NAD(P)H oxidase and peroxidase activities in purified plasma membranes from cauliflower inflorescences

An NAD(P)H oxidase activity stimulated by phenolic compounds has been investigated in purified plasma membranes (pm) and in an intracellular membrane (icm) fraction depleted in plasma membranes, both obtained from a microsomal fraction from cauliflower inflorescences ( Brassica oleracea L.). The phenolic compounds salicylhydroxamic acid (SHAM), ferulic acid, coniferyl alcohol, n -propyl gallate, naringenin, kaempferol and caffeic acid all strongly stimulated the activity. Peroxidase (EC 1.11.1.7), or a peroxidase-like enzyme, was responsible for the NAD(P)H oxidase activity, which proceeded through a free-radical chain reaction and was inhibited by catalase (EC 1.11.1.6), superoxide dismutase (EC 1.15.1.1) and KCN. Most of the total activity was soluble; however, the membrane-bound activity was highly enriched in the pm compared to the icm. The catalase activity was 6 times higher in the icm-fraction than in the pm-fraction, but this was not the reason for the much lower phenol-stimulated NADH oxidase activity in the icm. Peroxidase activity measured with o -dianisidine and H₂O₂ had about the same specific activities in the pm-and icm-fractions.
Neither the phenol-stimulated NADH oxidase nor the peroxidase activity could be washed away from the pm even by 0.7 M NaCl, indicating that these activities are truly membrane-bound. SHAM as well as the other phenolic compounds capable of stimulating the NADH oxidase reaction were potent inhibitors of blue light-induced cytochrome b -reduction in the pm fraction.     

Assessing the complex sponge microbiota: core,variable and species-specific bacterial communities in marine sponges

Marine sponges are well known for their associations with highly diverse, yet very specific and often highly similar microbiota. The aim of this study was to identify potential bacterial sub-populations in relation to sponge phylogeny and sampling sites and to define the core bacterial community. 16S ribosomal RNA gene amplicon pyrosequencing was applied to 32 sponge species from eight locations around the world''s oceans, thereby generating 2567 operational taxonomic units (OTUs at the 97% sequence similarity level) in total and up to 364 different OTUs per sponge species. The taxonomic richness detected in this study comprised 25 bacterial phyla with Proteobacteria, Chloroflexi and Poribacteria being most diverse in sponges. Among these phyla were nine candidate phyla, six of them found for the first time in sponges. Similarity comparison of bacterial communities revealed no correlation with host phylogeny but a tropical sub-population in that tropical sponges have more similar bacterial communities to each other than to subtropical sponges. A minimal core bacterial community consisting of very few OTUs (97%, 95% and 90%) was found. These microbes have a global distribution and are probably acquired via environmental transmission. In contrast, a large species-specific bacterial community was detected, which is represented by OTUs present in only a single sponge species. The species-specific bacterial community is probably mainly vertically transmitted. It is proposed that different sponges contain different bacterial species, however, these bacteria are still closely related to each other explaining the observed similarity of bacterial communities in sponges in this and previous studies. This global analysis represents the most comprehensive study of bacterial symbionts in sponges to date and provides novel insights into the complex structure of these unique associations.     

The DEK Oncogene Is a Target of Steroid Hormone Receptor Signaling in Breast Cancer

