Cathepsin B localizes to plasma membrane caveolae of differentiating myoblasts and is secreted in an active form at physiological pH

Our in vitro studies support a functional link between the induction of cathepsin B gene expression and the catabolic restructuring associated with myotube formation during myogenesis in vivo. We have tested two predictions that are basic to this hypothesis: (1) that active cathepsin B is localized to plasma membrane caveolae of fusing myoblasts; and (2) that active cathepsin B is secreted from fusing myoblasts at physiological pH. During differentiation, L6 rat myoblasts demonstrated a fusion-related increase in activity associated with the 25/26-kDa, fully processed, active form of cathepsin B. Immunocytochemical studies demonstrated a redistribution of lysosomal cathepsin B protein toward the membrane of fusing myoblasts, and a colocalization of cathepsin B with caveolin-3, the muscle-specific structural protein of membrane caveolae. Sucrose density fractionation and Western blot analysis demonstrated that an active form of cathepsin B localizes to caveolar fractions along with caveolin-3, annexin-VII, beta-dystroglycan and dystrophin. Finally, 'real-time' activity assays and Western blot analysis demonstrated that active cathepsin B is secreted from fusing myoblasts at physiological pH. Collectively, these studies support an association of active cathepsin B with plasma membrane caveolae and the secretion of active cathepsin B from differentiating myoblasts during myoblast fusion.     

Purinergic junctional transmission and propagation of calcium waves in spinal cord astrocyte networks

Micro-photolithographic methods have been employed to form discrete patterns of spinal cord astrocytes that allow quantitative measurements of Ca(2+) wave propagation. Astrocytes were confined to lanes 20-100 microm wide and Ca(2+) waves propagated from a point of mechanical stimulation or of application of adenosine triphosphate; all Ca(2+) wave propagation was blocked by simultaneous application of purinergic P2Y(1) and P2Y(2) antagonists. Stimulation of an astrocyte at one end of a lane, followed by further stimulation of this astrocyte, gave rise to Ca(2+) transients in the same astrocytes; however, if the second stimulation was applied to an astrocyte at the other end of the lane, then this gave rise to a different but overlapping set of astrocytes generating a Ca(2+) signal. Both the amplitude and velocity of the Ca(2+) wave decreased over 270 microm from the point of initiation, and thereafter remained, on average, constant with random variations for at least a further 350 microm. Also, the percentage of astrocytes that gave a Ca(2+) transient decreased with distance along lanes. All the above observations were quantitatively predicted by our recent theoretical model of purinergic junctional transmission, as was the Ca(2+) wave propagation along and between parallel lanes of astrocytes different distances apart. These observations show that a model in which the main determinants are the diffusion of adenosine triphosphates regeneratively released from a stimulated astrocyte, together with differences in the properties and density of the purinergic P2Y receptors on astrocytes, is adequate to predict a wide range of Ca(2+) wave transmission and propagation phenomena.     

Effects of successive seasons of genetically modified herbicide-tolerant maize cropping on weeds and invertebrates

The use of genetically modified herbicide‐tolerant (GMHT) crops influences the abundance of weeds and some invertebrate groups because the associated herbicide regime contrasts with that of conventional systems. However, it is not clear to what extent these effects might be cumulative; should GMHT crops be grown continuously. In northern Europe, in the near future, this situation is most likely to apply to maize crops. Here, we consider the effects of continuous GMHT maize cropping on plant and invertebrate taxa using a split‐field experiment. Half of each field was managed using GMHT and the other half with a conventional variety, with the treatments retained for two seasons. The treatment effects were broadly consistent with those found in the larger sample of non‐continuous maize sites within the Farm Scale Evaluations. There was little evidence of effects being significantly more pronounced in the second year; any cumulative differences in above‐ground biodiversity between GMHT and conventional cropping were too variable to be readily detected.     

New insights on the mechanism of acid degradation of pea starch

The degradation of pea starch granules by acid hydrolysis has been investigated using a range of chemical and structural methods, namely through measuring changes in amylose content by both the iodine binding and concanavalin A precipitation methods, along with small angle X-ray scattering (SAXS), wide angle X-ray diffraction (XRD) and field emission scanning electron microscopy (FE-SEM). The relative crystallinity, intensity of the lamellar peak and the low-q scattering increased during the initial stages of acid hydrolysis, indicating early degradation of the amorphous regions (growth rings and lamellae). In the first 2 days of hydrolysis, there was a rapid decline in amylose content, a concomitant loss of precipitability of amylopectin by concanavalin A, and damage to the surface and internal granular structures was evident. These observations are consistent with both amylose and amylopectin being located on the surface of the granules and attacked simultaneously in the early stages of acid hydrolysis. The results are also consistent with amylose being more concentrated at the core of the granules. More extensive hydrolysis resulted in the simultaneous disruption of amorphous and crystalline regions, which was indicated by a decrease in lamellar peak intensity, decrease in interhelix peak intensity and no further increase in crystallinity. These results provide new insights into the organization of starch granules.     

