Inhibition of oral cancer cell growth by adenovirusMnSOD plus BCNU treatment

We hypothesized that inhibitors of peroxide removal, such as BCNU, an indirect inhibitor of glutathione peroxidase (GPx), and 3-amino-1,2,4-triazole (AT), a direct inhibitor of catalase (CAT), should cause toxicity to cancer cells after manganese superoxide dismutase (MnSOD) overexpression due to elevated peroxide levels. In vitro, hamster cheek pouch carcinoma cells (HCPC-1) and human oral squamous carcinoma cells (SCC-25) were infected with various combinations of adenovirus containing MnSOD cDNA (AdMnSOD). Cells were then treated with or without BCNU and assayed for viability using Annexin/PI staining and flow cytometry. In AdMnSOD plus BCNU-treated SCC-25 and HCPC-1 cells, a 30-60% decrease in cell viability was observed compared to BCNU alone. In vivo, HCPC-1 and SCC-25 xenografts were allowed to grow to approximately 70 mm(3) and 10(9) plaque forming units (pfu) of AdMnSOD were injected directly into the tumors. Two days later, 15 or 30 mg/kg BCNU was injected intratumorally. Tumor growth was greatly inhibited (4- to 20-fold) by this combined treatment, as well as increasing animal survival. Tumor volume could be decreased further by giving multiple doses of AdMnSOD or inhibiting catalase activity with AT. These results suggest that, by using these combination therapies, a significant decrease in tumor mass can be achieved.     

Identification of an expanded binding surface on the FADD death domain responsible for interaction with CD95/Fas

The initiation of programmed cell death at CD95 (Fas, Apo-1) is achieved by forming a death-inducing signaling complex (DISC) at the cytoplasmic membrane surface. Assembly of the DISC has been proposed to occur via homotypic interactions between the death domain (DD) of FADD and the cytoplasmic domain of CD95. Previous analysis of the FADD/CD95 interaction led to the identification of a putative CD95 binding surface within FADD DD formed by alpha helices 2 and 3. More detailed analysis of the CD95/FADD DD interaction now demonstrates that a bimodal surface exists in the FADD DD for interaction with CD95. An expansive surface on one side of the domain is composed of elements in alpha helices 1, 2, 3, 5, and 6. This major surface is common to many proteins harboring this motif, whether or not they are associated with programmed cell death. A secondary surface resides on the opposite face of the domain and involves residues in helices 3 and 4. The major surface is topologically similar to the protein interaction surface identified in Drosophila Tube DD and the death effector domain of hamster PEA-15, two physiologically unrelated proteins which interact with structurally unrelated binding partners. These results demonstrate the presence of a structurally conserved surface within the DD which can mediate protein recognition with homo- and heterotypic binding partners, whereas a second surface may be responsible for stabilizing the higher order complex in the DISC.     

Shrimps remove ectoparasites from fishes in temperate waters

We have found that two very common species of North Atlantic shallow water shrimp, Palaemon adspersus and Palaemon elegans, remove and feed on ectoparasites on plaice (Pleuronectes platessa L.). The relationship could be mutualistic, as we did not observe any attempts by the fishes to feed on the shrimps. The ectoparasites removed included monogenean worms (Gyrodactylus sp.) and sea lice (Lepeophtheirus pectoralis). An experiment showed that there were 65% more Gyrodactylus parasites on the fishes that had been apart from compared with those that had been together with shrimps for 48h. Shrimps on coral reefs are known for cleaning fishes, but that shrimps in temperate waters show parasite-cleaning behaviour is, to our knowledge, a new observation.     

Ultrastructural Observations on the Spermatozoa of Two Temnocephalids (Platyhelminthes)

The spermatozoa of two Temnocephalidae collected in Uruguay, Temnocephala iheringi Haswell, 1893 (Host: Pomacea canaliculata) and Temnocephala axenos Monticelli, 1899 (Host: Parastacus varicosus), were studied with a transmission electron microscope. In both species the spermatozoon is made up of a long sperm body which bears at one extremity two free flagella of the 9+‘1’ flatworm pattern. The sperm body contains the nucleus, mitochondria, dense bodies and parallel, cortical, longitudinal singlet microtubules. Along a part of the sperm body the palissade of the microtubules displays a spiral pattern in transverse sections. A part of the perimeter of the cell is thus lined by two overlapping rows of microtubules. This spiral pattern of the singlets is considered as a synapomorphy of the family Temnocephalidae. The singlet microtubules are interconnected by two kinds of links: tangential links between neighbouring singlets in the same row and radial links between singlets belonging to two rows. The presence of these links suggests that this structure could be a motile system of singlets.     

