Preclinical Evaluation of Zn(II) Self-Assemblies with Selective Cytotoxic Activity Against Cancer Cells In Vitro and In Ovo.

Dipodal pyridylthiazole amine ligands L1 and L2 both form different metallo-supramolecular self-assemblies with Zn2+ and Cu2+ and these are shown to be toxic and selective towards cancer cell lines in vitro. Furthermore, potency and selectivity are highly dependent upon the metal ions, ligand system and bound anion, with significant changes in chemosensitivity and selectivity dependent upon which species are employed. Importantly, significant anti-tumor activity was observed in ovo at doses that are non-toxic.

Photochemistry of Heteroleptic 1,4,5,8-Tetraazaphenanthrene- and Bi-1,2,3-triazolyl-Containing Ruthenium(II) Complexes.

Diimine metal complexes have significant relevance in the development of photodynamic therapy (PDT) and photoactivated chemotherapy (PACT) applications. In particular, complexes of the TAP ligand (1,4,5,8-tetraazaphenanthrene) are known to lead to photoinduced oxidation of DNA, while TAP- and triazole-based complexes are also known to undergo photochemical ligand release processes relevant to PACT. The photophysical and photochemical properties of heteroleptic complexes [Ru(TAP)n(btz)3-n]2+ (btz = 1,1'-dibenzyl-4,4'-bi-1,2,3-triazolyl, n = 1 (1), 2 (2)) have been explored. Upon irradiation in acetonitrile, 1 displays analogous photochemistry to that previously observed for [Ru(bpy)(btz)2]2+ (bpy = 2,2'-bipyridyl) and generates trans-[Ru(TAP)(btz)(NCMe)2]2+ (5), which has been crystallographically characterized, with the observation of the ligand-loss intermediate trans-[Ru(TAP)(κ2-btz)(κ1-btz)(NCMe)]2+ (4). Complex 2 displays more complicated photochemical behavior with not only preferential photorelease of btz to form cis-[Ru(TAP)2(NCMe)2]2+ (6) but also competitive photorelease of TAP to form 5. Free TAP is then taken up by 6 to form [Ru(TAP)3]2+ (3) with the proportion of 5 and 3 observed to progressively increase during prolonged photolysis. Data suggest a complex set of reversible photochemical ligand scrambling processes in which 2 and 3 are interconverted. Computational DFT calculations have enabled optimization of geometries of the pro-trans 3MCcis states with repelled btz or TAP ligands crucial for the formation of 5 from 1 and 2, respectively, lending weight to recent evidence that such 3MCcis states play an important mechanistic role in the rich photoreactivity of Ru(II) diimine complexes.

Malondialdehyde-Acetaldehyde Adduct Formation Decreases Immunoglobulin A Transport across Airway Epithelium in Smokers Who Abuse Alcohol.

Alcohol misuse and smoking are risk factors for pneumonia, yet the impact of combined cigarette smoke and alcohol on pneumonia remains understudied. Smokers who misuse alcohol form lung malondialdehyde-acetaldehyde (MAA) protein adducts and have decreased levels of anti-MAA secretory IgA (sIgA). Transforming growth factor-ß (TGF-ß) down-regulates polymeric Ig receptor (pIgR) on mucosal epithelium, resulting in decreased sIgA transcytosis to the mucosa. It is hypothesized that MAA-adducted lung protein increases TGF-ß, preventing expression of epithelial cell pIgR and decreasing sIgA. Cigarette smoke and alcohol co-exposure on sIgA and TGF-ß in human bronchoalveolar lavage fluid and in mice instilled with MAA-adducted surfactant protein D (SPD-MAA) were studied herein. Human bronchial epithelial cells (HBECs) and mouse tracheal epithelial cells were treated with SPD-MAA and sIgA and TGF-ß was measured. Decreased sIgA and increased TGF-ß were observed in bronchoalveolar lavage from combined alcohol and smoking groups in humans and mice. CD204 (MAA receptor) knockout mice showed no changes in sIgA. SPD-MAA decreased pIgR in HBECs. Conversely, SPD-MAA stimulated TGF-ß release in both HBECs and mouse tracheal epithelial cells, but not in CD204 knockout mice. SPD-MAA stimulated TGF-ß in alveolar macrophage cells. These data show that MAA-adducted surfactant protein stimulates lung epithelial cell TGF-ß, down-regulates pIgR, and decreases sIgA transcytosis. These data provide a mechanism for the decreased levels of sIgA observed in smokers who misuse alcohol.

