograde tracer-identified corticospinal neurons and Cornu Ammonis (CA)1 and CA3 pyramidal neurons were significantly reduced while the shape or length of the dendritic arbors was not altered. Three days of rest restored the spine loss and the degraded spatial learning and weight-loaded forced swimming performances to control levels. In conclusion, although we could not rule out additional non-hypothalamic-pituitary-adrenal stress, the apparent fatigue induced following a few days of sleep deprivation could change brain structurally and functionally and the effects were reversible with a few days of rest.

The cytoarchitecture and soma-dendritic arbors of the pyramidal neurons of aged rat sensorimotor cortex: an intracellular dye injection study.

We studied the cytoarchitecture and dendritic arbors of the output neurons of the sensorimotor cortex of aged rats and found that although individual cortical layer became thinner, the overall cytoarchitecture and neuron densities remained comparable to those of young adults. To find out whether aging affects cortical outputs we studied the soma-dendritic arbors of layers III and V pyramidal neurons, main output neurons of the cerebral cortex, using brain slice intracellular dye injection technique. With a fluorescence microscope, selected neurons were filled with fluorescence dye under visual guidance. Injected slices were resectioned into thinner sections for converting the injected dye into non-fading material immunohistochemically. The long apical dendritic trunk and branches could be routinely revealed. This allowed us to reconstruct and study the dendritic arbors of these neurons in isolation in 300-microm-thick dimension. Analysis shows that their cell bodies did not shrink, but the densities of spines on dendrites and the total dendritic length significantly reduced. Among spines, those with long thin stalks thought to be involved in memory acquisition appeared to be reduced. These could underlie the compromise of sensorimotor functions following aging.

Comparison of two-phase (201)Tl SPECT with chest CT to differentiate thoracic malignancies from benign lesions.

AIM: This study was conducted to compare the performance of (201)Tl single photon emission computed tomography ((201)Tl SPECT) with chest computed tomography (CT) in differentiating thoracic malignancies from benign lesions. METHODS: One hundred and seventy patients with confirmed diagnostic thoracic lesions found in chest radiographs were prospectively examined by (201)Tl SPECT. The performance of (201)Tl SPECT in differentiating thoracic malignancies from benign lesions was evaluated in 161 patients with a measurable retention index (RI), using the region-of-interest method. Chest CT scans were retrospectively collected from 165 patients and were interpreted by two independent observers. RESULTS: The areas under the receiver operating characteristics curves were 0.85 using the RI value to differentiate thoracic malignancies from benign lesions. The sensitivity, specificity, and accuracy were 71.9%, 83.1%, and 76.4%, respectively, with a cutoff level for the RI set at 20%. Similarly, the sensitivity, specificity and accuracy of chest CT scans to differentiate malignancies from benign lesions were 78.2%, 69.7% and 74.9%, respectively. Focusing on patients with concordant results in both (201)Tl SPECT and chest CT scans, we can differentiate thoracic malignancies from benign lesions with a sensitivity of 89.1%, a specificity of 90%, and an accuracy of 89.4%. CONCLUSION: Both (201)Tl SPECT and chest CT scans are useful imaging tools in differentiating thoracic malignancies from benign lesions, with an accuracy of around 75%. By combining these two image modalities, the accuracy improves to 89.4%, which may circumvent the need for invasive procedures for certain equivocal cases, using either single image alone.

Recurrence of peptic ulcer in uraemic and non-uraemic patients after Helicobacter pylori eradication: a 2-year study.

BACKGROUND: The role of Helicobacter pylori in the pathogenesis of peptic ulcer disease in patients with uraemia remains unclear. AIM: To evaluate the long-term effect of H. pylori eradication in these patients. METHODS: Uraemic and non-uraemic patients with peptic ulcer were enrolled in this study. Patients having history of non-steroidal anti-inflammatory drugs use or cardiovascular disease that need aspirin use were excluded. After confirmation of H. pylori infection, they received a triple therapy and were followed up for 2 years. RESULTS: Between September 1999 and December 2005, 34 patients (41%) of the end-stage renal disease [H. pylori (+) group] and 67 (84%) of the non-uraemic patients with peptic ulcer disease (PU group) received anti-H. pylori therapy. After triple therapy, 32 (94%) from the end-stage renal disease group and 64 (96%) from the peptic ulcer group obtained successful eradication. During the 2-year follow-up, three patients in the end-stage renal disease group were excluded because of the presence of cardiovascular disease and aspirin use in two cases and died of heart failure in one case; two patients in peptic ulcer group refused follow-up. Finally, 29 uraemic and 62 non-uraemic patients had achieved the follow-up. Recurrence of peptic ulcer was more in the end-stage renal disease group than in the peptic ulcer group with intention-to-treat analysis (eight of 32, 25% vs. two of 64, 3%, P = 0.001, OR: 10.0, 95% CI: 1.979-50.540) or per-protocol analysis (eight of 29, 28% vs. two of 62, 3%, P < 0.001, OR: 11.4, 95% CI: 2.245-58.168). CONCLUSIONS: Peptic ulcer recurrence after H. pylori eradication is higher in end-stage renal disease patients with peptic ulcer than in peptic ulcer patients without renal disease. Factors aside from H. pylori play an important role in peptic ulcer recurrence in end-stage renal disease patients.

