Antioxidant activity of synthetic cytokinin analogues: 6-alkynyl- and 6-alkenylpurines as novel 15-Lipoxygenase inhibitors

Synthetic cytokinin analogues as well as the well known CKs 6-benzylaminopurine (BAP), kinetin and trans-zeatin were examined for antioxidant activity. The compounds were tested as potential diphenylpicrylhydrazyl (DPPH) scavengers and as inhibitors of 15-lipoxygenase (15-LO). The natural plant hormones were essentially inactive in both assays, but several synthetic analogues have a profound inhibiting effect on 15-lipoxygenase from soybeans. The same compounds were only weak DPPH scavengers and they may therefore be regarded as so-called non antioxidant inhibitors of 15-LO.     

The Frequencies of IFNγ+IL2+TNFα+ PPD-Specific CD4+CD45RO+ T-Cells Correlate with the Magnitude of the QuantiFERON? Gold In-Tube Response in a Prospective Study of Healthy Indian Adolescents

Background

QuantiFERON-TB Gold In-Tube (QFT) is an IFNγ-release assay used in the diagnosis of Mycobacterium tuberculosis (MTB) infection. The risk of TB progression increases with the magnitude of the MTB-specific IFNγ-response. QFT reversion, also associated with low Tuberculin Skin Test responses, may therefore represent a transient immune response with control of M. tuberculosis infection. However, studies at the single cell level have suggested that the quality (polyfunctionality) of the T-cell response is more important than the quantity of cytokines produced.

Objective

To explore the quality and/or magnitude of mycobacteria-specific T-cell responses associated with QFT reversion and persistent QFT-positivity.

Methods

Multi-color flowcytometry on prospectively collected peripheral blood mononuclear cells was applied to assess mycobacteria-specific T-cell responses in 42 QFT positive Indian adolescents of whom 21 became QFT negative (reverters) within one year. Ten QFT consistent negatives were also included as controls.

Results

There was no difference in the qualitative PPD-specific CD4+ T-cell response between QFT consistent positives and reverters. However, compared with QFT consistent positives, reverters displayed lower absolute frequencies of polyfunctional (IFNγ+IL2+TNFα+) CD4+ T-cells at baseline, which were further reduced to the point where they were not different to QFT negative controls one year later. Moreover, absolute frequencies of these cells correlated well with the magnitude of the QFT-response.

Conclusion

Whereas specific polyfunctional CD4+ T-cells have been suggested to protect against TB progression, our data do not support that higher relative or absolute frequencies of PPD-specific polyfunctional CD4+ T-cells in peripheral blood can explain the reduced risk of TB progression observed in QFT reverters. On the contrary, absolute frequencies of these cells correlated with the QFT-response, suggesting that this readout reflects antigenic load.     

Toxicological Profile of Ultrapure 2,2′,3,4,4′,5,5′-Heptachlorbiphenyl (PCB 180) in Adult Rats

PCB 180 is a persistent non-dioxin-like polychlorinated biphenyl (NDL-PCB) abundantly present in food and the environment. Risk characterization of NDL-PCBs is confounded by the presence of highly potent dioxin-like impurities. We used ultrapure PCB 180 to characterize its toxicity profile in a 28-day repeat dose toxicity study in young adult rats extended to cover endocrine and behavioral effects. Using a loading dose/maintenance dose regimen, groups of 5 males and 5 females were given total doses of 0, 3, 10, 30, 100, 300, 1000 or 1700 mg PCB 180/kg body weight by gavage. Dose-responses were analyzed using benchmark dose modeling based on dose and adipose tissue PCB concentrations. Body weight gain was retarded at 1700 mg/kg during loading dosing, but recovered thereafter. The most sensitive endpoint of toxicity that was used for risk characterization was altered open field behavior in females; i.e. increased activity and distance moved in the inner zone of an open field suggesting altered emotional responses to unfamiliar environment and impaired behavioral inhibition. Other dose-dependent changes included decreased serum thyroid hormones with associated histopathological changes, altered tissue retinoid levels, decreased hematocrit and hemoglobin, decreased follicle stimulating hormone and luteinizing hormone levels in males and increased expression of DNA damage markers in liver of females. Dose-dependent hypertrophy of zona fasciculata cells was observed in adrenals suggesting activation of cortex. There were gender differences in sensitivity and toxicity profiles were partly different in males and females. PCB 180 adipose tissue concentrations were clearly above the general human population levels, but close to the levels in highly exposed populations. The results demonstrate a distinct toxicological profile of PCB 180 with lack of dioxin-like properties required for assignment of WHO toxic equivalency factor. However, PCB 180 shares several toxicological targets with dioxin-like compounds emphasizing the potential for interactions.     

