Improved automated monitoring and new analysis algorithm for circadian phototaxis rhythms in Chlamydomonas

Automated monitoring of circadian rhythms is an efficient way of gaining insight into oscillation parameters like period and phase for the underlying pacemaker of the circadian clock. Measurement of the circadian rhythm of phototaxis (swimming towards light) exhibited by the green alga Chlamydomonas reinhardtii has been automated by directing a narrow and dim light beam through a culture at regular intervals and determining the decrease in light transmittance due to the accumulation of cells in the beam. In this study, the monitoring process was optimized by constructing a new computer-controlled measuring machine that limits the test beam to wavelengths reported to be specific for phototaxis and by choosing an algal strain, which does not need background illumination between test light cycles for proper expression of the rhythm. As a result, period and phase of the rhythm are now unaffected by the time a culture is placed into the machine. Analysis of the rhythm data was also optimized through a new algorithm, whose robustness was demonstrated using virtual rhythms with various noises. The algorithm differs in particular from other reported algorithms by maximizing the fit of the data to a sinusoidal curve that dampens exponentially. The algorithm was also used to confirm the reproducibility of rhythm monitoring by the machine. Machine and algorithm can now be used for a multitude of circadian clock studies that require unambiguous period and phase determinations such as light pulse experiments to identify the photoreceptor(s) that reset the circadian clock in C. reinhardtii.     

A phosphatidylinositol 3-kinase class III sub-complex containing VPS15, VPS34, Beclin 1, UVRAG and BIF-1 regulates cytokinesis and degradative endocytic traffic

The mammalian class III phosphatidylinositol 3-kinase (PI3K-III) complex regulates fundamental cellular functions, including growth factor receptor degradation, cytokinesis and autophagy. Recent studies suggest the existence of distinct PI3K-III sub-complexes that can potentially confer functional specificity. While a substantial body of work has focused on the roles of individual PI3K-III subunits in autophagy, functional studies on their contribution to endocytic receptor downregulation and cytokinesis are limited. We therefore sought to elucidate the specific nature of the PI3K-III complexes involved in these two processes. High-content microscopy-based assays combined with siRNA-mediated depletion of individual subunits indicated that a specific sub-complex containing VPS15, VPS34, Beclin 1, UVRAG and BIF-1 regulates both receptor degradation and cytokinesis, whereas ATG14L, a PI3K-III subunit involved in autophagy, is not required. The unanticipated role of UVRAG and BIF-1 in cytokinesis was supported by a strong localisation of these proteins to the midbody. Importantly, while the tumour suppressive functions of Beclin 1, UVRAG and BIF-1 have previously been ascribed to their roles in autophagy, these results open the possibility that they may also contribute to tumour suppression via downregulation of mitogenic signalling by growth factor receptors or preclusion of aneuploidy by ensuring faithful completion of cell division.     

Expression and regulation of resistin in osteoblasts and osteoclasts indicate a role in bone metabolism

The adipose tissue is the site of expression and secretion of a range of biologically active proteins, called adipokines, for example, leptin, adiponectin, and resistin. Leptin has previously been shown to be expressed in osteoblasts and to promote bone mineralization, whereas adiponectin expression is enhanced during osteoblast differentiation. In the present study we explored the possible role of resistin in bone metabolism. We found that resistin is expressed in murine preosteoclasts and preosteoblasts (RAW 264.7, MC3T3-E1), in primary human bone marrow stem cells and in mature human osteoblasts. The expression of resistin mRNA in RAW 264.7 was increased during differentiation and seemed to be regulated through PKC- and PKA-dependent mechanisms. Recombinant resistin increased the number of differentiated osteoclasts and stimulated NFkappaB promoter activity, indicating a role in osteoclastogenesis. Resistin also enhanced the proliferation of MC3T3-E1 cells in a PKA and PKC-dependent manner, but only weakly interfered with genes known to be upregulated during differentiation of MC3T3-E1 into osteoblasts. All together, our results indicate that resistin may play a role in bone remodeling.     

