Dual mechanisms contribute to enhanced voltage dependence of an electric fish potassium channel.

The voltage dependence of different voltage-gated potassium channels, described by the voltage at which half of the channels are open (V1/2), varies over a range of 80 mV and is influenced by factors such as the number of positive gating charges and the identity of the hydrophobic amino acids in the channel's voltage sensor (S4). Here we explore by experimental manipulations and molecular dynamics simulation the contributions of two derived features of an electric fish potassium channel (Kv1.7a) that is among the most voltage-sensitive Shaker family potassium channels known. These are a patch of four contiguous negatively charged glutamates in the S3-S4 extracellular loop and a glutamate in the S3b helix. We find that these negative charges affect V1/2 by separate, complementary mechanisms. In the closed state, the S3-S4 linker negative patch reduces the membrane surface charge biasing the channel to enter the open state while, upon opening, the negative amino acid in the S3b helix faces the second (R2) gating charge of the voltage sensor electrostatically biasing the channel to remain in the open state. This work highlights two evolutionary novelties that illustrate the potential influence of negatively charged amino acids in extracellular loops and adjacent helices to voltage dependence.

Single Channel Analysis of Isoflurane and Ethanol Enhancement of Taurine-Activated Glycine Receptors.

The amino acid taurine is an endogenous ligand acting on glycine receptors (GlyRs), which is released by astrocytes in many brain regions, such as the nucleus accumbens and prefrontal cortex. Taurine is a partial agonist with an efficacy significantly lower than that of glycine. Allosteric modulators such as ethanol and isoflurane produce leftward shifts of glycine concentration-response curves but have no effects at saturating glycine concentrations. In contrast, in whole-cell electrophysiology studies these modulators increase the effects of saturating taurine concentrations. A number of possible mechanisms may explain these enhancing effects, including modulator effects on conductance, channel open times, or channel closed times. We used outside-out patch-clamp single channel electrophysiology to investigate the mechanism of action of 200 mM ethanol and 0.55 mM isoflurane in enhancing the effects of a saturating concentration of taurine. Neither modulator enhanced taurine-mediated conductance. Isoflurane increased the probability of channel opening. Isoflurane also increased the lifetimes of the two shortest open dwell times while both agents decreased the likelihood of occurrence of the longest-lived intracluster channel-closing events. The mechanism of enhancement of GlyR functioning by these modulators is dependent on the efficacy of the agonist activating the receptor and the concentration of agonist tested.

Disruption of an intersubunit electrostatic bond is a critical step in glycine receptor activation.

Proper regulation of neurotransmission requires that ligand-activated ion channels remain closed until agonist binds. How channels then open remains poorly understood. Glycine receptor (GlyR) gating is initiated by agonist binding at interfaces between adjacent subunits in the extracellular domain. Aspartate-97, located at the alpha1 GlyR interface, is a conserved residue in the cys-loop receptor superfamily. The mutation of D97 to arginine (D97R) causes spontaneous channel opening, with open and closed dwell times similar to those of maximally activated WT GlyR. Using a model of the N-terminal domain of the alpha1 GlyR, we hypothesized that an arginine-119 residue was forming intersubunit electrostatic bonds with D97. The D97R/R119E charge reversal restored this interaction, stabilizing channels in their closed states. Cysteine substitution shows that this link occurs between adjacent subunits. This intersubunit electrostatic interaction among GlyR subunits thus contributes to the stabilization of the closed channel state, and its disruption represents a critical step in GlyR activation.

A story told by a single nanowire: optical properties of wurtzite GaAs.

The optical properties of the wurtzite (WZ) GaAs crystal phase found in nanowires (NWs) are a highly controversial topic. Here, we study high-quality pure WZ GaAs/AlGaAs core-shell NWs grown by Au-assisted molecular beam epitaxy (MBE) with microphotoluminescence spectroscopy (µ-PL) and (scanning) transmission electron microscopy on the very same single wire. We determine the room temperature (294 K) WZ GaAs bandgap to be 1.444 eV, which is ∼20 meV larger than in zinc blende (ZB) GaAs, and show that the free exciton emission at 15 K is at 1.516 eV. On the basis of time- and temperature-resolved µ-PL results, we propose a Γ(8) conduction band symmetry in WZ GaAs. We suggest a method for quantifying the optical quality of NWs, taking into consideration the difference between the room and low temperature integrated PL intensity, and demonstrate that Au-assisted GaAs/AlGaAs core-shell NWs can have high PL brightness up to room temperature.