Hot-stage microscopy - Direct Analysis in Real-time mass spectrometry (HDM) as a novel tool for monitoring thermally-driven reactions on a small scale.

There is a requirement for reliable real-time analytical tools for reaction monitoring to optimise chemical syntheses. We have developed a new technique which combines thermal analysis, digital microscopy and chemical identification using ambient ionisation mass spectrometry. We term this hot-stage microscopy-Direct Analysis in Real-Time mass spectrometry (HDM). The technique provides optical data as a function of temperature coupled with chemical characterisation of evolved species, including reactants, intermediates and products throughout the course of a reaction. In addition, only a few milligrams of sample are required with analyte detection down to the nanogram range. We demonstrate the benefits of HDM using a series of solvent-free reactions. Our results confirm the suitability of the technique as the reactions studied follow the same pathways as published previously. The accurate temperature control achieved with HDM could also be used to assess the optimum temperature at which thermally-driven reactions can proceed efficiently.

What's eating you? cat flea (Ctenocephalides felis) revisited.

Cat fleas (Ctenocephalides felis) often leave cats and dogs to bite humans, causing pruritic lesions of the lower extremities. Once flea bites are confirmed, various efforts can be made to eradicate these pests from the home or pets. Cat fleas also play a role in transmission of vector-borne diseases, especially endemic typhus.

Application of hot-stage microscopy direct analysis in real time mass spectrometry (HDM) to the analysis of polymers.

RATIONALE: Polymers are ubiquitous, and characterisation of their chemical, thermal and mechanical properties is important in many applications. Hot-stage microscopy Direct Analysis in Real Time mass spectrometry (HDM) is a new technique which combines optical measurements with the benefits of ambient ionisation mass spectrometry. Physical and chemical information can be obtained as a function of sample temperature, in real time. METHODS: Samples were placed on a miniaturised hot-stage between a custom-made Direct Analysis in Real Time (DART) source and the inlet of an ion trap mass spectrometer, and subjected to both linear and cycled temperature programmes. Optical images were collected using a digital microscope and mass spectra (positive and negative ion) were recorded simultaneously. RESULTS: Mass spectra and optical images were used to monitor the thermal expansion and release of volatile oligomers from both medical and domestic grades of silicone. Series of ions separated by 74 m/z units were observed, consistent with the SiOMe2 monomer; the median mass of these increased with increasing temperature up to the decomposition point (340-400°C). The abundance of volatile material produced decreased with repeated thermal cycling. The coefficients of thermal expansion were calculated from optical data and were in agreement with conventional measurements (2.7-3.6 × 10-4 °C-1 ). Two samples of beach sand analysed for the presence of microplastics were found to contain polyethylene and polystyrene, respectively. CONCLUSIONS: Results indicate that the novel technique of HDM can be successfully applied to the characterisation of a wide range of polymers including those in complex matrices.

Aquatic antagonists: stingray injury update.

Stingrays cause more sting-related injuries than any other fish. Their venom and mechanism of injury lead to painful, poor-healing wounds that often become infected. Stingray injuries are further complicated by retained barbs and foreign bodies, which also may lead to considerable morbidity. Most stingray injuries can be treated with hot water immersion, wound debridement, and prophylactic antibiotics, while some may require more extensive treatment and surgical intervention at a tertiary care center. Educating patients about avoidance strategies, such as wading through water with a shuffling gait and wearing protective leg guards, may help decrease stingray injuries.