Expression of estrogen and progesterone hormone receptors indicates a favorable prognosis due to the successful use of hormonal therapies such as tamoxifen and aromatase inhibitors. Unfortunately, 15–20% of patients will experience breast cancer recurrence despite continued use of tamoxifen. Drug resistance to hormonal therapies is of great clinical concern so it is imperative to identify novel molecular factors that contribute to tumorigenesis in hormone receptor positive cancers and/or mediate drug sensitivity. The hope is that targeted therapies, in combination with hormonal therapies, will improve survival and prevent recurrence. We have previously shown that the DEK oncogene, which is a chromatin remodeling protein, supports breast cancer cell proliferation, invasion and the maintenance of the breast cancer stem cell population. In this report, we demonstrate that DEK expression is associated with positive hormone receptor status in primary breast cancers and is up-regulated in vitro following exposure to the hormones estrogen, progesterone, and androgen. Chromatin immunoprecipitation experiments identify DEK as a novel estrogen receptor α (ERα) target gene whose expression promotes estrogen-induced proliferation. Finally, we report for the first time that DEK depletion enhances tamoxifen-induced cell death in ER+ breast cancer cell lines. Together, our data suggest that DEK promotes the pathogenesis of ER+ breast cancer and that the targeted inhibition of DEK may enhance the efficacy of conventional hormone therapies.     

cGMP-mediated signaling via cGKIalpha is required for the guidance and connectivity of sensory axons

Previous in vitro studies using cGMP or cAMP revealed a cross-talk between signaling mechanisms activated by axonal guidance receptors. However, the molecular elements modulated by cyclic nucleotides in growth cones are not well understood. cGMP is a second messenger with several distinct targets including cGMP-dependent protein kinase I (cGKI). Our studies indicated that the alpha isoform of cGKI is predominantly expressed by sensory axons during developmental stages, whereas most spinal cord neurons are negative for cGKI. Analysis of the trajectories of axons within the spinal cord showed a longitudinal guidance defect of sensory axons within the developing dorsal root entry zone in the absence of cGKI. Consequently, in cGKI-deficient mice, fewer axons grow within the dorsal funiculus of the spinal cord, and lamina-specific innervation, especially by nociceptive sensory neurons, is strongly reduced as deduced from anti-trkA staining. These axon guidance defects in cGKI-deficient mice lead to a substantial impairment in nociceptive flexion reflexes, shown using electrophysiology. In vitro studies revealed that activation of cGKI in embryonic dorsal root ganglia counteracts semaphorin 3A-induced growth cone collapse. Our studies therefore reveal that cGMP signaling is important for axonal growth in vivo and in vitro.     

Receptor Binding Sites and Antigenic Epitopes on the Fiber Knob of Human Adenovirus Serotype 3

The adenovirus fiber knob causes the first step in the interaction of adenovirus with cell membrane receptors. To obtain information on the receptor binding site(s), the interaction of labeled cell membrane proteins to synthetic peptides covering the adenovirus type 3 (Ad3) fiber knob was studied. Peptide P6 (amino acids [aa] 187 to 200), to a lesser extent P14 (aa 281 to 294), and probably P11 (aa 244 to 256) interacted specifically with cell membrane proteins, indicating that these peptides present cell receptor binding sites. Peptides P6, P11, and P14 span the D, G, and I β-strands of the R-sheet, respectively. The other reactive peptides, P2 (aa 142 to 156), P3 (aa 153 to 167), and P16 (aa 300 to 319), probably do not present real receptor binding sites. The binding to these six peptides was inhibited by Ad3 virion and was independent of divalent cations. We have also screened the antigenic epitopes on the knob with recombinant Ad3 fiber, recombinant Ad3 fiber knob, and Ad3 virion-specific antisera by enzyme-linked immunosorbent assay. The main antigenic epitopes were presented by P3, P6, P12 (aa 254 to 269), P14, and especially the C-terminal P16. Peptides P14 and P16 of the Ad3 fiber knob were able to inhibit Ad3 infection of cells.     

