The synergistic anti-inflammatory effects of lycopene, lutein, β-carotene, and carnosic acid combinations via redox-based inhibition of NF-κB signaling

Inflammatory mediators and cytokines play important roles in the pathogenesis of a vast number of human diseases; therefore much attention is focused on blunting their proinflammatory modes of action. The aims of the present research were to assess the effectiveness of combinations of carotenoids and phenolics, at concentrations that can be achieved in blood, to inhibit the release of inflammatory mediators from macrophages exposed to lipopolysaccharide (LPS) and to determine what the anti-inflammatory effect of the phytonutrient combinations was in an in vivo mouse model of peritonitis. Preincubation of mouse peritoneal macrophages with lycopene (1μM) or Lyc-O-Mato (1μM) and carnosic acid (2μM), lutein (1μM), and/or β-carotene (2μM) 1h before addition of LPS for 24h caused a synergistic inhibition of NO, prostaglandin E(2), and superoxide production derived from downregulation of iNOS, COX-2, and NADPH oxidase protein and mRNA expression and synergistic inhibition of TNFα secretion. We surmise that the anti-inflammatory action of the phytonutrient combinations used probably resides in their antioxidant properties, because they caused an immediate, efficient, and synergistic inhibition of LPS-induced internal superoxide production leading to a marked decrease in ERK and NF-κB activation. The anti-inflammatory effects of the selected phytonutrient combinations were also demonstrated in a mouse model of peritonitis: their supplementation in drinking water resulted in attenuation of neutrophil recruitment to the peritoneal cavity and in inhibition of inflammatory mediator production by peritoneal neutrophils and macrophages.     

ApoE isoform‐dependent deficits in extinction of contextual fear conditioning

The three major human apoE isoforms (apoE2, apoE3 and apoE4) are encoded by distinct alleles (?2, ?3 and ?4). Compared with ?3, ?4 is associated with increased risk to develop Alzheimer's disease (AD), cognitive impairments in Parkinson's disease (PD), and other conditions. In contrast, a recent study indicated an increased susceptibility to the recurring and re‐experiencing symptom cluster of Post‐Traumatic Stress Disorder (PTSD), as well as related memory impairments, in patients carrying at least one ?2 allele. Contextual fear conditioning and extinction are used in human and animal models to study this symptom cluster. In this study, acquisition (day 1, training), consolidation (day 2, first day of re‐exposure) and extinction (days 2–5) of conditioned contextual fear in human apoE2, apoE3 and apoE4 targeted replacement and C57BL/6J wild‐type (WT) mice was investigated. Male and female apoE2 showed acquisition and retrieval of conditioned fear, but failed to exhibit extinction. In contrast, WT, apoE3 and apoE4 mice showed extinction. While apoE2 mice exhibited lower freezing in response to the context on day 2 than apoE3 and apoE4 mice, this cannot explain their extinction deficit as WT mice exhibited similar freezing levels as apoE2 mice on day 2 but still exhibited extinction. Elevating freezing through extended training preserved extinction in controls, but failed to ameliorate extinction deficits in apoE2 animals. These data along with clinical data showing an association of apoE2 with susceptibility to specific symptom clusters in PTSD supports an important role for apoE isoform in the extinction of conditioned fear.     

Genetic and chemical characterization of an EMS induced mutation in Cucumis melo CRTISO gene

In order to broaden the available genetic variation of melon, we developed an ethyl methanesulfonate mutation library in an orange-flesh ‘Charentais’ type melon line that accumulates β-carotene. One mutagenized M₂ family segregated for a novel recessive trait, a yellow–orange fruit flesh (‘yofI’). HPLC analysis revealed that ‘yofI’ accumulates pro-lycopene (tetra-cis-lycopene) as its major fruit pigment. The altered carotenoid composition of ‘yofI’ is associated with a significant change of the fruit aroma since cleavage of β-carotene yields different apocarotenoids than the cleavage of pro-lycopene. Normally, pro-lycopene is further isomerized by CRTISO (carotenoid isomerase) to yield all-trans-lycopene, which is further cyclized to β-carotene in melon fruit. Cloning and sequencing of ‘yofI’ CRTISO identified two mRNA sequences which lead to truncated forms of CRTISO. Sequencing of the genomic CRTISO identified an A–T transversion in ‘yofI’ which leads to a premature STOP codon. The early carotenoid pathway genes were up regulated in yofI fruit causing accumulation of other intermediates such as phytoene and ζ-carotene. Total carotenoid levels are only slightly increased in the mutant. Mutants accumulating pro-lycopene have been reported in both tomato and watermelon fruits, however, this is the first report of a non-lycopene accumulating fruit showing this phenomenon.     

