A FINE STRUCTURAL ANALYSIS OF CLEAVAGE INDUCTION AND FURROWING IN THE EGGS OF ARBACIA PUNCTULATA

A fine structural study has been carried out on the various formed elements present before, during, and after the first cleavage division, not only in normally developing Arbacia eggs, but also in eggs which have been induced to cleave prematurely by high-pressure centrifugation. The aim has been to ascertain whether or not any of the morphologically identifiable components may be involved in initiating the furrowing process. Also, attention has been given to the fine structure of the cytoplasmic cortex, particulary in the walls of the furrow, in the hope of reaching a better understanding of the mechanics of cleavage. The annulate lamellae and the membranous envelope of the nucleus are the only formed elements which disappear shortly before cleavage, not only in eggs undergoing normal division, but also in eggs which have been induced to cleave ahead of schedule by high-pressure, high-force centrifugation. Therefore, it is suggested as a tentative hypothesis that materials liberated upon disintegration of the nuclear membrane and the annulate lamellae play an essential role in initiating and effecting the furrowing reaction, especially since the stratification of these elements in experimentally induced eggs corresponds to the position of the developing furrow. Another of the membranous elements in the egg, the Golgi complex, shows considerable modification as a result of high-pressure centrifugation, but these structures do not undergo disintegration. Rather, they become curled into rounded bodies. The vacuole population is not greatly affected by inducing treatments. During cleavage, both naturally occurring and experimentally induced, a considerable number of 50 A filaments appear in the denser cytoplasmic cortex, but only in the walls of the furrow. These filaments are similar to those which have been demonstrated in a number of contractile cells. Accordingly, it is suggested that this fibrillar system may be actively involved in the development of the cleavage force.     

Characterization of Wolbachia host cell range via the in vitro establishment of infections

Maternally transmitted bacteria of the genus Wolbachia are obligate, intracellular symbionts that are frequently found in insects and cause a diverse array of reproductive manipulations, including cytoplasmic incompatibility, male killing, parthenogenesis, and feminization. Despite the existence of a broad range of scientific interest, many aspects of Wolbachia research have been limited to laboratories with insect-rearing facilities. The inability to culture these bacteria outside of the invertebrate host has also led to the existing bias of Wolbachia research toward infections that occur in host insects that are easily reared. Here, we demonstrate that Wolbachia infections can be simply established, stably maintained, and cryogenically stored in vitro using standard tissue culture techniques. We have examined Wolbachia host range by introducing different Wolbachia types into a single tissue culture. The results show that an Aedes albopictus (Diptera: Culicidae) cell line can support five different Wolbachia infection types derived from Drosophila simulans (Diptera: Drosophilidae), Culex pipiens (Culicidae), and Cadra cautella (Lepidoptera: Phycitidae). These bacterial types include infection types that have been assigned to two of the major Wolbachia clades. As an additional examination of Wolbachia host cell range, we demonstrated that a Wolbachia strain from D. simulans could be established in host insect cell lines derived from A. albopictus, Spodoptera frugiperda (Lepidoptera: Noctuidae), and Drosophila melanogaster. These results will facilitate the development of a Wolbachia stock center, permitting novel approaches for the study of Wolbachia infections and encouraging Wolbachia research in additional laboratories.     

Flow-induced alterations to fish assemblages, habitat and fish–habitat associations in a regulated lowland river