Exploring the Human Plasma Proteome for Humoral Mediators of Remote Ischemic Preconditioning - A Word of Caution

Despite major advances in early revascularization techniques, cardiovascular diseases are still the leading cause of death worldwide, and myocardial infarctions contribute heavily to this. Over the past decades, it has become apparent that reperfusion of blood to a previously ischemic area of the heart causes damage in and of itself, and that this ischemia reperfusion induced injury can be reduced by up to 50% by mechanical manipulation of the blood flow to the heart. The recent discovery of remote ischemic preconditioning (RIPC) provides a non-invasive approach of inducing this cardioprotection at a distance. Finding its endogenous mediators and their operative mode is an important step toward increasing the ischemic tolerance. The release of humoral factor(s) upon RIPC was recently demonstrated and several candidate proteins were published as possible mediators of the cardioprotection. Before clinical applicability, these potential biomarkers and their efficiency must be validated, a task made challenging by the large heterogeneity in reported data and results. Here, in an attempt to reproduce and provide more experimental data on these mediators, we conducted an unbiased in-depth analysis of the human plasma proteome before and after RIPC. From the 68 protein markers reported in the literature, only 28 could be mapped to manually reviewed (Swiss-Prot) protein sequences. 23 of them were monitored in our untargeted experiment. However, their significant regulation could not be reproducibly estimated. In fact, among the 394 plasma proteins we accurately quantified, no significant regulation could be confidently and reproducibly assessed. This indicates that it is difficult to both monitor and reproduce published data from experiments exploring for RIPC induced plasma proteomic regulations, and suggests that further work should be directed towards small humoral factors. To simplify this task, we made our proteomic dataset available via ProteomeXchange, where scientists can mine for novel potential targets.     

MX 2 is a novel regulator of cell cycle in melanoma cells

MX2 protein is a dynamin‐like GTPase2 that has recently been identified as an interferon‐induced restriction factor of HIV‐1 and other primate lentiviruses. A single nucleotide polymorphism (SNP), rs45430, in an intron of the MX2 gene, was previously reported as a novel melanoma susceptibility locus in genome‐wide association studies. Functionally, however, it is still unclear whether and how MX2 contributes to melanoma susceptibility and tumorigenesis. Here, we show that MX2 is differentially expressed in melanoma tumors and cell lines, with most metastatic cell lines showing lower MX2 expression than primary melanoma cell lines and melanocytes. Furthermore, high expression of MX2 RNA in primary melanoma tumors is associated with better patient survival. Overexpression of MX2 reduces in vivo proliferation partially through inhibition of AKT activation, suggesting that it can act as a tumor suppressor in melanoma. However, we have also identified a subset of melanoma cell lines with high endogenous MX2 expression where downregulation of MX2 leads to reduced proliferation. In these cells, MX2 downregulation interfered with DNA replication and cell cycle processes. Collectively, our data for the first time show that MX2 is functionally involved in the regulation of melanoma proliferation but that its function is context‐dependent.     

