Protein kinases mediate nitric oxide-induced apoptosis in the insect cell line IPLB-LdFB

The involvement of protein kinases (PKA, PKC and PKB) in nitric oxide (NO)-induced apoptosis with sodium nitroprusside plus N-acetyl-L-cysteine in the IPLB-LdFB cell line from the insect Lymantria dispar was investigated. The presence of protein kinase-like molecules was demonstrated by western blot analysis. The role of the kinases in programmed cell death was analysed in cytofluorimetric experiments by incubating the insect cells with H-89 (a specific inhibitor of PKA), calphostin C (an inhibitor of PKC) or wortmannin (an inhibitor of phosphatidylinositol 3-kinase). The results show that PKA is correlated with the induction and PKC and PKB with the prevention of NO-induced insect cell death. Moreover, NO-induced apoptosis involves the release of cytochrome c. Received 15 March 2002; accepted 25 March 2002     

RAGE is a multiligand receptor of the immunoglobulin superfamily: implications for homeostasis and chronic disease

Receptor for AGE (RAGE) is a member of the immunoglobulin superfamily that engages distinct classes of ligands. The biology of RAGE is driven by the settings in which these ligands accumulate, such as diabetes, inflammation, neurodegenerative disorders and tumors. In this review, we discuss the context of each of these classes of ligands, including advance glycation end-products, amyloid beta peptide and the family of beta sheet fibrils, S100/calgranulins and amphoterin. Implications for the role of these ligands interacting with RAGE in homeostasis and disease will be considered.     

Morphine 6 glucuronide stimulates nitric oxide release in mussel neural tissues: evidence for a morphine 6 glucuronide opiate receptor subtype

We have previously demonstrated that Mytilus edulis pedal ganglia contain opiate alkaloids, i.e., morphine and morphine 6 glucuronide (M6G), as well as mu opiate receptor subtype fragments exhibiting high sequence similarity to those found in mammals. Now we demonstrate that M6G stimulates pedal ganglia constitutive nitric oxide (NO) synthase (cNOS)-derived NO release at identical concentrations and to similar peak levels as morphine. However, the classic opiate antagonist, naloxone, only blocked the ability of morphine to stimulate cNOS-derived NO release and not that of M6G. CTOP, a mu-specific antagonist, blocked the ability of M6G to induce cNOS-derived NO release as well as that of morphine, suggesting that a novel mu opiate receptor was present and selective toward M6G. In examining a receptor displacement analysis, both opiate alkaloids displaced [³H]-dihydromorphine binding to the mu opiate receptor subtype. However, morphine exhibited a twofold higher affinity, again suggesting that a novel mu opiate receptor may be present. Received 1 November 2001; received after revision 1 February 2002; accepted 1 February 2002     

HIV-1 superinfection despite broad CD8+ T-cell responses containing replication of the primary virus

Early treatment of acute HIV-1 infection followed by treatment interruptions has shown promise for enhancing immune control of infection. A subsequent loss of control, however, allows the correlates of protective immunity to be assessed. Here we show that sudden breakthrough of plasma viraemia occurred after prolonged immune containment in an individual infected with HIV-1 at a time when 25 distinct CD8+ T-cell epitopes in the viral proteins Gag, RT, Integrase, Env, Nef, Vpr, Vif and Rev were being targeted. Sequencing of the virus in plasma and cells showed that superinfection with a second clade-B virus was coincident with the loss of immune control. This sudden increase in viraemia was associated with a decline in half of the CD8+ T-cell responses. The declining CD8+ T-cell responses were coupled with sequence changes relative to the initial virus that resulted in impaired recognition. Our data show that HIV-1 superinfection can occur in the setting of a strong and broadly directed virus-specific CD8+ T-cell response. The lack of cross-protective immunity for closely related HIV-1 strains, despite persistent recognition of multiple CD8 epitopes, has important implications for public health and vaccine development.     