Bioavailability and antioxidant effect of epigallocatechin gallate administered in purified form versus as green tea extract in healthy individuals

Tea polyphenols have strong in vitro antioxidant activity. Due to their limited bioavailability, however, their contribution to in vivo antioxidant activity may depend on the form of administration. A human intervention study was performed to evaluate the bioavailability and antioxidant capacity of (-)-epigallocatechin-3-gallate (EGCG) administered as a single large dose in the form of either purified EGCG or as green tea extract (Polyphenon E). Plasma concentrations of tea polyphenols were determined by high-performance liquid chromatography (HPLC) analysis combined with coulometric array electrochemical detection (ECD). We found no differences in plasma EGCG concentrations and trolox equivalents determined by the trolox equivalent antioxidant capacity assay after administration of either form of EGCG. However, we found that the plasma antioxidant activity was significantly affected by changes in the plasma urate concentration, which may have interfered with the effect of tea polyphenols on the antioxidant activity. In addition, lymphocyte 8-hydroxydeoxyguanosine to deoxyguanosine (8-OHdG/10(6)dG) ratios were determined by HPLC with ECD. The 8-OHdG/10(6)dG ratios did not change significantly during the 24 h following both EGCG interventions but correlated significantly within individuals determined during the two interventions separated by 1 week. In summary, changes in plasma uric acid due to dietary intake were significantly correlated to the plasma antioxidant activity and exerted a stronger influence on the plasma antioxidant activity compared with the EGCG intervention. In future studies of dietary effects on the plasma antioxidant capacity, changes in plasma uric acid will need to be closely monitored.     

Neuropeptide S-mediated facilitation of synaptic transmission enforces subthreshold theta oscillations within the lateral amygdala

The neuropeptide S (NPS) receptor system modulates neuronal circuit activity in the amygdala in conjunction with fear, anxiety and the expression and extinction of previously acquired fear memories. Using in vitro brain slice preparations of transgenic GAD67-GFP (Δneo) mice, we investigated the effects of NPS on neural activity in the lateral amygdala as a key region for the formation and extinction of fear memories. We are able to demonstrate that NPS augments excitatory glutamatergic synaptic input onto both projection neurons and interneurons of the lateral amygdala, resulting in enhanced spike activity of both types of cells. These effects were at least in part mediated by presynaptic mechanisms. In turn, inhibition of projection neurons by local interneurons was augmented by NPS, and subthreshold oscillations were strengthened, leading to their shift into the theta frequency range. These data suggest that the multifaceted effects of NPS on amygdaloid circuitry may shape behavior-related network activity patterns in the amygdala and reflect the peptide's potent activity in various forms of affective behavior and emotional memory.     

Nicastrin, presenilin, APH-1, and PEN-2 form active gamma-secretase complexes in mitochondria

Mitochondria are central in the regulation of cell death. Apart from providing the cell with ATP, mitochondria also harbor several death factors that are released upon apoptotic stimuli. Alterations in mitochondrial functions, increased oxidative stress, and neurons dying by apoptosis have been detected in Alzheimer's disease patients. These findings suggest that mitochondria may trigger the abnormal onset of neuronal cell death in Alzheimer's disease. We previously reported that presenilin 1 (PS1), which is often mutated in familial forms of Alzheimer's disease, is located in mitochondria and hypothesized that presenilin mutations may sensitize cells to apoptotic stimuli at the mitochondrial level. Presenilin forms an active gamma-secretase complex together with Nicastrin (NCT), APH-1, and PEN-2, which among other substrates cleaves the beta-amyloid precursor protein (beta-APP) generating the amyloid beta-peptide and the beta-APP intracellular domain. Here we have identified dual targeting sequences (for endoplasmic reticulum and mitochondria) in NCT and showed expression of NCT in mitochondria by immunoelectron microscopy. We also showed that NCT together with APH-1, PEN-2, and PS1 form a high molecular weight complex located in mitochondria. gamma-secretase activity in isolated mitochondria was demonstrated using C83 (alpha-secretase-cleaved C-terminal 83-residue beta-APP fragment from BD8 cells lacking presenilin and thus gamma-secretase activity) or recombinant C100-Flag (C-terminal 100-residue beta-APP fragment) as substrates. Both systems generated an APP intracellular domain, and the activity was inhibited by the gamma-secretase inhibitors l-685,458 or Compound E. This novel localization of NCT, PS1, APH-1, and PEN-2 expands the role and importance of gamma-secretase activity to mitochondria.