An Integrated Hot-Stage Microscope-Direct Analysis in Real Time-Mass Spectrometry System for Studying the Thermal Behavior of Materials.

This paper describes a new analytical instrument that combines a precisely temperature-controlled hot-stage with digital microscopy and Direct Analysis in Real Time-mass spectrometry (DART-MS) detection. The novelty of the instrument lies in its ability to monitor processes as a function of temperature through the simultaneous recording of images, quantitative color changes, and mass spectra. The capability of the instrument was demonstrated through successful application to four very varied systems including profiling an organic reaction, decomposition of silicone polymers, and the desorption of rhodamine B from an alumina surface. The multidimensional, real-time analytical data provided by this instrument allow for a much greater insight into thermal processes than could be achieved previously.

Reporting of Lost to Follow-Up and Treatment Discontinuation in Pharmacotherapy and Device Trials in Chronic Heart Failure: A Systematic Review.

BACKGROUND: Premature treatment discontinuation and loss to follow-up (LTFU) with unknown outcomes leave uncertainty about the true efficacy and safety of a treatment and a lack of confidence in the results of any trial. We reviewed the extent of (and trends over time in) reporting LTFU and treatment discontinuation in large studies in chronic heart failure published since 1990. METHODS AND RESULTS: Online databases were systematically reviewed to identify randomized controlled clinical trials (RCTs) in chronic heart failure with >400 participants and utilizing all-cause mortality as a component of the primary or secondary end point. Assessments were made of documentation of treatment discontinuation, LTFU, inclusion of and completeness of a Consolidated Standards Of Reporting Trials (CONSORT) diagram, and whether LTFU was differentiated from withdrawal of consent. Sixty-eight trials were identified, with >154 000 participants. Reasons for treatment discontinuation in pharmacotherapy trials were infrequently reported (35%), particularly in a CONSORT diagram (20%). Eighty-three percent of trials reported LTFU, although only 34% of these differentiated LTFU for vital status from withdrawal of consent. Use of a CONSORT diagram increased over time, although reporting of LTFU in the CONSORT diagram remained low overall at 35%. CONCLUSIONS: Participant flow through RCTs in chronic heart failure has not been uniformly reported, and the use of a complete CONSORT diagram has been low, although it seems to be improving. All study participants should be accounted for within a CONSORT diagram in any RCT to enable the practicing cardiologist to interpret how the results should influence his/her clinical practice.

A role for the 2' residue in the second transmembrane helix of the GABA A receptor gamma2S subunit in channel conductance and gating.

GABA(A) receptors composed of alpha, beta and gamma subunits display a significantly higher single-channel conductance than receptors comprised of only alpha and beta subunits. The pore of GABA(A) receptors is lined by the second transmembrane region from each of its five subunits and includes conserved threonines at the 6', 10' and 13' positions. At the 2' position, however, a polar residue is present in the gamma subunit but not the alpha or beta subunits. As residues at the 2', 6' and 10' positions are exposed in the open channel and as such polar channel-lining residues may interact with permeant ions by substituting for water interactions, we compared both the single-channel conductance and the kinetic properties of wild-type alpha1beta1 and alpha1beta1gamma2S receptors with two mutant receptors, alphabetagamma(S2'A) and alphabetagamma(S2'V). We found that the single-channel conductance of both mutant alphabetagamma receptors was significantly decreased with respect to wild-type alphabetagamma, with the presence of the larger valine side chain having the greatest effect. However, the conductance of the mutant alphabetagamma receptors remained larger than wild-type alphabeta channels. This reduction in the conductance of mutant alphabetagamma receptors was observed at depolarized potentials only (E(Cl) = -1.8 mV), which revealed an asymmetry in the ion conduction pathway mediated by the gamma2' residue. The substitutions at the gamma2' serine residue also altered the gating properties of the channel in addition to the effects on the conductance with the open probability of the mutant channels being decreased while the mean open time increased. The data presented in this study show that residues at the 2' position in M2 of the gamma subunit affects both single-channel conductance and receptor kinetics.