UBE3A and transsynaptic complex NRXN1-CBLN1-GluD1 in a hypothalamic VMHvl-arcuate feedback circuit regulates aggression.

The circuit origins of aggression in autism spectrum disorder remain undefined. Here we report Tac1-expressing glutamatergic neurons in ventrolateral division of ventromedial hypothalamus (VMHvl) drive intermale aggression. Aggression is increased due to increases of Ube3a gene dosage in the VMHvl neurons when modeling autism due to maternal 15q11-13 triplication. Targeted deletion of increased Ube3a copies in VMHvl reverses the elevated aggression adult mice. VMHvl neurons form excitatory synapses onto hypothalamic arcuate nucleus AgRP/NPY neurons through a NRXN1-CBLN1-GluD1 transsynaptic complex and UBE3A impairs this synapse by decreasing Cbln1 gene expression. Exciting AgRP/NPY arcuate neurons leads to feedback inhibition of VMHvl neurons and inhibits aggression. Asymptomatic increases of UBE3A synergize with a heterozygous deficiency of presynaptic Nrxn1 or postsynaptic Grid1 (both ASD genes) to increase aggression. Targeted deletions of Grid1 in arcuate AgRP neurons impairs the VMHvl to AgRP/NPY neuron excitatory synapses while increasing aggression. Chemogenetic/optogenetic activation of arcuate AgRP/NPY neurons inhibits VMHvl neurons and represses aggression. These data reveal that multiple autism genes converge to regulate the VMHvl-arcuate AgRP/NPY glutamatergic synapse. The hypothalamic circuitry implicated by these data suggest impaired excitation of AgRP/NPY feedback inhibitory neurons may explain the increased aggression behavior found in genetic forms of autism.

Positive allosteric modulators differentially affect full versus partial agonist activation of the glycine receptor.

Taurine acts as a partial agonist at the glycine receptor (GlyR) in some brain regions such as the hippocampus, striatum, and nucleus accumbens. Ethanol, volatile anesthetics, and inhaled drugs of abuse are all known positive allosteric modulators of GlyRs, but their effects on taurine-activated GlyRs remain poorly understood, especially their effects on the high concentrations of taurine likely to be found after synaptic release. Two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes was used to compare the enhancing effects of ethanol, anesthetics, and inhalants on human homomeric α1-GlyR activated by saturating concentrations of glycine versus taurine. Allosteric modulators had negligible effects on glycine-activated GlyR while potentiating taurine-activated currents. In addition, inhaled anesthetics markedly enhanced desensitization rates of taurine- but not glycine-activated receptors. Our findings suggest that ethanol, volatile anesthetics, and inhalants differentially affect the time courses of synaptic events at GlyR, depending on whether the receptor is activated by a full or partial agonist.

Polymorphisms and haplotypes in VDR gene are associated with female idiopathic infertility.

Unexplained infertility refers to the absence of a definable cause of reproductive failure. Vitamin D receptor (VDR) acts as a transcription factor and regulates a number of vitamin D-responsive genes, including those involved in the immune system. Recent finding that VDR is expressed in reproductive tissues suggests a possible importance of vitamin D in pregnancy. We conducted a case-control study to examine the association of polymorphisms in VDR gene with reproductive success. DNA from 117 female patients with unexplained infertility and 130 fertile controls was isolated from peripheral blood and VDR genotypes (FokI, BsmI, ApaI and TaqI) were detected by PCR-RFLP. Haplotypes were determined using Haploview software. Our results show significant association of FokI and BsmI polymorphisms with infertility (p < 0.05). The haplotype analysis confirmed strong linkage disequilibrium between closely positioned BsmI, ApaI and TaqI polymorphisms. Two haplotypes were associated with infertility: (i) haplotype bAT was increasing the risk for secondary infertility; while (ii) haplotype BAT had a protective role against primary infertility (p < 0.05). By changing the expression and the activity of VDR gene, which leads to the change in expression of vitamin D-responsive genes, these polymorphisms and haplotypes could possibly have an effect on immune system in the female reproductive tract.

