Validation of endogenous controls for gene expression studies in peripheral lymphocytes from war veterans with and without PTSD

Background  

Selection of appropriate endogenous control is a critical step in gene expression analysis. The aim of this study was to evaluate expression stability of four frequently used endogenous controls: β-actin, glyceraldehyde-3-phosphate dehydrogenase, β₂-microglobulin and RNA polymerase II polypeptide A in peripheral blood mononuclear cells from war veterans with and without posttraumatic stress disorder (PTSD). The study was designed as to identify suitable reference gene(s) for normalization of gene expression in peripheral blood mononuclear cells in response to war trauma and/or PTSD.     

Design and synthesis of peptides that bind α-bungarotoxin with high affinity and mimic the three-dimensional structure of the binding-site of acetylcholine receptor

α-Bungarotoxin (α-BTX) is a highly toxic snake neurotoxin that binds to acetylcholine receptor (AChR) at the neuromuscular junction, and is a potent inhibitor of this receptor. In the following we review multi-phase research of the design, synthesis and structure analysis of peptides that bind α-BTX and inhibit its binding to AChR. Structure-based design concomitant with biological information of the α-BTX/AChR system yielded 13-mer peptides that bind to α-BTX with high affinity and are potent inhibitors of α-BTX binding to AChR (IC₅₀ of 2 nM). X-Ray and NMR spectroscopy reveal that the high-affinity peptides fold into an anti-parallel β-hairpin structure when bound to α-BTX. The structures of the bound peptides and the homologous loop of acetylcholine binding protein, a soluble analog of AChR, are remarkably similar. Their superposition indicates that the toxin wraps around the binding-site loop, and in addition, binds tightly at the interface of two of the receptor subunits and blocks access of acetylcholine to its binding site. The procedure described in this article may serve as a paradigm for obtaining high-affinity peptides in biochemical systems that contain a ligand and a receptor molecule.     

Death-associated protein (DAP) kinase plays a central role in ceramide-induced apoptosis in cultured hippocampal neurons.

Treatment of cultured hippocampal neurons with high concentrations of short-chain acyl ceramide derivatives, such as N-hexanoyl-D-sphingosine (C(6)-Cer), results in apoptotic cell death. We now show that death-associated protein (DAP) kinase plays an important role in mediating this effect. Upon incubation with C(6)-Cer, DAP kinase levels are elevated as early as 1 h after treatment, reaching levels 2-3-fold higher than untreated cells after 4 h. Neurons cultured from DAP kinase-deficient mice were significantly less sensitive to apoptosis induced by C(6)-Cer or by ceramide generated by high concentrations of nerve growth factor. A peptide corresponding to the 17 amino acids at the C terminus of DAP kinase protected wild type neurons from C(6)-Cer-induced death and from death induced by the addition of exogenous bacterial neutral sphingomyelinase, whereas a scrambled peptide had no protective effect, implying that the DAP kinase C-terminal tail inhibits the function of DAP kinase. Together, these data demonstrate that DAP kinase plays a central role in ceramide-induced cell death in neurons, but the pathway in which DAP kinase is involved is not the only one via which ceramide can induce apoptosis.     

Genetic architecture of seed longevity in bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

The deterioration in the quality of ex situ conserved seed over time reflects a combination of both physical and chemical changes. Intraspecific variation for longevity is, at least in part, under genetic control. Here, the grain of 183 bread wheat accessions maintained under low-temperature storage at the IPK-Gatersleben genebank over some decades have been tested for their viability, along with that of fresh grain subjected to two standard artificial ageing procedures. A phenotype–genotype association analysis, conducted to reveal the genetic basis of the observed variation between accessions, implicated many regions of the genome, underling the genetic complexity of the trait. Some, but not all, of these regions were associated with variation for both natural and experimental ageing, implying some non-congruency obtains between these two forms of testing for longevity. The genes underlying longevity appear to be independent of known genes determining dormancy and pre-harvest sprouting.     

A single MIU motif of MINDY‐1 recognizes K48‐linked polyubiquitin chains

The eight different types of ubiquitin (Ub) chains that can be formed play important roles in diverse cellular processes. Linkage‐selective recognition of Ub chains by Ub‐binding domain (UBD)‐containing proteins is central to coupling different Ub signals to specific cellular responses. The motif interacting with ubiquitin (MIU) is a small UBD that has been characterized for its binding to monoUb. The recently discovered deubiquitinase MINDY‐1/FAM63A contains a tandem MIU repeat (tMIU) that is highly selective at binding to K48‐linked polyUb. We here identify that this linkage‐selective binding is mediated by a single MIU motif (MIU2) in MINDY‐1. The crystal structure of MIU2 in complex with K48‐linked polyubiquitin chains reveals that MIU2 on its own binds to all three Ub moieties in an open conformation that can only be accommodated by K48‐linked triUb. The weak Ub binder MIU1 increases overall affinity of the tMIU for polyUb chains without affecting its linkage selectivity. Our analyses reveal new concepts for linkage selectivity and polyUb recognition by UBDs.     