Dengue virus M protein C-terminal peptide (DVM-C) forms ion channels.

A chemically synthesized peptide consisting of the C-terminus of the M protein of the Dengue virus type 1 strain Singapore S275/90 (DVM-C) produced ion channel activity in artificial lipid bilayers. The channels had a variable conductance and were more permeable to sodium and potassium ions than to chloride ions and more permeable to chloride ions than to calcium ions. Hexamethylene amiloride (100 microM) and amantadine (10 microM), blocked channels formed by DVM-C. Ion channels may play an important role in the life cycle of many viruses and drugs that block these channels may prove to be useful antiviral agents.

An amino-acid substitution in the influenza-B NB protein affects ion-channel gating.

The effects of site-directed mutations in NB, a protein encoded by the influenza B virus that has been shown to form cation-selective ion channels at pH 6.0, were studied on ion channel characteristics in artificial lipid bilayers. It was thought that the residues in the hydrophobic region of NB we selected for mutation might be involved in the transport of cations across the channel and that changes in these residues might affect channel properties such as gating and ion-selectivity. Serine residues at positions 20 and 28, threonine at position 24 and cysteine at position 26 were replaced by alanine. We found that the mutation S20A gave channels that did not gate and that remained open most of the time. Proton permeability of NB channels, as detected by fluorescence quenching, was also altered by the mutation S20A: channels were no longer proton-permeable. The other mutations, S28A, T24A and C26A, did not have any detectable effect on the activity or proton permeability of channels formed by NB. The results indicate that serine 20 may have an important role in normal function of NB channels.

Cation-selective ion channels formed by p7 of hepatitis C virus are blocked by hexamethylene amiloride.

A 63 residue peptide, p7, encoded by hepatitis C virus was synthesised and tested for ion channel activity in lipid bilayer membranes. Ion channels formed by p7 had a variable conductance: some channels had conductances as low as 14 pS. The reversal potential of currents flowing through the channels formed by p7 showed that they were permeable to potassium and sodium ions and less permeable to calcium ions. Addition of Ca(2+) to solutions made channels formed by p7 less potassium- or sodium-selective. Hexamethylene amiloride, a drug previously shown to block ion channels formed by Vpu encoded by HIV-1, blocked channels formed by p7. In view of the increasing number of peptides encoded by viruses that have been shown to form ion channels, it is suggested that ion channels may play an important role in the life cycle of many viruses and that drugs that block these channels may prove to be useful antiviral agents.

GABA concentration sets the conductance of delayed GABAA channels in outside-out patches from rat hippocampal neurons.

GABAA channels were activated by GABA in outside-out patches from rat cultured hippocampal neurons. They were blocked by bicuculline and potentiated by diazepam. In 109 of 190 outside-out patches, no channels were active before exposure to GABA (silent patches). The other 81 patches showed spontaneous channel activity. In patches containing spontaneous channel activity, rapid application of GABA rapidly activated channels. In 93 of the silent patches, channels could be activated by GABA but only after a delay that was sometimes as long as 10 minutes. The maximum channel conductance of the channels activated after a delay increased with GABA concentration from less than 10 pS (0.5 microm GABA) to more than 100 pS (10 mm GABA). Fitting the data with a Hill-type equation gave an EC50 value of 33 microm and a Hill coefficient of 0.6. The channels showed outward rectification and were chloride selective. In the presence of 1 microm diazepam, the GABA EC50 decreased to 0.2 microm but the maximum conductance was unchanged. Diazepam decreased the average latency for channel opening. Bicuculline, a GABA antagonist, caused a concentration-dependent decrease in channel conductance. In channels activated with 100 microm GABA the bicuculline IC50 was 19 microm. The effect of GABA on channel conductance shows that the role of the ligand in GABAA receptor channel function is more complex than previously thought.

The glutathione transferase structural family includes a nuclear chloride channel and a ryanodine receptor calcium release channel modulator.