Hemostasis-related gene polymorphisms and their epistatic relationship in women with idiopathic infertility.

: A numerous factor can cause infertility, but around one of four reproductive failure cases remain unexplained and diagnosed as idiopathic infertility. In the past few decades, analysis of gene polymorphisms takes a significant place in pathogenesis of infertility. The aim of this study was to evaluate the possible role of hemostasis-related gene polymorphisms in unexplained infertility. The study includes 117 female patients with idiopathic infertility and 130 fertile women with at least one born child. Eight polymorphisms important for hemostasis (ITGB3 1565T>C, FV 1691G>A, FII 20210G>A, MTHFR 677C>T and 1298A>C, ATIII 786G>A, PAI-14G/5G and ACE I/D) were genotyped by real-time PCR system. The frequencies of alleles and genotypes of examined polymorphisms were analyzed in SPSS statistical program, whereas gene interactions were identified using the GMDR software. Examination of etiological factors has shown that family history is a significant factor in assessing individual risk for infertility. The alleles and genotypes frequency of FV 1691G>A and FII 20210G>A polymorphisms were statistically different between control and patient group leading to a greater risk for infertility. The analysis of epistatic relationship between examined hemostasis-related gene polymorphisms identified more complex high-risk genotypes associated with infertility. Our results suggest that positive family history could be important predictive factor for fertility problems, pointing to the potential hereditary basis of this condition. Polymorphisms FVL and FII prothrombin are independent risk factors for idiopathic infertility, whereas multilocus interactions approach should be taken into consideration for the future research.

Toxicity of leachate from bottom ash in a road construction.

A test road constructed with municipal solid waste incineration (MSWI) bottom ash was monitored over a period of 36 months. Using chemical and toxicological characterisation, the environmental impact of leachates from bottom ash was evaluated and compared with leachates from gravel used as reference. Initial leaching of Cl, Cu, K, Na, NH4-N and TOC from bottom ash was of major concern. However, the quality of the bottom ash leachate approached that of the gravel leachate with time. Leachates from the two materials were compared regarding the concentration of pollutants using multivariate data analyses (MVDA). A standardized luminescent bacteria assay using Vibrio fischeri did not show any toxicity, most likely because saline contamination can mask the toxic response and stimulate luminescence in these marine bacteria. A mung bean assay using Phaseolus aureus revealed that the toxicity of bottom ash leachate collected at the very beginning of the experimental period (October 2001 and May 2002) might be attributed to the following components and their respective concentrations in mg l(-1): Al (34.2-39.2), Cl (2914-16,446), Cu (0.48-1.92), K (197-847), Na (766-4180), NH4-N (1.80-8.47), total-N (12.0-18.5), and TOC (34.0-99.0). The P. aureus assay was judged as a promising environmental tool in assessing the toxicity of bottom ash leachate.

Disruption of a putative intersubunit electrostatic bond enhances agonist efficacy at the human α1 glycine receptor.

Partial agonists have lower efficacies than compounds considered 'full agonists', eliciting submaximal responses even at saturating concentrations. Taurine is a partial agonist at the glycine receptor (GlyR), a member of the cys-loop ligand-gated ion channel superfamily. The molecular mechanisms responsible for agonism are not fully understood but evidence suggests that efficacy at these receptors is determined by conformational changes that occur early in the process of receptor activation. We previously identified a residue located near the human α1 glycine binding site (aspartate-97; D97) that, when mutated to arginine (D97R), results in GlyR channels opening spontaneously with a high open probability, mimicking the effects of saturating glycine concentrations on wildtype GlyR. This D97 residue is hypothesized to form an electrostatic interaction with arginine-119 on an adjacent subunit, stabilizing the channel in a shut state. Here we demonstrate that the disruption of this putative bond increases the efficacy of partial agonists including taurine, as well as two other ß-amino acid partial agonists, ß-aminobutyric acid (ß-ABA) and ß-aminoisobutyric acid (ß-AIBA). Even the subtle charge-conserving mutation of D97 to glutamate (D97E) markedly affects partial agonist efficacy. Mutation to the neutral alanine residue in the D97A mutant mimics the effects seen with D97R, indicating that charge repulsion does not significantly affect these findings. Our findings suggest that the determination of efficacy following ligand binding to the glycine receptor may involve the disruption of an intersubunit electrostatic interaction occurring near the agonist binding site.

