T-cell recognition of chemicals, protein allergens and drugs: towards the development of in vitro assays

Chemicals can elicit T-cell-mediated diseases such as allergic contact dermatitis and adverse drug reactions. Therefore, testing of chemicals, drugs and protein allergens for hazard identification and risk assessment is essential in regulatory toxicology. The seventh amendment of the EU Cosmetics Directive now prohibits the testing of cosmetic ingredients in mice, guinea pigs and other animal species to assess their sensitizing potential. In addition, the EU Chemicals Directive REACh requires the retesting of more than 30,000 chemicals for different toxicological endpoints, including sensitization, requiring vast numbers of animals. Therefore, alternative methods are urgently needed to eventually replace animal testing. Here, we summarize the outcome of an expert meeting in Rome on 7 November 2009 on the development of T-cell-based in vitro assays as tools in immunotoxicology to identify hazardous chemicals and drugs. In addition, we provide an overview of the development of the field over the last two decades.     

Deficiency of UBR1, a ubiquitin ligase of the N-end rule pathway, causes pancreatic dysfunction, malformations and mental retardation (Johanson-Blizzard syndrome)

Johanson-Blizzard syndrome (OMIM 243800) is an autosomal recessive disorder that includes congenital exocrine pancreatic insufficiency, multiple malformations such as nasal wing aplasia, and frequent mental retardation. We mapped the disease-associated locus to chromosome 15q14-21.1 and identified mutations, mostly truncating ones, in the gene UBR1 in 12 unrelated families with Johanson-Blizzard syndrome. UBR1 encodes one of at least four functionally overlapping E3 ubiquitin ligases of the N-end rule pathway, a conserved proteolytic system whose substrates include proteins with destabilizing N-terminal residues. Pancreas of individuals with Johanson-Blizzard syndrome did not express UBR1 and had intrauterine-onset destructive pancreatitis. In addition, we found that Ubr1(-/-) mice, whose previously reported phenotypes include reduced weight and behavioral abnormalities, had an exocrine pancreatic insufficiency, with impaired stimulus-secretion coupling and increased susceptibility to pancreatic injury. Our findings indicate that deficiency of UBR1 perturbs the pancreas' acinar cells and other organs, presumably owing to metabolic stabilization of specific substrates of the N-end rule pathway.     

Apoptosis initiated by Bcl-2-regulated caspase activation independently of the cytochrome c/Apaf-1/caspase-9 apoptosome

Apoptosis is an evolutionarily conserved cell suicide process executed by cysteine proteases (caspases) and regulated by the opposing factions of the Bcl-2 protein family. Mammalian caspase-9 and its activator Apaf-1 were thought to be essential, because mice lacking either of them display neuronal hyperplasia and their lymphocytes and fibroblasts seem resistant to certain apoptotic stimuli. Because Apaf-1 requires cytochrome c to activate caspase-9, and Bcl-2 prevents mitochondrial cytochrome c release, Bcl-2 is widely believed to inhibit apoptosis by safeguarding mitochondrial membrane integrity. Our results suggest a different, broader role, because Bcl-2 overexpression increased lymphocyte numbers in mice and inhibited many apoptotic stimuli, but the absence of Apaf-1 or caspase-9 did not. Caspase activity was still discernible in cells lacking Apaf-1 or caspase-9, and a potent caspase antagonist both inhibited apoptosis and retarded cytochrome c release. We conclude that Bcl-2 regulates a caspase activation programme independently of the cytochrome c/Apaf-1/caspase-9 'apoptosome', which seems to amplify rather than initiate the caspase cascade.     

A nebula of gases from Io surrounding Jupiter

Several planetary missions have reported the presence of substantial numbers of energetic ions and electrons surrounding Jupiter; relativistic electrons are observable up to several astronomical units (au) from the planet. A population of energetic (>30[?]keV) neutral particles also has been reported, but the instrumentation was not able to determine the mass or charge state of the particles, which were subsequently labelled energetic neutral atoms. Although images showing the presence of the trace element sodium were obtained, the source and identity of the neutral atoms---and their overall significance relative to the loss of charged particles from Jupiter's magnetosphere---were unknown. Here we report the discovery by the Cassini spacecraft of a fast (>103[?]km[?]s-1) and hot magnetospheric neutral wind extending more than 0.5[?]au from Jupiter, and the presence of energetic neutral atoms (both hot and cold) that have been accelerated by the electric field in the solar wind. We suggest that these atoms originate in volcanic gases from Io, undergo significant evolution through various electromagnetic interactions, escape Jupiter's magnetosphere and then populate the environment around the planet. Thus a 'nebula' is created that extends outwards over hundreds of jovian radii.     