Identical TCR beta-chain rearrangements in streptococcal angina and skin lesions of patients with psoriasis vulgaris

Tonsillar infection with Streptococcus pyogenes may induce several nonsuppurative autoimmune sequelae. The precise pathogenetic mechanisms behind this clinically well-established association are still unresolved. Using TCR analysis, we sought to identify a link between streptococcal tonsillitis and the T cell-mediated autoimmune response in psoriasis. Three patients with streptococcal-induced psoriasis underwent tonsillectomy. Using size spectratyping and sequencing of TCR beta-chain variable region gene (TCRBV) rearrangements, we compared the TCR usage of psoriatic skin lesions, blood, tonsils, and tonsillar T cells fractionated according to the expression of the skin address in "cutaneous lymphocyte-associated Ag" (CLA). TCRBV-size spectratype analysis of the blood lymphocytes, tonsils, and the CLA-negative tonsillar T cells revealed largely unselected T cell populations. Instead, TCRBV gene families of the psoriatic lesions and skin-homing CLA-positive tonsillar T cells displayed highly restricted spectratypes. Sequencing of TCRBV cDNA identified various clonal TCRBV rearrangements within the psoriatic lesions that indicated Ag-driven T cell expansion. Several of these clonotypes were also detected within the tonsils and, in one of the patients, within the small subset of CLA-positive tonsillar T cells, suggesting that T cells from the same T cell clones were simultaneously present within skin and tonsillar tissue. Because after tonsillectomy psoriasis cleared in all three patients our observations indicate that T cells may connect psoriatic inflammation to streptococcal angina. They suggest that the chronic streptococcal immune stimulus within the tonsils could act as a source for pathogenic T cells in poststreptococcal disorders, and they may help to explain why eliminating this source with tonsillectomy may improve streptococcal-induced sequelae.     

Mechanisms of cell death induction by L-amino acid oxidase, a major component of ophidian venom

L-amino acid oxidase (LAAO) from the Malayan pit viper induces both necrosis and apoptosis in Jurkat cells. Cell death by necrosis is attributed to H₂O₂ produced by oxidation of α-amino acids. In the presence of catalase that effectively scavenges H₂O_2, a switch to apoptosis is observed. The major factors contributing to apoptosis are proposed to be: (i) generation of toxic intermediates from fetal calf serum (ii) binding and internalization of LAAO. The latter process appears to be mediated by the glycan moiety of the enzyme as desialylation reduces cytotoxicity. D-amino acid oxidase (DAAO), which catalyzes the same reaction as LAAO but lacks glycosylation, triggers necrosis as a consequence of H₂O₂ production but not apoptosis in the presence of catalase. Thus induction of cell death by LAAO appears to involve both the generation of H₂O₂ and the molecular interaction of the glycan moiety of the enzyme with structures at the cell surface. S. R. Ande, P. R. Kommoju contributed equally to this work.     

Light environment under Rhododendron maximum thickets and estimated carbon gain of regenerating forest tree seedlings

Canopy tree recruitment is inhibited by evergreen shrubs in many forests. In the southern Appalachian mountains of the USA, thickets of Rhododendron maximum L. restrict dominant canopy tree seedling survival and persistence. Using R. maximum as a model system, we examined available light under the thickets and the photosynthetic responses of seedlings of canopy tree species. We tested the hypothesis that the additional shading from under R. maximum drives carbon gain in seedlings below the threshold for growth and survival. A reduction in light under the thicket was found where canopy openness (derived from canopy photographs) under R. maximum was half the amount measured in forest without R. maximum. R.␣maximum also reduced direct radiation by 50% and diffuse radiation by 12–29% compared to forest without the shrub layer. Mean mid-day PPFD (photosynthetically active photon flux density between 1000 and 1400 h) under R. maximum (obtained from quantum sensors) was below 10 mol m⁻² s⁻¹ on both clear and overcast days and the amount of sunflecks greater than 10 mol m⁻² s⁻¹ PPFD was only 0–20 min per day. In contrast, forest without R. maximum received a mean PPFD of 18–25 mol m⁻² s⁻¹ on clear days and a cumulative sunfleck duration of 100–220 min per day in all sky conditions. Consistent with light availability between the sites, daily carbon gain in Quercus rubra L. seedlings was lower in forest with R. maximum compared to forest where the shrub was absent. The presence of the shrub layer also significantly suppressed average mid-day photosynthesis of both Q. rubra and Prunus serotina Ehrt. seedlings on 8 out of 11 measurement dates. However, parameters derived from light response curves between seedlings growing in forest sites with or without a thicket of R. maximum was significantly different only in A _max (maximum photosynthetic rate), indicating a lack of further acclimation to the deeper shade under R. maximum. While the additional shade cast by R. maximum is sufficient to prevent the regeneration of tree seedlings under this shrub, there was sufficient heterogeneity in light under the thicket to imply that deep shade only partially explains the complete inhibition of regenerating canopy trees under R. maximum.     

