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.     

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.     

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.     

Pollination and reproductive biology of twelve species of neotropical Malpighiaceae: stigma morphology and its implications for the breeding system

BACKGROUND AND AIMS: This study on reproductive biology examines the stigmatic morphology of 12 Brazilian Malpighiaceae species with regard to their pollination and breeding system. METHODS: The species were studied in natural populations of a semi-deciduous forest fragment. Style tips were processed for observation by SEM and pollen-tube growth was analyzed under fluorescence microscopy. The breeding system was investigated by isolating flowers within waterproof bags. Floral visitors were recorded through notes and photographs. KEY RESULTS: Flowers are yellow, pink or white, protogynous, herkogamous and sometimes lack oil glands. While Banisteriopsis pubipetala has functional female flowers (with indehiscent anthers), 11 species present hermaphrodite flowers. Stigmas of these species may be terminal, with a slightly concave surface, or internal, consisting of a circular cavity with a large orifice, and are covered with a thin, impermeable cuticle that prevents pollen from adhering, hydrating, or germinating. Malpighiaceae have a special type of 'wet' stigma, where a secretion accumulates under the cuticle and is released by mechanical means-mainly rupture by pollinators. Even though six species show a certain degree of self-compatibility, four of them present a form of late-acting self-incompatibility, and the individual of B. pubipetala is agamospermous. Species of Centris, Epicharis and Monoeca bees pollinate these flowers, mainly collecting oil. Some Epicharis and Monoeca species collected pollen by vibration. Paratetrapedia and Tetrapedia bees are pollen and oil thieves. CONCLUSIONS: The Malpiguiaceae species studied are pollinator-dependent, as spontaneous self-pollination is limited by herkogamy, protogyny and the stigmatic cuticle. Both the oil- and pollen-collecting behaviours of the pollinators favour the rupture of the stigmatic cuticle and the deposition of pollen on or inside the stigmas. As fruit-set rates in natural conditions are low, population fragmentation may have limited the sexual reproduction of these species.