Phylogenetic relationships within the polyopisthocotylean monogeneans (Platyhelminthes) inferred from partial 28S rDNA sequences

Recent studies based on molecular data (18S rDNA and partial 28S rDNA) and morphology did not resolve a terminal polytomy within the Polyopisthocotylea. Here, we have used sequences from the full domain D2 of the 28S rDNA for 24 species (18 new sequences) with three phylogenetic methods, maximum parsimony, neighbour-joining and maximum likelihood, to infer the relationships among the Polyopisthocotylea. The analysis of the domain D2 of the 28S rDNA has been performed on two data sets. The first one, complete, included the Polystomatidae as the outgroup in order to infer general relationships, and the second one, reduced, excluded the Polystomatidae and the polyopisthocotylean parasites of chondrichthyans, but used the Mazocraeidae as the outgroup in order to resolve the relationships between the terminal groups. The topology found, sustained by high bootstrap and decay index value, is: (outgroup (Chimaericolidae (Mazocraeidae (Gastrocotylinea, other Polyopisthocotylea)))). The polyopisthocotylean parasites of chondrichthyans are the sister-group of the polyopisthocotylean parasites of teleosts. In the latter, the Mazocraeidae, essentially parasites of Clupeidae, have a basal position. The polytomy between Gastrocotylinea, Discocotylinea and Microcotylinea is partially resolved in this study for the first time: the Gastrocotylinea are the sister-group of an unresolved group including the Microcotylinea, Discocotylinea and Plectanocotylidae. Inclusion of the Plectanocotylidae in the suborder Mazocraeinea is rejected. Monophyly of the Microcotylinea and Plectanocotylidae is confirmed, but monophyly of the Discocotylinea is questioned by the exclusion of Diplozoon.     

On the structure and function of the phytoene desaturase CRTI from Pantoea ananatis, a membrane-peripheral and FAD-dependent oxidase/isomerase

CRTI-type phytoene desaturases prevailing in bacteria and fungi can form lycopene directly from phytoene while plants employ two distinct desaturases and two cis-tans isomerases for the same purpose. This property renders CRTI a valuable gene to engineer provitamin A-formation to help combat vitamin A malnutrition, such as with Golden Rice. To understand the biochemical processes involved, recombinant CRTI was produced and obtained in homogeneous form that shows high enzymatic activity with the lipophilic substrate phytoene contained in phosphatidyl-choline (PC) liposome membranes. The first crystal structure of apo-CRTI reveals that CRTI belongs to the flavoprotein superfamily comprising protoporphyrinogen IX oxidoreductase and monoamine oxidase. CRTI is a membrane-peripheral oxidoreductase which utilizes FAD as the sole redox-active cofactor. Oxygen, replaceable by quinones in its absence, is needed as the terminal electron acceptor. FAD, besides its catalytic role also displays a structural function by enabling the formation of enzymatically active CRTI membrane associates. Under anaerobic conditions the enzyme can act as a carotene cis-trans isomerase. In silico-docking experiments yielded information on substrate binding sites, potential catalytic residues and is in favor of single half-site recognition of the symmetrical C(40) hydrocarbon substrate.     

Exosomal Lipids Impact Notch Signaling and Induce Death of Human Pancreatic Tumoral SOJ-6 Cells

Exosomes are of increasing interest as alternative mode of cell-to-cell communication. We previously reported that exosomes secreted by human SOJ-6 pancreatic tumor cells induce (glyco)protein ligand-independent cell death and inhibit Notch-1 pathway, this latter being particularly active during carcinogenesis and in cancer stem cells. Therefore, we asked whether exosomal lipids were key-elements for cell death and hypothesized that cholesterol-rich membrane microdomains were privileged sites of exosome interactions with tumor cells. To address these questions and based on the lipid composition of exosomes from SOJ-6 cells (Ristorcelli et al. (2008) FASEB J. 22; 3358–3369) enriched in cholesterol and sphingomyelin (lipids forming liquid-ordered phase, Lo) and depleted in phospholipids (lipids forming liquid-disordered phase, Ld), we designed Synthetic Exosome-Like Nanoparticles (SELN) with ratios Lo/Ld from 3.0 to 6.0 framing that of SOJ-6 cell exosomes. SELN decreased tumor cell survival, the higher the Lo/Ld ratio, the lower the cell survival. This decreased survival was due to activation of cell death with inhibition of Notch pathway. FRET analyses indicated fusions/exchanges of SELN with cell membranes. Fluorescent SELN co-localized with the ganglioside GM1 then with Rab5A, markers of lipid microdomains and of early endosomes, respectively. These interactions occurred at lipid microdomains of plasma and/or endosome membranes where the Notch-1 pathway matures. We thus demonstrated a major role for lipids in interactions between SELN and tumor cells, and in the ensued cell death. To our knowledge this is the first report on such effects of lipidic nanoparticles on tumor cell behavior. This may have implications in tumor progression.     