Lysine binding to activated human platelets and its similarity to fibrinogen binding

Platelet surface glycoproteins IIb-IIIa are considered to function as the binding site for fibrinogen. Fibrinogen binding is essential for platelet aggregation and several amines have been shown to inhibit this binding. The present study compares the binding properties of 125I-fibrinogen and [3H]lysine with platelets activated by the Ca2+ ionophore A23187. Many lines of similarities in the binding properties are apparent; however, several differences were also found. The similarities are listed below and the differences are pointed out in parentheses. Marked enhancement by platelet activation; deficiency of binding by thrombasthenic platelets lacking the glycoproteins IIb-IIIa; saturability (fibrinogen binding approaches saturation at more than 12 microM, within 10 min; lysine binding at more than 100 mM within 1 min); Ca2+-dependence (at 1 mM Ca2+ lysine binding is minute and fibrinogen binding is half-saturated); reversibility; the binding achieved within 10 min is exchangeable; dissociation depends upon time and external ligand concentration; inhibition by the oligoamines His-Lys and Lys4; inhibition by serum from a thrombasthenic patient who developed anti-glycoproteins IIb-IIIa antibodies; specificity; alanine neither binds to activated platelets nor inhibits fibrinogen binding; it thus appears that the lysine which associates with activated platelets is mostly bound onto the surface of the cells rather than being incorporated. Moreover, the major site of lysine binding seems to be the complexed glycoproteins IIb-IIIa.     

Effects of carbanilates on the growth and development of cell suspension cultures of Acer pseudoplatanus

The effects of three isopropyl carbanilate derivatives (propham, chlorpropham, 3,4-diCl isopropyl carbanilate) were studied on Acer pseudoplatanus L. cell suspension cultures (Lamport's strain). At 0.1 m M , three types of effects were observed: the 3,4-diCl derivative rapidly killed the cells; chlorpropham inhibited mitosis, but the cells remained alive without any significant increase in fresh or dry matter; and propham strongly inhibited mitosis, but not cell growth.
Ultrastructural analysis indicated that no observable changes in mitochondrial or plastid density occurred during the cytoplasmic expansion in the presence of propham. Size increase was also accompanied by vacuolar expansion and morphological changes of the nuclei. In Acer cell cultures, isopropyl carbanilates clearly show selective inhibition of mitosis only when the phenyl ring is not substituted by chlorine.     

Donor-Derived Brain Tumor Following Neural Stem Cell Transplantation in an Ataxia Telangiectasia Patient

Background

Neural stem cells are currently being investigated as potential therapies for neurodegenerative diseases, stroke, and trauma. However, concerns have been raised over the safety of this experimental therapeutic approach, including, for example, whether there is the potential for tumors to develop from transplanted stem cells.

Methods and Findings

A boy with ataxia telangiectasia (AT) was treated with intracerebellar and intrathecal injection of human fetal neural stem cells. Four years after the first treatment he was diagnosed with a multifocal brain tumor. The biopsied tumor was diagnosed as a glioneuronal neoplasm. We compared the tumor cells and the patient''s peripheral blood cells by fluorescent in situ hybridization using X and Y chromosome probes, by PCR for the amelogenin gene X- and Y-specific alleles, by MassArray for the ATM patient specific mutation and for several SNPs, by PCR for polymorphic microsatellites, and by human leukocyte antigen (HLA) typing. Molecular and cytogenetic studies showed that the tumor was of nonhost origin suggesting it was derived from the transplanted neural stem cells. Microsatellite and HLA analysis demonstrated that the tumor is derived from at least two donors.

Conclusions

This is the first report of a human brain tumor complicating neural stem cell therapy. The findings here suggest that neuronal stem/progenitor cells may be involved in gliomagenesis and provide the first example of a donor-derived brain tumor. Further work is urgently needed to assess the safety of these therapies.     

Concepts and schemes for the re-engineering of physical protein modules: generating nanodevices via targeted replacements with constrained amino acids

Physically building complex multi-molecular structures from naturally occurring biological macromolecules has aroused a great deal of interest. Here we focus on nanostructures composed of re-engineered, natural 'foldamer' building blocks. Our aim is to provide some of the underlying concepts and schemes for crafting structures utilizing such conformationally relatively stable molecular components. We describe how, via chemical biology strategies, it is further possible to chemically manipulate the foldamer building blocks toward specific shape-driven structures, which in turn could be used toward potential-designed functions. We outline the criteria in choosing candidate foldamers from the vast biological repertoire, and how to enhance their stability through selected targeted replacements by non-proteinogenic conformationally constrained amino acids. These approaches combine bioinformatics, high performance computations and mathematics with synthetic organic chemistry. The resulting artificially engineered self-organizing molecular scale structures take advantage of nature's nanobiology toolkit and at the same time improve on it, since their new targeted function differs from that optimized by evolution. The major challenge facing nanobiology is to be able to exercise fine control over the performance of these target-specific molecular machines.     