Caspase-containing complexes in the regulation of cell death and inflammation

Caspases are a family of cysteine proteases that are essential in the initiation and execution of apoptosis and the proteolytic maturation of inflammatory cytokines such as IL-1beta and IL-18. Caspases can be subdivided into those that have a large prodomain and those that have not. In general, apoptotic and inflammatory signalling pathways are initiated when large-prodomain caspases are recruited to large protein complexes via homotypic interactions involving death domain folds. The formation of these specialised multimeric platforms involves three major functions: (1) the sensing of cellular stress, damage, infection or inflammation; (2) multimerisation of the platform; and (3) recruitment and conformational activation of caspases. In this overview we discuss the complexes implicated in the regulation of cell death and inflammatory processes such as the death-inducing signalling complex (DISC), the apoptosome, the inflammasomes and the PIDDosome. We describe their sensing functions, compositions and functional outcomes. Inhibitory protein families such as FLIPs and CARD-only proteins prevent the recruitment of caspases in these sensing complexes, avoiding inappropriate initiation of cell death or inflammation.     

Molecular basis of RNA recognition by the human alternative splicing factor Fox-1

The Fox-1 protein regulates alternative splicing of tissue-specific exons by binding to GCAUG elements. Here, we report the solution structure of the Fox-1 RNA binding domain (RBD) in complex with UGCAUGU. The last three nucleotides, UGU, are recognized in a canonical way by the four-stranded beta-sheet of the RBD. In contrast, the first four nucleotides, UGCA, are bound by two loops of the protein in an unprecedented manner. Nucleotides U1, G2, and C3 are wrapped around a single phenylalanine, while G2 and A4 form a base-pair. This novel RNA binding site is independent from the beta-sheet binding interface. Surface plasmon resonance analyses were used to quantify the energetic contributions of electrostatic and hydrogen bond interactions to complex formation and support our structural findings. These results demonstrate the unusual molecular mechanism of sequence-specific RNA recognition by Fox-1, which is exceptional in its high affinity for a defined but short sequence element.     

Migratory connectivity and temporal segregation of dunlin (Calidris alpina) in Portugal: evidence from morphology, ringing recoveries and mtDNA

Migratory connectivity plays an important role in conservation of long-distance migrant birds. Here, we study migratory links of dunlin (Calidris alpina), focusing on a stopover and wintering region (Portugal) where it is known that migration routes of dunlin from a broad geographic range (three subspecies) converge, and populations occur simultaneously or separated in time. We combine three methods (ringing recoveries, morphometrics and molecular genetics) to assess breeding origins and extent of temporal segregation of dunlin assemblages. Ringing recoveries show temporal separation of dunlin from different migration routes. Birds found in Portugal during August and September, migrating via Britain, reveal links to breeding areas in Iceland and Greenland. In October, a clear shift to more eastern migration routes occurs, with most Portuguese winter records from stopover sites along migration routes of populations from northern Scandinavia and Russia. Mitochondrial DNA (mtDNA) of Portuguese dunlin was compared with breeding populations. Spring and autumn migrants in Portugal corresponded to C. a. schinzii and C. a. arctica populations, while the Portuguese winter population clearly differs by including mtDNA haplotypes of C. a. alpina. For genetically sexed individuals, we found significant differences in morphology (bill and tarsus length) supporting the temporal separation of populations/subspecies revealed by recoveries and mtDNA. Our results give evidence for migratory connectivity of dunlin populations between geographic areas previously not considered connected. They confirm the existence of clear differences in breeding origin between birds in Portugal at different times of year. These results are important in the consideration of future long-term conservation plans.