In Macrophages,Caspase-1 Activation by SopE and the Type III Secretion System-1 of S. Typhimurium Can Proceed in the Absence of Flagellin

The innate immune system is of vital importance for protection against infectious pathogens. Inflammasome mediated caspase-1 activation and subsequent release of pro-inflammatory cytokines like IL-1β and IL-18 is an important arm of the innate immune system. Salmonella enterica subspecies 1 serovar Typhimurium (S. Typhimurium, SL1344) is an enteropathogenic bacterium causing diarrheal diseases. Different reports have shown that in macrophages, S. Typhimurium may activate caspase-1 by at least three different types of stimuli: flagellin, the type III secretion system 1 (T1) and the T1 effector protein SopE. However, the relative importance and interdependence of the different factors in caspase-1 activation is still a matter of debate. Here, we have analyzed their relative contributions to caspase-1 activation in LPS-pretreated RAW264.7 macrophages. Using flagellar mutants (fliGHI, flgK) and centrifugation to mediate pathogen-host cell contact, we show that flagellins account for a small part of the caspase-1 activation in RAW264.7 cells. In addition, functional flagella are of key importance for motility and host cell attachment which is a prerequisite for mediating caspase-1 activation via these three stimuli. Using site directed mutants lacking several T1 effector proteins and flagellin expression, we found that SopE elicits caspase-1 activation even when flagellins are absent. In contrast, disruption of essential genes of the T1 protein injection system (invG, sipB) completely abolished caspase-1 activation. However, a robust level of caspase-1 activation is retained by the T1 system (or unidentified T1 effectors) in the absence of flagellin and SopE. T1-mediated inflammasome activation is in line with recent work by others and suggests that the T1 system itself may represent the basic caspase-1 activating stimulus in RAW264.7 macrophages which is further enhanced independently by SopE and/or flagellin.     

Expression of VASP and zyxin in cochlear pillar cells: indication for actin-based dynamics?

Vasodilator-stimulated phosphoprotein (VASP) is a member of the ENA/VASP-protein family. VASP is considered to be a crucial factor in the regulation of actin dynamics, which involves processes such as motility and cell adhesion, e.g. in filopodia or growth cones. In these processes zyxin acts as an important partner of VASP and is particularly concentrated at sites where VASP-dependent actin dynamics occur. Based on indirect evidence that actin-mediated dynamics may effect the mechanical properties of the cochlea, we have investigated expression of VASP and zyxin in the postnatal and adult rat cochlea using polymerase chain reaction and Western blot approaches, as well as immunohistochemistry and confocal microscopy. Besides an expected expression in vessels and fibroblasts, VASP and zyxin expression was also observed in pillar cells. Here, the staining was restricted to the head and foot plate of the pillar cells. Onset of VASP expression in pillar cells coincided with the beginning of hearing. In pillar cells, VASP and zyxin were co-localised with pan-actin, suggesting actin-based dynamics in these cochlear cells, which until now were rather presumed to form a highly rigid bridge between the inner and outer sensory cells. Thus, pillar cells may be more dynamically involved in controlling longer-lasting mechanical properties of the cochlea as hitherto presumed.     

Protein kinase CK2 is coassembled with small conductance Ca(2+)-activated K+ channels and regulates channel gating

Small conductance Ca(2+)-activated K+ channels (SK channels) couple the membrane potential to fluctuations in intracellular Ca2+ concentration in many types of cells. SK channels are gated by Ca2+ ions via calmodulin that is constitutively bound to the intracellular C terminus of the channels and serves as the Ca2+ sensor. Here we show that, in addition, the cytoplasmic N and C termini of the channel protein form a polyprotein complex with the catalytic and regulatory subunits of protein kinase CK2 and protein phosphatase 2A. Within this complex, CK2 phosphorylates calmodulin at threonine 80, reducing by 5-fold the apparent Ca2+ sensitivity and accelerating channel deactivation. The results show that native SK channels are polyprotein complexes and demonstrate that the balance between kinase and phosphatase activities within the protein complex shapes the hyperpolarizing response mediated by SK channels.     

ECVAM's role in the implementation of the Three Rs concept in the field of biologicals

Biologicals can be defined as products that are derived from living organisms or are produced by them. They include vaccines, hormones, monoclonal and polyclonal antibodies, blood products and rDNA products. The production of conventionally produced biologicals requires an extensive batch-related quality control, to ensure that these products are both safe and potent. As several of the control tests rely on animal models, it is inevitable that the large numbers of animals are used. Many initiatives have been undertaken in the last few decades to reduce and refine the use of animals in this area. ECVAM has been involved in many activities to support the development, validation and implementation of these Three Rs methods. The role that ECVAM has played in a number of validation studies is summarised. It is concluded that ECVAM should continue to support the activities that have been shown to be successful, preferably in collaboration with the regulatory bodies.     