The ubiquitous glutathione transferases (GSTs) catalyze glutathione conjugation to many compounds and have other diverse functions that continue to be discovered. We noticed sequence similarities between Omega class GSTs and a nuclear chloride channel, NCC27 (CLIC1), and show here that NCC27 belongs to the GST structural family. The structural homology prompted us to investigate whether the human Omega class glutathione transferase GSTO1-1 forms or modulates ion channels. We find that GSTO1-1 modulates ryanodine receptors (RyR), which are calcium channels in the endoplasmic reticulum of various cells. Cardiac RyR2 activity was inhibited by GSTO1-1, whereas skeletal muscle RyR1 activity was potentiated. An enzymatically active conformation of GSTO1-1 was required for inhibition of RyR2, and mutation of the active site cysteine (Cys-32 --> Ala) abolished the inhibitory activity. We propose a novel role for GSTO1-1 in protecting cells containing RyR2 from apoptosis induced by Ca(2+) mobilization from intracellular stores.

Oxygen-sensing persistent sodium channels in rat hippocampus.

1. Persistent sodium channel activity was recorded before and during hypoxia from cell-attached and inside-out patches obtained from cultured hippocampal neurons at a pipette potential (Vp) of +30 mV. Average mean current (IU) of these channels was very low under normoxic conditions and was similar in cell-attached and excised inside-out patches (-0.018 +/- 0.010 and -0.025 +/- 0.008 pA, respectively, n = 24). 2. Hypoxia increased the activity of persistent sodium channels in 10 cell-attached patches (IU increased from -0. 026 +/- 0.016 pA in control to -0.156 +/- 0.034 pA during hypoxia, n = 4, P = 0.013). The increased persistent sodium channel activity was most prominent at a VP between +70 and +30 mV (membrane potential, Vm = -70 to -30 mV) and could be blocked by lidocaine, TTX or R56865 (n = 5). Sodium cyanide (NaCN, 5 mM; 0.5-5 min) increased persistent sodium channel activity in cell-attached patches (n = 3) in a similar manner. 3. Hypoxia also increased sodium channel activity in inside-out patches from hippocampal neurons. Within 2-4 min of exposure to hypoxia, I had increased 9-fold to -0. 18 +/- 0.04 pA (n = 21, P = 0.001). Sodium channel activity increased further with longer exposures to hypoxia. 4. The hypoxia-induced sodium channel activity in inside-out patches could be inhibited by exposure to 10-100 microM lidocaine applied via the bath solution (I = -0.03 +/- 0.01 pA, n = 8) or by perfusion of the pipette tip with 1 microM TTX (I = -0.01 +/- 0.01 pA, n = 3). 5. The reducing agent dithiothreitol (DTT, 2-5 mM) rapidly abolished the increase in sodium channel activity caused by hypoxia in excised patches (I = -0.01 +/- 0.01 pA, n = 4). Similarly, reduced glutathione (GSH, 5-20 mM) also reversed the hypoxia-induced increase in sodium channel activity (IU = -0.02 +/- 0.02 pA, n = 5). 6. These results suggest that persistent sodium channels in neurons can sense O2 levels in excised patches of plasma membrane. Hypoxia triggers an increase in sodium channel activity. The redox reaction involved in increasing the sodium channel activity probably occurs in an auxiliary regulatory protein, co-localized in the plasma membrane.

Bicuculline, pentobarbital and diazepam modulate spontaneous GABA(A) channels in rat hippocampal neurons.