Genetics of suspected thrombophilia in Serbian females with infertility, including three cases, homozygous for FII 20210A or FV 1691A mutations.

Reproductive failure (recurrent foetal loss, unexplained infertility and IVF implantation failure) may be, in a number of cases, explained by thrombophilia, either acquired or inherited. Several genes contribute to thrombophilia, some with major effect (Factor V, Factor II), and some with minor effect (MTHFR, PAI-1, ATIII, etc.). The aim of this study was to estimate frequency of thrombophilia-associated genotypes (FII20210G > A, FV1691G > A, MTHFR677C > T and PAI-1 -675 4G/5G) in a group of 1631 Serbian women experiencing reproductive failure, and compare it with a healthy, female control group. Our results showed marginally significant (p = 0.050) differences in allele frequencies between patients and controls for the FV1691 mutations. For the FII20210G > A, although the statistical significance was not achieved (p = 0.076), we found higher frequency of variant allele in patients compared to controls (1.87% vs. 0.38%, respectively) which may point to a possible role of this polymorphism in thrombotic events. For the MTHFR677C > T and PAI-1 -675 4G/5G, we found no difference in distributions of genotype or allele frequencies between these two groups (p > 0.05). For three subjects with very rare genotypes (two patients homozygous for FV1691G > A and one patient homozygous for FII20210G > A) we performed additional biochemical analyses for haemostasis, as well as genotyping of two polymorphisms (MTHFR1298A > C and ATIII786G > A).

Demobilisation of critical contaminants in four typical waste-to-energy ashes by carbonation.

Two bottom ashes, one air pollution control (APC) residue and one fly ash from three different Swedish municipal solid waste incineration (MSWI) plants were characterised regarding the leaching of environmentally relevant components. Characterisation was performed using a diffusion tank leaching test. The impact of carbonation on the release of eight critical components, i.e., Cl(-), Cr, Cu, Mo, Pb, Sb, Se, SO(4)(2-) and Zn, was assessed at a lab-scale and showed carbonation to have a more pronounced demobilising effect on critical components in bottom ashes than in APC residue and fly ash. From grate type incinerator bottom ash, the release of Cr decreased by 97%, by 63% for Cu and by 45% for Sb. In the investigated APC residue, the releases of Cr, Se and Pb were defined as critical, although they either remained unaffected or increased after carbonation. Cl(-) and SO(4)(2-) remained mobile after carbonation in all investigated residues.

Co-expression of TTF-1 and neuroendocrine markers in the human fetal lung and pulmonary neuroendocrine tumors.

The expression pattern of thyroid transcription factor 1 (TTF-1) and neuroendocrine markers, neuron cell adhesion molecule (NCAM; CD56), chromogranin A (CgA) and synaptophysin (Syp), of different lung cell lineages was histologically analyzed in 15 normal human fetal lungs and 12 neuroendocrine tumors (NETs) using immunohistochemical methods. During pseudoglandular phase strong nuclear TTF-1 staining was detected in the columnar nonciliated epithelial cells, while NCAM, CgA and Syp had a moderate expression in the proximal airways and mild expression in the distal airways. Neuroendocrine cells (NECs) in proximal lung airway were co-localizing TTF-1 and other neuroendocrine markers while neuroendocrine bodies (NEBs) exhibit only staining with NCAM and Syp. In the canalicular phase TTF-1 nuclear staining was expressed only in several epithelial cells in proximal airways, while budding airways epithelium showed strong TTF-1 expression. Expression of NCAM, CgA and Syp in this phase equals the one in pseudoglandular phase. NEBs cells were co-localizing TTF-1 and NCAM in proximal airways and few NECs in distal airway were co-localizing TTF-1 and Syp. TTF-1 staining in the saccular phase was limited to subsets of epithelial cells in the proximal airways with stronger positivity in the distal airways. NCAM expression is moderate only in proximal airways, while Syp and CgA show mild expression in proximal and distal airways. NECs were co-localizing TTF-1 and NCAM in proximal lung airway. With regard to NECs, all small cell lung cancer (SCLC) cells had strong TTF-1, NCAM, Syp and CgA positivity and TTF-1 co-localized with other neuroendocrine markers. All pulmonary typical carcinoids were TTF-1 negative, while pulmonary atypical carcinoids were focal positive for TTF-1 and some neoplastic cells co-localized TTF-1 with neuroendocrine markers. Our results indicate that TTF-1 expression in NECs suggests a possible role in their normal development and differentiation. Our results also indicate that possible cell of origin for poorly differentiated SCLC and some atypical carcinoid could be a progenitor cell in neuroendocrine lineage while in typical carcinoids possible cell of origin is localized in terminally differentiated NECs.