APETx1 from sea anemone Anthopleura elegantissima is a gating modifier peptide toxin of the human ether-a-go-go- related potassium channel.

We studied the mechanism of action and the binding site of APETx1, a peptide toxin purified from sea anemone, on the human ether-a-go-go-related gene (hERG) channel. Similar to the effects of gating modifier toxins (hanatoxin and SGTx) on the voltage-gated potassium (Kv) 2.1 channel, APETx1 shifts the voltage-dependence of hERG activation in the positive direction and suppresses its current amplitudes elicited by strong depolarizing pulses that maximally activate the channels. The APETx1 binding site is distinctly different from that of a pore-blocking peptide toxin, BeKm-1. Mutations in the S3b region of hERG have dramatic impact on the responsiveness to APETx1: G514C potentiates whereas E518C abolishes the APETx1 effect. Restoring the negative charge at position 518 (methanethiosulfonate ethylsulfonate modification of 518C) partially restores APETx1 responsiveness, supporting an electrostatic interaction between E518 and APETx1. Among the three hERG isoforms, hERG1 and hERG3 are equally responsive to APETx1, whereas hERG2 is insensitive. The key feature seems to be an arginine residue uniquely present at the 514-equivalent position in hERG2, where the other two isoforms possess a glycine. Our data show that APETx1 is a gating modifier toxin of the hERG channel, and its binding site shares characteristics with those of gating modifier toxin binding sites on other Kv channels.

Lack of serotonin1B receptor expression leads to age-related motor dysfunction, early onset of brain molecular aging and reduced longevity.

Normal aging of the brain differs from pathological conditions and is associated with increased risk for psychiatric and neurological disorders. In addition to its role in the etiology and treatment of mood disorders, altered serotonin (5-HT) signaling is considered a contributing factor to aging; however, no causative role has been identified in aging. We hypothesized that a deregulation of the 5-HT system would reveal its contribution to age-related processes and investigated behavioral and molecular changes throughout adult life in mice lacking the regulatory presynaptic 5-HT(1B) receptor (5-HT(1B)R), a candidate gene for 5-HT-mediated age-related functions. We show that the lack of 5-HT(1B)R (Htr1b(KO) mice) induced an early age-related motor decline and resulted in decreased longevity. Analysis of life-long transcriptome changes revealed an early and global shift of the gene expression signature of aging in the brain of Htr1b(KO) mice. Moreover, molecular changes reached an apparent maximum effect at 18-months in Htr1b(KO) mice, corresponding to the onset of early death in that group. A comparative analysis with our previous characterization of aging in the human brain revealed a phylogenetic conservation of age-effect from mice to humans, and confirmed the early onset of molecular aging in Htr1b(KO) mice. Potential mechanisms appear independent of known central mechanisms (Bdnf, inflammation), but may include interactions with previously identified age-related systems (IGF-1, sirtuins). In summary, our findings suggest that the onset of age-related events can be influenced by altered 5-HT function, thus identifying 5-HT as a modulator of brain aging, and suggesting age-related consequences to chronic manipulation of 5-HT.

Oxidative stress in nonallergic nasal polyps associated with bronchial hyperresponsiveness.

BACKGROUND: Nasal polyposis (NP) is a chronic inflammatory disease of upper airway with unknown etiology. NP is frequently associated with asthma; the interaction between these comorbidities remains interesting. Oxidative stress has been implicated in the pathophysiology of NP and asthma. The aim of this study is to investigate the significance of oxidative stress in sinonasal microenvironments by evaluating its association with clinopathological parameters and its impacts on the pathogenesis of bronchial hyperresponsiveness (BHR) in NP. METHODS: Polyp biopsy specimens were obtained from 20 nonallergic patients; control mucosas were obtained from 20 volunteers. The levels of free radicals in the tissues and in blood were determined by a sensitive chemiluminescence (CL) method. NP patients were substratified into three subgroups, NP without BHR, NP with asymptomatic BHR, and NP with BHR and asthma by the results of provocative testing. Four histological characteristics of NP, inflammatory cells, eosinophil infiltration, edema and fibrosis were estimated and applied to correlate with the tissue-CL. RESULTS: The mean CL level in polyp-tissues, but not in blood, was higher than in the control specimens. In NP patients, tissue-CL was associated with endoscopy score; high tissue-CL levels were positively correlated with the abundance of inflammatory cells and eosinophils. Tissue-CL and endoscopy score were associated with BHR/asthma phenotype. CONCLUSION: These results suggest an important role for oxidative stress in the pathophysiology of NP and a causal relation between oxidative stress and inflammatory cells, especially the eosinophils. Free radical levels in polyp-tissues associated with NP severity and with BHR/asthma phenotype in nonallergic NP patients.