Understanding the cause–effect response of aquatic biota to hydrological variability is fundamental to the restoration of regulated rivers. Spatio-temporal variation in fish assemblage structure, microhabitat cover and fish–habitat associations were investigated in the main channel of the regulated lower River Murray, Australia, during a prolonged period of low within-channel flows and following a high flow event and flood. Several small-bodied species (e.g. carp gudgeon, Hypseleotris spp.), were abundant and significantly associated with submerged macrophytes during low flows, but were absent or significantly less abundant following flooding, and the loss of these microhabitats. Large-bodied riverine species that spawn in response to increases in flow (e.g. golden perch, Macquaria ambigua ambigua) or spawn and recruit in inundated floodplain habitats (e.g. common carp, Cyprinus carpio), exhibited flexible microhabitat use and were significantly more abundant following flooding. In the lower River Murray, high flow events appear integral in structuring fish assemblages, indirectly influencing the abundance of small-bodied fish by re-structuring macrophyte cover and directly influencing the abundance of large-bodied species by facilitating critical life history processes (e.g. recruitment). These results highlight species-specific differences in cause–effect responses to flow variability and have implications for managing flow in regulated rivers.     

Protocol of a prospective study on the diagnostic value of transcranial duplex scanning of the substantia nigra in patients with parkinsonian symptoms

Background  

Parkinson's disease (PD) is the second most common neurodegenerative disorder. As there is no definitive diagnostic test, its diagnosis is based on clinical criteria. Recently transcranial duplex scanning (TCD) of the substantia nigra in the brainstem has been proposed as an instrument to diagnose PD. We and others have found that TCD scanning of substantia nigra duplex is a relatively accurate diagnostic instrument in patients with parkinsonian symptoms. However, all studies on TCD so far have involved well-defined, later-stage PD patients, which will obviously lead to an overestimate of the diagnostic accuracy of TCD.     

Floristic variation and willow carr development within a southwest England wetland

Abstract. Woodland colonization on wetlands is considered to have a detrimental effect on their ecological value, even though detailed analysis of this process is lacking. This paper provides an evaluation of the ecological changes resulting from succession of poor fen (base‐poor mire) to willow wet woodland on Goss Moor NNR in Cornwall, UK. Different ages of willow carr were associated with eight understorey communities. During willow colonization, in the ground flora, there was a progressive decrease in poor fen species and an associated increase in woodland species, which appeared to be related to an increase in canopy cover and therefore shade. The most diverse community was found to be the most recent willow and was dominated by poor fen species. The oldest willow was the second most diverse and was associated with a reduction in poor fen species and an increase in woodland species. Architectural features were used successfully to assess the general condition and structure of willow. Tree height and DBH were identified as useful parameters to accurately assess willow age in the field. The implications of active intervention to remove willow in order to conserve the full range of communities within the hydrosere are discussed.     

The Kinase Activity of Rip2 Determines Its Stability and Consequently Nod1- and Nod2-mediated Immune Responses