Multi‐scale characterization of the energy landscape of proteins with application to the C3D/Efb‐C complex

We present a novel multi‐level methodology to explore and characterize the low energy landscape and the thermodynamics of proteins. Traditional conformational search methods typically explore only a small portion of the conformational space of proteins and are hard to apply to large proteins due to the large amount of calculations required. In our multi‐scale approach, we first provide an initial characterization of the equilibrium state ensemble of a protein using an efficient computational conformational sampling method. We then enrich the obtained ensemble by performing short Molecular Dynamics (MD) simulations on selected conformations from the ensembles as starting points. To facilitate the analysis of the results, we project the resulting conformations on a low‐dimensional landscape to efficiently focus on important interactions and examine low energy regions. This methodology provides a more extensive sampling of the low energy landscape than an MD simulation starting from a single crystal structure as it explores multiple trajectories of the protein. This enables us to obtain a broader view of the dynamics of proteins and it can help in understanding complex binding, improving docking results and more. In this work, we apply the methodology to provide an extensive characterization of the bound complexes of the C3d fragment of human Complement component C3 and one of its powerful bacterial inhibitors, the inhibitory domain of Staphylococcus aureus extra‐cellular fibrinogen‐binding domain (Efb‐C) and two of its mutants. We characterize several important interactions along the binding interface and define low free energy regions in the three complexes. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Variations in the number of pituitary LHRH receptors correlated with altered responsiveness to LHRH

Isolated pituitary cells from metestrous, ovariectomized (OVX), and ovariectomized-estradiol treated (OVX-EB) rats were employed to study the gonadotropin response to luteinizing hormone-releasing hormone (LHRH) challenge and to quantitate LHRH receptors, using a labeled LHRH analog. Ovariectomy (3–4 weeks post castration) resulted in a reduction of LHRH receptor concentration from 34.4 ± 2.1 in metestrous females to 14.3 ± 0.9 fmoles/10⁶ cells. Concomitantly, the luteinizing hormone (LH) response to a near-maximal dose of LHRH (5 ng/ml) decreased from a 3-fold stimulation in intact females to 1.13-fold stimulation in cells from OVX rats. Replacement therapy with EB (50 ug/rat for 2 days) to OVX rats restored LH response and LHRH binding sites (a 2.5-fold stimulation in LH secretion and 32.0 ± 2.1 fmoles/10⁶ cells, respectively). The LH response to LHRH stimulation was not altered after one day of EB treatment although the number of LHRH binding sites was increased. The changes in the number of LHRH binding sites were not accompanied by any alterations in the affinity of the LHRH analog ($\text{K}{}_{\mathrm{d}}\text{? 0.5 × 10}{}^{\mathrm{?9}}\text{M}$). It is concluded that variations in LHRH receptor number reflect the degree of pituitary sensitivity to LHRH and it may suggest that LHRH and estradiol modulation of gonadotropin release is mediated by these receptors.     

Primary Coenzyme Q deficiency Due to Novel ADCK3 Variants,Studies in Fibroblasts and Review of Literature

Primary deficiency of coenzyme Q10 (CoQ10 ubiquinone), is classified as a mitochondrial respiratory chain disorder with phenotypic variability. The clinical manifestation may involve one or multiple tissue with variable severity and presentation may range from infancy to late onset. ADCK3 gene mutations are responsible for the most frequent form of hereditary CoQ10 deficiency (Q10 deficiency-4 OMIM #612016) which is mainly associated with autosomal recessive spinocerebellar ataxia (ARCA2, SCAR9). Here we provide the clinical, biochemical and genetic investigation for unrelated three nuclear families presenting an autosomal form of Spino-Cerebellar Ataxia due to novel mutations in the ADCK3 gene. Using next generation sequence technology we identified a homozygous Gln343Ter mutation in one family with severe, early onset of the disease and compound heterozygous mutations of Gln343Ter and Ser608Phe in two other families with variable manifestations. Biochemical investigation in fibroblasts showed decreased activity of the CoQ dependent mitochondrial respiratory chain enzyme succinate cytochrome c reductase (complex II?+?III). Exogenous CoQ slightly improved enzymatic activity, ATP production and decreased oxygen free radicals in some of the patient’s cells. Our results are presented in comparison to previously reported mutations and expanding the clinical, molecular and biochemical spectrum of ADCK3 related CoQ10 deficiencies.