Proliferation, apoptosis and expression of matrix metalloproteinase-9 in human fetal lung.

Expression pattern of the Ki-67, caspase-3 and matrix metalloproteinases-9 (MMP-9) factors were immunohistochemically analyzed in 48 human fetal lungs from 12 to 40 weeks of gestation. The number of Ki-67 positive cells in the epithelium of canaliculare (88cells/mm(2)) and sacculare stage (93cells/mm(2)) were significantly higher than in the epithelium of pseudoglandular stage (12cells/mm(2)) (p=0.0008 vs. p=0.003). The number of Ki-67 positive cells in the mesenchyme of canaliculare stage (132cells/mm(2)) was significantly higher than in the mesenchyme of pseudoglandular stage (37cells/mm(2)) (p=0.001). The proliferation of mesenchymal cells was higher than the epithelial cells in all developmental stages, especially in the canaliculare stage (p=0.007). Similarly, the number of caspase-3 positive cells in the epithelium of canalicular stage (13cells/mm(2)) was significantly higher than in the epithelium of pseudoglandular stage (6cells/mm(2)) (p=0.002) with peaks in the conductive epithelium of canalicular stage. The number of caspase-3 positive cells in the mesenchyme of canaliculare stage (3cells/mm(2)) was significantly higher than in the mesenchyme of saccular stage (0cells/mm(2)) (p=0.05). There were no caspase-3 positive cells in the mesenchyme of pseudoglandular stage. However, unlike the Ki-67 expression, mesenchymal cells in comparison to epithelial cells express substantially less caspase-3 in all developmental stages. Up to the saccular stage, the expression of MMP-9 in mesenchymal cells showed a linear increase with most pronounced expression in that stage. The number of MMP-9 positive cells in the mesenchyme of canaliculare (20cells/mm(2)) and sacculare (39cells/mm(2)) stage were significantly higher than in the mesenchyme of pseudoglandular stage (12cells/mm(2)) (p=0.04 vs. p=0.004). The first epithelial cells that express MMP-9 were present only at the alveolar stage. Increased proliferation and apoptosis of the mesenchymal cells of canalicular stage is important for formation of definite structures within the stroma of the lung parenchyma. Although apoptosis in the epithelium is not pronounced as proliferation, it is important for thinning of the epithelium and consequent spread of respiratory tract. However in the saccular stage when mesenchyme disappears, MMP-9 expression is more important for primitive alveoli differentiation.

Solidification with water as a treatment method for air pollution control residues.

The process of solidification with water was studied on air pollution control (APC) residues from incineration of refuse-derived fuel (RDF) regarding mechanical strength and leaching behaviour of solidified material. Factorial design in two levels was applied to investigate the impact of water addition, time, and temperature to mechanical strength of solidified material. Factors time and temperature, as well as the interaction between the addition of water and time significantly (alpha=0.05) influenced the mechanical strength of solidified material. The diffusion-leaching test NEN 7345 was performed to investigate if the leaching behaviour of elements from solidified material was determined by diffusion. Since it was found that leaching is not diffusion controlled, the long-term leaching behaviour was not assessed. However, the investigation showed that some of the studied components (Al, Hg, Mn, Pb, Si, and Zn) could be considerably demobilised by solidification with water. Concentrations of As, Cd, Co, Cu, Fe, and Ni were either below or not quite above the detection limits to be included in the analysis of leaching behaviour. The elements least demobilised by solidification were Cl, Cr, K, and Na.

Physiological concentrations of zinc reduce taurine-activated GlyR responses to drugs of abuse.

