Special-interest subgroups at the ASCB: Gap junctions

The annual meeting of the American Society for Cell Biology (ASCB) is a large and diverse gathering. At last year's meeting**The American Society for Cell Biology 38th Annual Meeting, San Francisco, USA; 12-16 December, 1998. Program chair: Jennifer Lippincott-Schwartz., there were over 8000 attendees, and the topics discussed covered many areas of cell biology. It would be impossible to cover the entire meeting within a trends in CELL BIOLOGY report, so instead we are focusing on an aspect of it that provided some of the most interesting and fruitful discussions. On Saturday afternoon, before the main symposia began, there were 11 special-interest subgroup meetings. The atmosphere at these meetings was informal, and they encouraged open and frank discussion of data and issues. This report provides a brief summary of the discussions at seven of the special-interest subgroup meetings.     

Life-history constraints on the success of the many small eggs reproductive strategy

The reproductive strategy of most fishes is to produce a large number of tiny eggs, leading to a huge difference between egg size and asymptotic body size. The viability of this strategy is examined by calculating the life-time reproductive success R₀ as a function of the asymptotic body size. A simple criterion for the optimality of producing small eggs is found, depending on the rate of predation relative to the specific rate of consumption. Secondly it is shown that the success of the reproductive strategy is increasing with asymptotic body size. Finally the existence of both upper and lower limits on the allowed asymptotic sizes is demonstrated. A metabolic upper limit to asymptotic body size for all higher animals is derived.     

Crystal structures of the pro-inflammatory cytokine interleukin-23 and its complex with a high-affinity neutralizing antibody

Interleukin (IL)-23 is a pro-inflammatory cytokine playing a key role in the pathogenesis of several autoimmune and inflammatory diseases. We have determined the crystal structures of the heterodimeric p19-p40 IL-23 and its complex with the Fab (antigen-binding fragment) of a neutralizing antibody at 2.9 and 1.9 Å, respectively. The IL-23 structure closely resembles that of IL-12. They share the common p40 subunit, and IL-23 p19 overlaps well with IL-12 p35. Along the hydrophilic heterodimeric interface, fewer charged residues are involved for IL-23 compared with IL-12. The binding site of the Fab is located exclusively on the p19 subunit, and comparison with published cytokine-receptor structures suggests that it overlaps with the IL-23 receptor binding site.     

Deciduous woodland conservation under heavy deer browsing on Devils Tower National Monument, USA

Native deciduous woodlands comprise <5% of the vegetation in the Black Hills and northern plains of the USA and appear to be in decline. Because deciduous woodlands comprise 18% of the land cover on Devils Tower National Monument (DTNM), this natural area is potentially important for the conservation of these plant communities. Lack of tree regeneration and accumulation of dead wood suggest that the native deciduous woodlands on DTNM may die out under persistent, heavy deer (Odocoileus virginianus, O. hemionus) browsing. We monitored vegetation changes for seven years (1989–1996) inside and outside 6 deer exclosures on DTNM to determine the resilience of native woodland communities when protected from browsing, and to parameterize models that evaluate specific fencing scenarios for managing woodland communities. The herbaceous woodland understory was not directly, but indirectly affected by deer browsing because graminoid biomass declined as tall shrubs recovered. Tall shrubs, dominated by Prunus spp. showed the most rapid and consistent recovery across sites. Models of shrub growth indicated that rotational fencing for short durations (10 years) produced the highest long-term increase in tall-shrub densities and the greatest evenness among size classes. Because deciduous woodlands currently are resilient and occur in small patches, fencing may provide a practical management alternative to deer culling programs.     

c-Cbl binding and ubiquitin-dependent lysosomal degradation of membrane-associated Notch1.

Regulation of Notch1 activity is critical for cell fate decisions and differentiation of skeletal myoblasts. We have employed the skeletal myoblast cell line C2C12 to study posttranslational regulation of Notch1 protein levels during myogenesis. Although the major degradation pathway of the activated intracellular Notch1 fragment appears to involve ubiquitination and degradation by the 26 S proteasome, we provide evidence for an alternative catalytic pathway where the endogenous, transmembrane form of Notch1 is targeted to the lysosomal compartment. Immunoprecipitation analysis revealed ubiquitin-dependent accumulation of transmembrane Notch1 protein after treatment with the lysosomal inhibitor chloroquine but not after treatment with various proteasome inhibitors. This finding was supported by the observation that the transmembrane form of Notch1 was tyrosine-phosphorylated and specifically coprecipitated with the ubiquitin ligase c-Cbl. Our data suggest a regulatory mechanism down-regulating Notch1 protein levels already at the cellular surface, possibly with consequences for Notch-dependent signal transduction during terminal differentiation processes.     