Uptake of Glycine into Synaptic Vesicles Isolated from Rat Spinal Cord

Glycine was taken up by a synaptic vesicle fraction from spinal cord in a Mg-ATP-dependent manner. The accumulation of glycine was inhibited by carbonyl cyanide-m-chlorophenylhydrazone (CCCP) and nigericin, agents known to destroy the proton gradient across the vesicle membrane. Vesicular uptake of glycine was clearly different from synaptosomal uptake, with respect to both the affinity constant and the effect of Na+, ATP, CCCP, and temperature. Oligomycin and strychnine did not inhibit the vesicular uptake, showing that neither mitochondrial H(+)-ATPase nor binding to strychnine-sensitive glycine receptors was involved. It is suggested that the vesicular uptake of glycine is driven by a proton gradient generated by a Mg2(+)-ATPase. A low concentration of Cl- had little effect on the uptake of glycine, whereas the uptake of glutamate in the same experiment was highly stimulated. High concentrations of gamma-amino-n-butyric acid and beta-alanine inhibited vesicular glycine uptake, but glutamate did not. Accumulation of glycine was found to be fourfold higher in a spinal cord synaptic vesicle fraction than in a vesicle fraction from cerebral cortex.     

Contributing mechanisms for cysteine excitotoxicity in cultured cerebellar granule cells

Several possible mechanisms for cysteine toxicity on rat cerebellar granule cells were studied and compared with the excitotoxic effect of glutamate. It was shown that the excitotoxic potency of both cysteine and glutamate increased in the presence of elevated concentrations, of bicarbonate or increased pH. Pharmacological studies showed that the cysteine toxicity was specifically coupled to the NMDA receptor, whereas the glutamate toxicity was mediated to a smaller extent also by non-NMDA receptors. Treatment of cerebellar granule cells with cysteine led to an increased extracellular level of glutamate. In addition, cysteine sensitized NMDA receptors by reducing disulfide bonds in the receptor to sulfhydryl groups. A mechanism for cysteine excitotoxicity may therefore be formation of cysteine-sensitized NMDA receptors that are stimulated either by cysteine and/or by endogenous glutamate. This mechanism may also be important for the effects observed during regulated physiological release of cysteine.     

L-[3H]Glutamate Binding to a Membrane Preparation from Crayfish Muscle

The binding of L-[3H]glutamate to an isolated membrane preparation from crayfish tail muscle has been studied. The muscle homogenate was osmotically shocked, frozen and thawed, and thoroughly washed before incubation with L-[3H]glutamate. The preparation showed high specific binding of L-glutamate with a KD of 0.12 microM and Bmax of 4.7 pmol/mg protein measured in Tris/HCl pH 7.3 and at 4 degrees C. Nonspecific binding was 5-10% of total binding. The glutamate binding was highly stereospecific [K0.5 (D-glutamate), 270 microM] and showed a high degree of discrimination between L-glutamate and L-aspartate [K0.5 (L-aspartate), 54 microM]. In mammalian CNS preparations potent agonists of L-glutamate such as kainate and N-methyl-D-aspartate had no effect at 1 mM, and quisqualate was a weak inhibitor of L-glutamate binding [K0.5 (quisqualate), 162 microM]. Ibotenate was the most potent inhibitor [K0.5 (ibotenate), 0.27 microM], and various esters of L-glutamate were of intermediate potency as displacers of L-[3H]glutamate binding (K0.5 values from 6 to 60 microM). The glutamate binding site from crayfish muscle is clearly different from any of the subclasses of glutamate receptors in mammalian CNS. A possible physiological function of the binding site is a postsynaptic receptor for glutamate, either an extra-junctional or a junctional receptor.     

Differences in Anionic Dependence of the Synaptic Efflux of d-Aspartic Acid and γ-Aminobutyric Acid

The synaptosomal effluxes of D-aspartate and gamma-aminobutyric acid (GABA) induced by a substitution of the Cl- ions with propionate in the incubation medium were measured in preparations of hippocampal tissue homogenates. The efflux of aspartate was significantly higher than spontaneous efflux at 125 mM Cl- (normal = 144 mM) and was increased with decreasing Cl- concentration. GABA efflux was much less sensitive to a reduction in Cl- concentration than D-aspartate. The efflux was Ca2+-dependent in both cases.