Spontaneously opening, chloride-selective channels that showed outward rectification were recorded in ripped-off patches from rat cultured hippocampal neurons and in cell-attached patches from rat hippocampal CA1 pyramidal neurons in slices. In both preparations, channels had multiple conductance states and the most common single-channel conductance varied. In the outside-out patches it ranged from 12 to 70 pS (Vp=40 mV) whereas in the cell-attached patches it ranged from 56 to 85 pS (-Vp=80 mV). Application of GABA to a patch showing spontaneous channel activity evoked a rapid, synchronous activation of channels. During prolonged exposure to either 5 or 100 microM GABA, the open probability of channels decreased. Application of GABA appeared to have no immediate effect on single-channel conductance. Exposure of the patches to 100 microM bicuculline caused a gradual decrease on the single-channel conductance of the spontaneous channels. The time for complete inhibition to take place was slower in the outside-out than in the cell-attached patches. Application of 100 microM pentobarbital or 1 microM diazepam caused 2 - 4 fold increase in the maximum channel conductance of low conductance (<40 pS) spontaneously active channels. The observation of spontaneously opening GABA(A) channels in cell-attached patches on neurons in slices suggests that they may have a role in neurons in vivo and could be an important site of action for some drugs such as benzodiazepines, barbiturates and general anaesthetics.

Pentobarbital modulates gamma-aminobutyric acid-activated single-channel conductance in rat cultured hippocampal neurons.

We examined the effect of a range of pentobarbital concentrations on 0.5 microM gamma-aminobutyric acid (GABA)-activated channels (10 +/- 1 pS) in inside-out or outside-out patches from rat cultured hippocampal neurons. The conductance increased from 12 +/- 4 to 62 +/- 9 pS as the pentobarbital concentration was raised from 10 to 500 microM and the data could be fitted by a Hill-type equation. At 100 microM pentobarbital plus 0.5 microM GABA, the conductance seemed to reach a plateau. The pentobarbital EC(50)(0.5 microM GABA) value was 22 +/- 4 microM and n was 1.9 +/- 0.5. In 1 mM pentobarbital plus 0.5 microM GABA, the single-channel conductance decreased to 34 +/- 8 pS. This apparent inhibition of channel conductance was relieved by 1 microM diazepam. The channel conductance was 64 +/- 6 pS in the presence of all three drugs. The channels were open more in the presence of both GABA and pentobarbital than in the presence of either drug alone. Pentobarbital alone (100 microM) activated channels with conductance (30 +/- 2 pS) and kinetic properties distinct from those activated by either GABA alone or GABA plus pentobarbital. Whether pentobarbital induces new conformations or promotes conformations observed in the presence of GABA alone cannot be determined from our study, but the results clearly show that it is the combination of drugs present that determines the single-channel conductance and the kinetic properties of the receptors.

Mutating the highly conserved second membrane-spanning region 9' leucine residue in the alpha(1) or beta(1) subunit produces subunit-specific changes in the function of human alpha(1)beta(1) gamma-aminobutyric Acid(A) receptors.

The properties of the human alpha(1)beta(1) gamma-aminobutyric acid (GABA)(A) receptors were investigated after mutation of a highly conserved leucine residue at the 9' position in the second membrane-spanning region (TM2). The role of this residue in alpha(1) and beta(1) subunits was examined by mutating the 9' leucine to phenylalanine, tyrosine, or alanine. The mutations were in either the alpha(1) subunit (alpha*beta), the beta(1) subunit (alphabeta*), or in both subunits (alpha*beta*), and the receptors were expressed in Sf9 cells. Our results show that the rate of desensitization is increased as the size and hydrophobicity of the 9' residue in the alpha(1) subunit is increased: Y, F > L > A, T. Mutation of L9' in only the beta(1) subunit (alphabeta*) to either phenylalanine or tyrosine increased the EC(50) value for GABA at least 100 times, but the EC(50) was unchanged in alphabeta* alanine mutants. In the 9' alpha(1) mutants (alpha*beta, alpha*beta*) the GABA EC(50) was minimally affected. In alpha*beta and alpha*beta*, but not alphabeta*, the peak currents evoked by millimolar concentrations of GABA were greatly reduced. The reduction in currents could only be partially accounted for by decreased expression of the receptors These findings suggest different roles for the two types of subunits in GABA activation and later desensitization of alpha(1)beta(1) receptors. In addition, an increase in the resting membrane conductance was recorded in alanine but not in phenylalanine and tyrosine mutants, indicating that the side chain size at the 9' position is a major determinant of current flow in the closed conformation.