Taurine is an endogenous ligand acting on glycine receptors in many brain regions, including the hippocampus, prefrontal cortex, and nucleus accumbens (nAcc). These areas also contain low concentrations of zinc, which is known to potentiate glycine receptor responses. Despite an increasing awareness of the role of the glycine receptor in the rewarding properties of drugs of abuse, the possible interactions of these compounds with zinc has not been thoroughly addressed. Two-electrode voltage-clamp electrophysiological experiments were performed on α1, α2 α1ß and α2ß glycine receptors expressed in Xenopus laevis oocytes. The effects of zinc alone, and zinc in combination with other positive modulators on the glycine receptor, were investigated when activated by the full agonist glycine versus the partial agonist taurine. Low concentrations of zinc enhanced responses of maximally-effective concentrations of taurine but not glycine. Likewise, chelation of zinc from buffers decreased responses of taurine- but not glycine-mediated currents. Potentiating concentrations of zinc decreased ethanol, isoflurane, and toluene enhancement of maximal taurine currents with no effects on maximal glycine currents. Our findings suggest that the concurrence of high concentrations of taurine and low concentrations of zinc attenuate the effects of additional modulators on the glycine receptor, and that these conditions are more representative of in vivo functioning than effects seen when these modulators are applied in isolation.

ZAP-70 expression and proliferative activity in chronic lymphocytic leukemia.

The expression of 70 kDa protein zeta-associated protein (ZAP-70) in chronic lymphocytic leukemia (CLL) has been used to detect those patients with more aggressive disease. The aim of this study was to determine the proliferative activity of ZAP-70(+) leukemic cells by immunocytochemical methods. The study was undertaken on native blood marrow (BM) and peripheral blood (PB) smears from 65 patients with CLL. ZAP-70 was expressed in leukemic cells of 35 patients (54%). We demonstrated that ZAP-70 immunoreactivity correlated with Rai 0-IV (p = 0.002) and Binet A-C stages (p < 0.001), total tumor mass (TTM score) (p < 0.001), ß2-microglobulin (p = 0.006), atypical lymphocytes (p < 0.001) and proliferative activity in bone marrow and peripheral blood (p = 0.014, p = 0.002, respectively) using χ(2) test and Mann-Whitney test. ZAP-70 protein expression is in direct correlation with the poorer prognostic parameters, which additionally confirms the successful method of detection of ZAP-70 expression. Higher Ki-67 expression in BM and PB smears of patients with ZAP-70(+) disease indicates higher proliferating compartments, which may contribute to poorer prognosis.

Identification and characterization of KCASH2 and KCASH3, 2 novel Cullin3 adaptors suppressing histone deacetylase and Hedgehog activity in medulloblastoma.

Medulloblastoma is the most common pediatric malignant brain tumor, arising from aberrant cerebellar precursors' development, a process mainly controlled by Hedgehog (Hh) signaling pathway. Histone deacetylase HDAC1 has been recently shown to modulate Hh signaling, deacetylating its effectors Gli1/2 and enhancing their transcriptional activity. Therefore, HDAC may represent a potential therapeutic target for Hh-dependent tumors, but still little information is available on the physiological mechanisms of HDAC regulation. The putative tumor suppressor REN(KCTD11) acts through ubiquitination-dependent degradation of HDAC1, thereby affecting Hh activity and medulloblastoma growth. We identify and characterize here two REN(KCTD11) homologues, defining a new family of proteins named KCASH, as "KCTD containing, Cullin3 adaptor, suppressor of Hedgehog." Indeed, the novel genes (KCASH2(KCTD21) and KCASH3(KCTD6)) share with REN(KCTD11) a number of features, such as a BTB domain required for the formation of a Cullin3 ubiquitin ligase complex and HDAC1 ubiquitination and degradation capability, suppressing the acetylation-dependent Hh/Gli signaling. Expression of KCASH2 and -3 is observed in cerebellum, whereas epigenetic silencing and allelic deletion are observed in human medulloblastoma. Rescuing KCASHs expression reduces the Hedgehog-dependent medulloblastoma growth, suggesting that loss of members of this novel family of native HDAC inhibitors is crucial in sustaining Hh pathway-mediated tumorigenesis. Accordingly, they might represent a promising class of endogenous "agents" through which this pathway may be targeted.