Florivory by the Crab Armases angustipes (Grapsidae) Influences Hummingbird Visits to Aechmea pectinata (Bromeliaceae)1

Armases angustipes (Grapsidae) crabs were recorded on 31.5 percent of Aechmea pectinata inflorescences, a common ornithofilous bromeliad in rain forests of southeastern Brazil. Crabs foraged mainly in the morning and used newly opened flowers, usually damaging the corolla, consuming the stamens and stigma, and interfering with hummingbird visits. This florivory may reduce the reproductive success of A. pectinata, both directly through consumption of flowers and indirectly by reducing pollinator visits.     

The genetics of coronary heart disease: the contribution of twin studies.

Despite the decline in coronary heart disease in many European countries, the disease remains an enormous public health problem. Although we know a great deal about environmental risk factors for coronary heart disease, a heritable component was recognized a long time ago. The earliest and best known examples of how our genetic constitution may determine cardiovascular risk relate to lipoprotein(a), familial hypercholesterolaemia and apolipoprotein E. In the past 20 years a fair number of polymorphisms assessed singly have shown strong associations with the disease but most are subject to poor repeatability. Twins constitute a compelling natural experiment to establish the genetic contribution to coronary heart disease and its risk factors. GenomEUtwin, a recently funded Framework 5 Programme of the European Community, affords the opportunity of comparing the heritability of risk factors in different European Twin Registries. As an illustration we present the heritabilities of systolic and diastolic blood pressure, based on data from over 4000 twin pairs from six different European countries and Australia. Heritabilities for systolic blood pressure are between 52 and 66% and for diastolic blood pressure between 44 and 66%. There is no evidence of sex differences in heritability estimates and very little to no evidence for a significant contribution of shared family environment. A non-twin based prospective case/cohort study of coronary heart disease and stroke (MORGAM) will allow hypotheses relating to cardiovascular disease, generated in the twin cohorts, to be tested prospectively in adult populations. Twin studies have also contributed to our understanding of the life course hypothesis, and GenomEUtwin has the potential to add to this.     

Studies on isoenzyme pattern during differentiation (spherulation) of Physarum polycephalum

Plasmodial homogenates of the true slime mold Physarum polycephalum grown on a liquid medium contain carbohydrates which form a complex with protein under conditions of acrylamide electrophoresis and thus make isoenzyme studies from those extracts impossible. A method, using mild homogenization and centrifugation on top of a 30% sucrose solution was developed. This treatment leaves most of the soluble cytoplasmic enzymes in the upper layer above the sucrose, which then can be used for successful isoenzyme or protein studies with polyacrylamide electrophoresis.The activity changes and isoenzyme pattern of 16 different enzymic activities were studied during differentiation (spherulation) of Physarum polycephalum, induced either by starvation or by mannitol. Only one enzyme, esterase, exhibited a conspicuous change in isoenzyme pattern during development.     

Large-scale purification of choline acetyltransferase and production of highly specific antisera

Choline acetyltransferase (ChAT) was purified by immunoaffinity chromatography using a covalently immobilized monoclonal antibody. In a two-step procedure, 10 kg porcine brain yielded 750 micrograms active enzyme of apparent homogeneity. This amount of ChAT was purified routinely. The purification factor was 18,000 and the yield of activity 4.3%. The affinity resin was stable under the experimental conditions applied and was used many times. The highly purified enzyme was subsequently employed to obtain a specific anti-ChAT antiserum of high titer.     

Increased noise sensitivity and altered inner ear MENA distribution in VASP−/− mice

Vasodilator-stimulated phosphoprotein (VASP) and mammalian-enabled protein (MENA) share similar cellular localisation and functions (signal transduction pathways, regulation of actin cytoskeleton dynamics). Functional substitution and compensation among Ena/VASP proteins have been proposed as the reason for the absence of major morphological and functional deficits in VASP–/– mice. The aim of this study was to investigate VASP expression in the mouse cochlea, to analyse cochlear function in VASP–/– mice compared with wildtype mice, and to analyse cochlear MENA distribution taking into account that MENA protein might compensate VASP loss in the cochlea of VASP–/– mice. We confirmed specific VASP expression in the pillar cells of the mice organ of Corti as previously reported for rat cochlea. By analysing the hearing function in VASP–/– mice, we found no differences in auditory brainstem responses and distortion product otoacoustic emissions from those of wildtype mice but evidence for an increased noise sensitivity at lower frequencies. When MENA protein levels in cochlea tissue were tested in mutant and wildtype mice by Western blot analysis, no significant differences were found, as was also seen with regard to MENA mRNA levels in laser-microdissected single pillar cells. Most surprisingly, however, MENA protein was absent in pillar cells of VASP–/– mice, whereas it was detected in other cochlear cells. The finding of a cell-specific, and not organ-specific, redundancy of MENA protein expression noted for the first time in VASP–/– mice is proposed as the reason for the observed distinct cochlear phenotype.