Neutrophils sustain effective CD8(+) T-cell responses in the respiratory tract following influenza infection

Neutrophils have an important role in early host protection during influenza A virus infection. Their ability to modulate the virus-specific adaptive immune response is less clear. Here, we have used a mouse model to examine the impact of neutrophils on CD8(+) T-cell responses during influenza virus infection. CD8(+) T-cell priming, expansion, migration, cytokine secretion and cytotoxic capacity were investigated in the virus-infected airways and secondary lymphoid organs. To do this, we utilised a Ly6G-specific monoclonal antibody (mAb; 1A8) that specifically depletes neutrophils in vivo. Neutrophil depletion early after infection with influenza virus strain HKx31 (H3N2) did not alter influenza virus-derived antigen presentation or na?ve CD8(+) T-cell expansion in the secondary lymphoid organs. Trafficking of virus-specific CD8(+) T cells into the infected pulmonary airways was also unaltered. Instead, early neutropenia reduced both the overall magnitude of influenza virus-specific CD8(+) T cells, together with impaired cytokine production and cytotoxic effector function. Therefore, neutrophils are important participants in anti-viral mechanisms that sustain effective CD8(+) T-cell responses in the respiratory tract of influenza virus-infected mice.     

Cognitive mechanisms of risky choice: is there an evaluation cost?

We contrast two classes of choice processes, those assuming time-consuming comparisons and those where stimuli for each option act independently, competing for expression by cross censorship. The Sequential Choice Model (SCM) belongs in the latter category, and has received empirical support in several procedures involving deterministic alternatives. Here we test this model in risky choices. In two treatments, each with five conditions, European starlings (Sturnus vulgaris) faced choices between options with unpredictable outcomes and risk-free alternatives. In the delay treatment the five conditions involved choices between a variable option offering two equiprobable delays to reward and a fixed option with delay differing between conditions. The amount treatment was structurally similar, but amount of reward rather than delay was manipulated. As assumed (and required) by the SCM, latency to respond in no-choice trials reflected each option's richness with respect to the background alternatives, and, crucially, preferences in simultaneous choices were predictable from latencies to each option in forced trials. However, we did not detect reliable differences in response times between forced and choice trials, neither the lengthening expected from evaluation models nor the shortening expected from the SCM.     

Spider Glue Proteins Have Distinct Architectures Compared with Traditional Spidroin Family Members

Adhesive spider glues are required to perform a variety of tasks, including web construction, prey capture, and locomotion. To date, little is known regarding the molecular and structural features of spider glue proteins, in particular bioadhesives that interconnect dragline or scaffolding silks during three-dimensional web construction. Here we use biochemical and structural approaches to identify and characterize two aggregate gland specific gene products, AgSF1 and AgSF2, and demonstrate that these proteins co-localize to the connection joints of both webs and wrapping silks spun from the black widow spider, Latrodectus hesperus. Protein architectures are markedly divergent between AgSF1 and AgSF2, as well as traditional spider silk fibroin family members, suggesting connection joints consist of a complex proteinaceous network. AgSF2 represents a nonglycosylated 40-kDa protein that has novel internal amino acid block repeats with the consensus sequence NVNVN embedded in a glycine-rich matrix. Analysis of the amino acid sequence of AgSF1 reveals pentameric QPGSG iterations that are similar to conserved modular elements within mammalian elastin, a rubber-like elastomeric protein that interfaces with collagen. Wet-spinning methodology using purified recombinant proteins show AgSF1 has the potential to self-assemble into fibers. X-ray fiber diffraction studies performed on these synthetic fibers reveal the presence of noncrystalline domains that resemble classical rubber networks. Collectively, these data support that the aggregate gland serves to extrude a protein mixture that contains substances that allow for the self-assembly of fiber-like structures that interface with dragline silks to mediate prey capture.