Rip2 (RICK, CARD3) has been identified as a key effector molecule downstream of the pattern recognition receptors, Nod1 and Nod2; however, its mechanism of action remains to be elucidated. In particular, it is unclear whether its kinase activity is required for signaling or for maintaining protein stability. We have investigated the expression level of different retrovirally expressed kinase-dead Rip2 mutants and the role of Rip2 kinase activity in the signaling events that follow Nod1 and Nod2 stimulation. We show that in primary cells expressing kinase-inactive Rip2, protein levels were severely compromised, and stability could not be reconstituted by the addition of a phospho-mimetic mutation in its autophosphorylation site. Consequently, inflammatory cytokine production in response to Nod1 and Nod2 ligands was abrogated both in vitro and in vivo in the absence of Rip2 kinase activity. Our results highlight the central role that Rip2 kinase activity plays in conferring stability to the protein and thus in the preservation of Nod1- and Nod2-mediated innate immune responses.A key step in the initiation of effector immune responses is the recognition of highly conserved molecules expressed by microbial pathogens. The immune system has developed specific receptors that sense these so-called pathogen-associated molecular patterns and initiate appropriate immune responses. One key family of pattern recognition receptors is the Nod-like receptor (NLR)² family (1–3), of which two members, Nod1 and Nod2, have been implicated in the recognition of bacterial peptidoglycan derivatives released into the cytosol upon bacterial infection (4–6). Several studies have shown that Nod1 plays a role in host defense against invasive pathogens such as Helicobacter pylori and Escherichia coli (7, 8), and Nod2 mutations have been associated with a higher incidence of Crohn disease (9, 10), thus highlighting these NLRs as important regulators of inflammatory immune responses.Rip2, also called CARD3, RICK, or CARDIAK, is a serine/threonine kinase, which was implicated in the induction of NF-κB activation and apoptosis (11–13). Rip2 has been described to be critical for responses against Toll-like receptor ligands such as LPS (14, 15), although findings from recent studies did not support this conclusion (16). Rip2 contains a caspase-recruitment domain (CARD), which mediates interaction with other CARD-containing proteins such as Nod1 and Nod2, in addition to an N-terminal kinase domain and an intermediate domain. Nod1 and Nod2 associate with Rip2 upon peptidoglycan ligation (17) leading to downstream signaling events that culminate in NF-κB and mitogen-activated protein kinase activation (15, 18–20). Recent reports have suggested that the mitogen-activated protein kinase kinase kinase family member TAK1 provides the link between Rip2 and NF-κB activation upon Nod1 and Nod2 stimulation (21–23). However, the exact role of Rip2 and in particular its kinase activity in mediating downstream effector activation in NLR signaling still remains unclear. Notably, in vitro investigations have suggested that Rip2 kinase activity may be dispensable for the induction of immune responses initiated by NLR-ligands (21, 24, 25) and that disruption of Rip2 kinase activity is associated with a loss in protein stability (23); however, such studies utilized protein overexpression in cell lines and are yet to be tested in primary cells or in vivo.In the current investigation we sought to elucidate the role of Rip2 kinase activity in transducing inflammatory signals upon NLR stimulation in vitro and in vivo. To this end, we utilized both Rip2 knock-out (15) and Rip2 kinase-dead knock-in mice (24) in addition to Rip2 deficient primary cells that were retrovirally reconstituted with different kinase-inactive mutants. We show here that in the absence of intact kinase activity, Rip2 protein is not stable and that insertion of a phospho-mimetic mutation is not sufficient to restore stability. Moreover, pharmacological abrogation of Rip2 kinase activity in primary cells similarly leads to destabilization of the molecule. As a consequence, signaling downstream of Nod1 and Nod2 and inflammatory cytokine production is impaired both in vivo and in vitro. Our results highlight Rip2 kinase activity as a central regulator of protein stability and consequently innate immune responses triggered by Nod1 and Nod2 ligands.     

Automated birdsong recognition in complex acoustic environments: a review

Conservationists are increasingly using autonomous acoustic recorders to determine the presence/absence and the abundance of bird species. Unlike humans, these recorders can be left in the field for extensive periods of time in any habitat. Although data acquisition is automated, manual processing of recordings is labour intensive, tedious, and prone to bias due to observer variations. Hence automated birdsong recognition is an efficient alternative. However, only few ecologists and conservationists utilise the existing birdsong recognisers to process unattended field recordings because the software calibration time is exceptionally high and requires considerable knowledge in signal processing and underlying systems, making the tools less user‐friendly. Even allowing for these difficulties, getting accurate results is exceedingly hard. In this review we examine the state‐of‐the‐art, summarising and discussing the methods currently available for each of the essential parts of a birdsong recogniser, and also available software. The key reasons behind poor automated recognition are that field recordings are very noisy, calls from birds that are a long way from the recorder can be faint or corrupted, and there are overlapping calls from many different birds. In addition, there can be large numbers of different species calling in one recording, and therefore the method has to scale to large numbers of species, or at least avoid misclassifying another species as one of particular interest. We found that these areas of importance, particularly the question of noise reduction, are amongst the least researched. In cases where accurate recognition of individual species is essential, such as in conservation work, we suggest that specialised (species‐specific) methods of passive acoustic monitoring are required. We also believe that it is important that comparable measures, and datasets, are used to enable methods to be compared.