Synthesis and Biological Evaluation of Novel 1‐(4‐(Hydroxy(1‐oxo‐1,3‐dihydro‐2H‐inden‐2‐ylidene)methyl)phenyl)‐3‐phenylurea Derivatives

A series of novel phenylurea containing 2‐benzoylindan‐1‐one derivatives 3a  –  3j were synthesized from the reaction of phenylurea‐substituted acetophenones 1a  –  1j with phthalaldehyde 2 under mild reaction conditions in good yields. All synthesized compounds were characterized by spectroscopic methods. The obtained compounds ( 3a  –  3j ) were evaluated for anticancer activity against HeLa and C6 cell lines. Antiproliferative activity was determined by the BrdU proliferation ELISA assay, 3f and 3g were found to be most active compounds. The compounds were also screened for antimicrobial activity and all compounds showed remarkable activity against used microorganisms.     

Langmuir probe study of an inductively coupled magnetic-pole-enhanced helium plasma

This study reports the effects of RF power and filling gas pressure variation on the plasma parameters, including the electron number density n _e , electron temperature T _e , plasma potential V _p , skin depth δ, and electron energy probability functions (EEPFs) in a low-pressure inductively coupled helium plasma source with magnetic pole enhancement. An RF compensated Langmuir probe is used to measure these plasma parameters. It is observed that the electron number density increases with both the RF power and the filling gas pressure. Conversely, the electron temperature decreases with increasing RF power and gas pressure. It is also noted that, at low RF powers and gas pressures, the EEPFs are non-Maxwellian, while at RF powers of ≥50 W, they evolve into a Maxwellian distribution. The dependences of the skin depth and plasma potential on the RF power are also studied and show a decreasing trend.     

Human health risk assessment of arsenic in groundwater aquifers of Lahore,Pakistan

The present study was conducted to estimate As concentration in groundwater and resulting human health risk in terms of chronic daily intake, hazard quotient (HQ), hazard index (HI), and carcinogenic risk (CR) both for oral and dermal exposure to As. Groundwater samples (n = 100) were collected from ten different towns of Lahore District (Pakistan). Arsenic concentration ranged from 2 to 111 µg L^?1 in groundwater samples of the study area, which was significantly greater than the safe limit of As (10 µg L^?1) in drinking water set by the World Health Organization. Health risk assessment of As showed that HQ (0.1–11) for oral exposure and HI (0.1–11) values also exceeded the typical toxic risk index value of 1. 9.75 × E-05–4.59 × E-03 and 5.89 × E-07–2.77 × E-05 for oral and dermal As exposure, respectively. Both CR and cancer index (CIs) values were higher than United States Environmental Protection Agency limit (10^?6), suggesting that people are at high risk of As-induced carcinogenicity from oral and dermal exposure to As in drinking water. It was concluded that As contamination of groundwater causes carcinogenic and noncarcinogenic health effects to the people; therefore, urgent management and remedial actions are required to protect people from As poisoning.     

Living on the edge: population ecology of <Emphasis Type="Italic">Phengaris teleius</Emphasis> in Serbia

Due to their complex and interesting life cycle and alarming conservation status the Large Blue butterflies have become one of the most studied group of insects in Europe. In Serbia, however, Phengaris teleius has recently been discovered (in the far north of the country) and, since this initial finding, significant efforts have been made to map the local distribution of this species and to implement conservation measures. A mark-release-recapture study was initiated to obtain a more detailed report about population size and structure in Serbia. Results have shown that localities and patches within these localities are well connected by migration of butterflies and gave some evidence for metapopulation organisation. The total number of individuals was estimated at 15,000, which makes it one of the largest known metapopulations in Europe. Unusual for populations at the edge of the distribution range, these are characterized with large population estimates and relative stability. Compared to other areas in Europe, butterflies in Serbia start to fly 2 weeks later in the summer, probably a consequence of a late mowing regime initiated during July. Some evidence of a negative relationship between the survival of P. teleius adults and local population density was also found. With favourable management, these populations could remain stable for the foreseeable future. The preservation of large, healthy populations of P. teleius supported by a favourable management and monitoring programme along with the sustainability of individual dispersal translocation between patches should be a prime aim.