Power requirement of the geodynamo from ohmic losses in numerical and laboratory dynamos

In the Earth's fluid outer core, a dynamo process converts thermal and gravitational energy into magnetic energy. The power needed to sustain the geomagnetic field is set by the ohmic losses (dissipation due to electrical resistance). Recent estimates of ohmic losses cover a wide range, from 0.1 to 3.5 TW, or roughly 0.3-10% of the Earth's surface heat flow. The energy requirement of the dynamo puts constraints on the thermal budget and evolution of the core through Earth's history. Here we use a set of numerical dynamo models to derive scaling relations between the core's characteristic dissipation time and the core's magnetic and hydrodynamic Reynolds numbers--dimensionless numbers that measure the ratio of advective transport to magnetic and viscous diffusion, respectively. The ohmic dissipation of the Karlsruhe dynamo experiment supports a simple dependence on the magnetic Reynolds number alone, indicating that flow turbulence in the experiment and in the Earth's core has little influence on its characteristic dissipation time. We use these results to predict moderate ohmic dissipation in the range of 0.2-0.5 TW, which removes the need for strong radioactive heating in the core and allows the age of the solid inner core to exceed 2.5 billion years.     

Mutations in VKORC1 cause warfarin resistance and multiple coagulation factor deficiency type 2

Coumarin derivatives such as warfarin represent the therapy of choice for the long-term treatment and prevention of thromboembolic events. Coumarins target blood coagulation by inhibiting the vitamin K epoxide reductase multiprotein complex (VKOR). This complex recycles vitamin K 2,3-epoxide to vitamin K hydroquinone, a cofactor that is essential for the post-translational gamma-carboxylation of several blood coagulation factors. Despite extensive efforts, the components of the VKOR complex have not been identified. The complex has been proposed to be involved in two heritable human diseases: combined deficiency of vitamin-K-dependent clotting factors type 2 (VKCFD2; Online Mendelian Inheritance in Man (OMIM) 607473), and resistance to coumarin-type anticoagulant drugs (warfarin resistance, WR; OMIM 122700). Here we identify, by using linkage information from three species, the gene vitamin K epoxide reductase complex subunit 1 (VKORC1), which encodes a small transmembrane protein of the endoplasmic reticulum. VKORC1 contains missense mutations in both human disorders and in a warfarin-resistant rat strain. Overexpression of wild-type VKORC1, but not VKORC1 carrying the VKCFD2 mutation, leads to a marked increase in VKOR activity, which is sensitive to warfarin inhibition.     

Increase in tropospheric nitrogen dioxide over China observed from space

Emissions from fossil fuel combustion and biomass burning reduce local air quality and affect global tropospheric chemistry. Nitrogen oxides are emitted by all combustion processes and play a key part in the photochemically induced catalytic production of ozone, which results in summer smog and has increased levels of tropospheric ozone globally. Release of nitrogen oxide also results in nitric acid deposition, and--at least locally--increases radiative forcing effects due to the absorption of downward propagating visible light. Nitrogen oxide concentrations in many industrialized countries are expected to decrease, but rapid economic development has the potential to increase significantly the emissions of nitrogen oxides in parts of Asia. Here we present the tropospheric column amounts of nitrogen dioxide retrieved from two satellite instruments GOME and SCIAMACHY over the years 1996-2004. We find substantial reductions in nitrogen dioxide concentrations over some areas of Europe and the USA, but a highly significant increase of about 50 per cent-with an accelerating trend in annual growth rate-over the industrial areas of China, more than recent bottom-up inventories suggest.     

Catalytic enantioselective reactions driven by photoinduced electron transfer

Photoinduced electron transfer is an essential step in the conversion of solar energy into chemical energy in photosystems I and II (ref. 1), and is also frequently used by chemists to build complex molecules from simple precursors. During this process, light absorption generates molecules in excited electronic states that are susceptible to accepting or donating electrons. But although the excited states are straightforward to generate, their short lifetimes makes it challenging to control electron transfer and subsequent product formation-particularly if enantiopure products are desired. Control strategies developed so far use hydrogen bonding, to embed photochemical substrates in chiral environments and to render photochemical reactions enantioselective through the use of rigid chiral complexing agents. To go beyond such stoichiometric chiral information transmission, catalytic turnover is required. Here we present a catalytic photoinduced electron transfer reaction that proceeds with considerable turnover and high enantioselectivity. By using an electron accepting chiral organocatalyst that enforces a chiral environment on the substrate through hydrogen bonding, we obtain the product in significant enantiomeric excess (up to 70%) and in yields reaching 64%. This performance suggests that photochemical routes to chiral compounds may find use in general asymmetric synthesis.     

Fracture surface energy of the Punchbowl fault, San Andreas system

Fracture energy is a form of latent heat required to create an earthquake rupture surface and is related to parameters governing rupture propagation and processes of slip weakening. Fracture energy has been estimated from seismological and experimental rock deformation data, yet its magnitude, mechanisms of rupture surface formation and processes leading to slip weakening are not well defined. Here we quantify structural observations of the Punchbowl fault, a large-displacement exhumed fault in the San Andreas fault system, and show that the energy required to create the fracture surface area in the fault is about 300 times greater than seismological estimates would predict for a single large earthquake. If fracture energy is attributed entirely to the production of fracture surfaces, then all of the fracture surface area in the Punchbowl fault could have been produced by earthquake displacements totalling <1 km. But this would only account for a small fraction of the total energy budget, and therefore additional processes probably contributed to slip weakening during earthquake rupture.