Multiple ionization of atom clusters by intense soft X-rays from a free-electron laser

Intense radiation from lasers has opened up many new areas of research in physics and chemistry, and has revolutionized optical technology. So far, most work in the field of nonlinear processes has been restricted to infrared, visible and ultraviolet light, although progress in the development of X-ray lasers has been made recently. With the advent of a free-electron laser in the soft-X-ray regime below 100 nm wavelength, a new light source is now available for experiments with intense, short-wavelength radiation that could be used to obtain deeper insights into the structure of matter. Other free-electron sources with even shorter wavelengths are planned for the future. Here we present initial results from a study of the interaction of soft X-ray radiation, generated by a free-electron laser, with Xe atoms and clusters. We find that, whereas Xe atoms become only singly ionized by the absorption of single photons, absorption in clusters is strongly enhanced. On average, each atom in large clusters absorbs up to 400 eV, corresponding to 30 photons. We suggest that the clusters are heated up and electrons are emitted after acquiring sufficient energy. The clusters finally disintegrate completely by Coulomb explosion.     

Inflammation and therapeutic vaccination in CNS diseases

The spectrum of inflammatory diseases of the central nervous system has been steadily expanding from classical autoimmune disorders such as multiple sclerosis to far more diverse diseases. Evidence now suggests that syndromes such as Alzheimer's disease and stroke have important inflammatory and immune components and may be amenable to treatment by anti-inflammatory and immunotherapeutic approaches. The notion of 'vaccinating' individuals against a neurodegenerative disorder such as Alzheimer's disease is a marked departure from classical thinking about mechanism and treatment, and yet therapeutic vaccines for both Alzheimer's disease and multiple sclerosis have been validated in animal models and are in the clinic. Such approaches, however, have the potential to induce unwanted inflammatory responses as well as to provide benefit.     

Active transport of Ca2+ by an artificial photosynthetic membrane

Transport of calcium ions across membranes and against a thermodynamic gradient is essential to many biological processes, including muscle contraction, the citric acid cycle, glycogen metabolism, release of neurotransmitters, vision, biological signal transduction and immune response. Synthetic systems that transport metal ions across lipid or liquid membranes are well known, and in some cases light has been used to facilitate transport. Typically, a carrier molecule located in a symmetric membrane binds the ion from aqueous solution on one side and releases it on the other. The thermodynamic driving force is provided by an ion concentration difference between the two aqueous solutions, coupling to such a gradient in an auxiliary species, or photomodulation of the carrier by an asymmetric photon flux. Here we report a different approach, in which active transport is driven not by concentration gradients, but by light-induced electron transfer in a photoactive molecule that is asymmetrically disposed across a lipid bilayer. The system comprises a synthetic, light-driven transmembrane Ca2+ pump based on a redox-sensitive, lipophilic Ca2+-binding shuttle molecule whose function is powered by an intramembrane artificial photosynthetic reaction centre. The resulting structure transports calcium ions across the bilayer of a liposome to develop both a calcium ion concentration gradient and a membrane potential, expanding Mitchell's concept of a redox loop mechanism for protons to include divalent cations. Although the quantum yield is relatively low (approximately 1 per cent), the Ca2+ electrochemical potential developed is significant.     

Endocytosis-mediated downregulation of LIN-12/Notch upon Ras activation in Caenorhabditis elegans

The coordination of signals from different pathways is important for cell fate specification during animal development. Here, we define a novel mode of crosstalk between the epidermal growth factor receptor/Ras/mitogen-activated protein kinase cascade and the LIN-12/Notch pathway during Caenorhabditis elegans vulval development. Six vulval precursor cells (VPCs) are initially equivalent but adopt different fates as a result of an inductive signal mediated by the Ras pathway and a lateral signal mediated by the LIN-12/Notch pathway. One consequence of activating Ras is a reduction of LIN-12 protein in P6.p (ref. 2), the VPC believed to be the source of the lateral signal. Here we identify a 'downregulation targeting signal' (DTS) in the LIN-12 intracellular domain, which encompasses a di-leucine-containing endocytic sorting motif. The DTS seems to be required for internalization of LIN-12, and on Ras activation it might mediate altered endocytic routing of LIN-12, leading to downregulation. We also show that if LIN-12 is stabilized in P6.p, lateral signalling is compromised, indicating that LIN-12 downregulation is important in the appropriate specification of cell fates in vivo.