Golden Rice--five years on the road--five years to go?

Provitamin A accumulates in the grain of Golden Rice as a result of genetic transformation. In developing countries, where vitamin A deficiency prevails, grain from Golden Rice is expected to provide this important micronutrient sustainably through agriculture. Since its original production, the prototype Golden Rice has undergone intense research to increase the provitamin A content, to establish the scientific basis for its carotenoid complement, and to better comply with regulatory requirements. Today, the current focus is on how to get Golden Rice effectively into the hands of farmers, which is a novel avenue for public sector research, carried out with the aid of international research consortia. Additional new research is underway to further increase the nutritional value of Golden Rice.     

The beta2 integrin CD11c distinguishes a subset of cytotoxic pulmonary T cells with potent antiviral effects in vitro and in vivo

Background

The integrin CD11c is known as a marker for dendritic cells and has recently been described on T cells following lymphotropic choriomeningitis virus infection, a systemic infection affecting a multitude of organs. Here, we characterise CD11c bearing T cells in a murine model of localised pulmonary infection with respiratory syncytial virus (RSV).

Methods

Mice were infected intranasally with RSV and expression of β2 integrins and T lymphocyte activation markers were monitored by flow cytometry. On day 8 post RSV infection CD11c⁺ CD8⁺ and CD11c^- CD8⁺ T cells were assessed for cytokine production, cytotoxic activity and migration. Expression of CD11c mRNA in CD8⁺ T cells was assessed by quantitative PCR.

Results

Following RSV infection CD11c⁺ CD8⁺ T cells were detectable in the lung from day 4 onwards and accounted for 45.9 ± 4.8% of CD8⁺ T cells on day 8 post infection, while only few such cells were present in mediastinal lymph nodes, spleen and blood. While CD11c was virtually absent from CD8⁺ T cells in the absence of RSV infection, its mRNA was expressed in CD8⁺ T cells of both naïve and RSV infected mice. CD11c⁺, but not CD11c^-, CD8⁺ T cells showed signs of recent activation, including up-regulation of CD11a and expression of CD11b and CD69 and were recruited preferentially to the lung. In addition, CD11c⁺ CD8⁺ T cells were the major subset responsible for IFNγ production, induction of target cell apoptosis in vitro and reduction of viral titres in vivo.

Conclusion

CD11c is a useful marker for detection and isolation of pulmonary antiviral cytotoxic T cells following RSV infection. It identifies a subset of activated, virus-specific, cytotoxic T cells that exhibit potent antiviral effects in vivo.     

Retinal biosynthesis in Eubacteria: in vitro characterization of a novel carotenoid oxygenase from Synechocystis sp. PCC 6803

Retinal and its derivatives represent essential compounds in many biological systems. In animals, they are synthesized through a symmetrical cleavage of beta-carotene catalysed by a monooxygenase. Here, we demonstrate that the open reading frame sll1541 from the cyanobacterium Synechocystis sp. PCC 6803 encodes the first eubacterial, retinal synthesizing enzyme (Diox1) thus far reported. In contrast to enzymes from animals, Diox1 converts beta-apo-carotenals instead of beta-carotene into retinal in vitro. The identity of the enzymatic product was proven by HPLC, GC-MS and in a biological test. Investigations, of the stereospecifity showed that Diox1 cleaved only the all-trans form of beta-apo-8'-carotenal, yielding all-trans-retinal. However, Diox1 exhibited wide substrate specificity with respect to chain-lengths and functional end-groups. Although with divergent Km and Vmax values, the enzyme converted beta-apo-carotenals, (3R)-3-OH-beta-apo-carotenals as well as apo-lycopenals into retinal, (3R)-3-hydroxy-retinal and acycloretinal respectively. In addition, the alcohols of these substrates were cleaved to yield the corresponding retinal derivatives.