Using hierarchical clustering of secreted protein families to classify and rank candidate effectors of rust fungi

Rust fungi are obligate biotrophic pathogens that cause considerable damage on crop plants. Puccinia graminis f. sp. tritici, the causal agent of wheat stem rust, and Melampsora larici-populina, the poplar leaf rust pathogen, have strong deleterious impacts on wheat and poplar wood production, respectively. Filamentous pathogens such as rust fungi secrete molecules called disease effectors that act as modulators of host cell physiology and can suppress or trigger host immunity. Current knowledge on effectors from other filamentous plant pathogens can be exploited for the characterisation of effectors in the genome of recently sequenced rust fungi. We designed a comprehensive in silico analysis pipeline to identify the putative effector repertoire from the genome of two plant pathogenic rust fungi. The pipeline is based on the observation that known effector proteins from filamentous pathogens have at least one of the following properties: (i) contain a secretion signal, (ii) are encoded by in planta induced genes, (iii) have similarity to haustorial proteins, (iv) are small and cysteine rich, (v) contain a known effector motif or a nuclear localization signal, (vi) are encoded by genes with long intergenic regions, (vii) contain internal repeats, and (viii) do not contain PFAM domains, except those associated with pathogenicity. We used Markov clustering and hierarchical clustering to classify protein families of rust pathogens and rank them according to their likelihood of being effectors. Using this approach, we identified eight families of candidate effectors that we consider of high value for functional characterization. This study revealed a diverse set of candidate effectors, including families of haustorial expressed secreted proteins and small cysteine-rich proteins. This comprehensive classification of candidate effectors from these devastating rust pathogens is an initial step towards probing plant germplasm for novel resistance components.     

Revealing the appetite of the marine aquarium fish trade: the volume and biodiversity of fish imported into the United States

The aquarium trade and other wildlife consumers are at a crossroads forced by threats from global climate change and other anthropogenic stressors that have weakened coastal ecosystems. While the wildlife trade may put additional stress on coral reefs, it brings income into impoverished parts of the world and may stimulate interest in marine conservation. To better understand the influence of the trade, we must first be able to quantify coral reef fauna moving through it. Herein, we discuss the lack of a data system for monitoring the wildlife aquarium trade and analyze problems that arise when trying to monitor the trade using a system not specifically designed for this purpose. To do this, we examined an entire year of import records of marine tropical fish entering the United States in detail, and discuss the relationship between trade volume, biodiversity and introduction of non-native marine fishes. Our analyses showed that biodiversity levels are higher than previous estimates. Additionally, more than half of government importation forms have numerical or other reporting discrepancies resulting in the overestimation of trade volumes by 27%. While some commonly imported species have been introduced into the coastal waters of the USA (as expected), we also found that some uncommon species in the trade have also been introduced. This is the first study of aquarium trade imports to compare commercial invoices to government forms and provides a means to, routinely and in real time, examine the biodiversity of the trade in coral reef wildlife species.     

Brief intervention to prevent hazardous drinking in young people aged 14--15 in a high school setting (SIPS JR-HIGH): study protocol for a randomized controlled trial

ABSTRACT: BACKGROUND: Whilst the overall proportion of young people drinking alcohol in the United Kingdom has decreased in recent years, those who do drink appear to drink a larger amount, and more frequently. Early and heavy drinking by younger adolescents is a significant public health problem linked to intellectual impairment, increased risk of injuries, mental health issues, unprotected or regretted sexual experience, violence, and sometimes accidental death, which leads to high social and economic costs. This feasibility pilot trial aims to explore the feasibility of delivering brief alcohol intervention in a school setting with adolescents aged 14 and 15 and to examine the acceptability of study measures to school staff, young people and parents.Methods and designSeven schools across one geographical area in the North East of England will be recruited. Schools will be randomly allocated to one of three conditions: provision of an advice leaflet (control condition, n = 2 schools); a 30-minute brief interactive session, which combines structured advice and motivational interviewing techniques delivered by the school learning mentor (level 1 condition, n = 2 schools); and a 60-minute session involving family members delivered by the school learning mentor (level 2 condition, n = 3 schools). Participants will be year 10 school pupils (aged 14 and 15) who screen positively on a single alcohol screening question and who consent to take part in the trial. Year 10 pupils in all seven schools will be followed up at 6 and 12 months. Secondary outcome measures include the ten-question Alcohol-Use Disorders Identification Test. The EQ-5D-Y and a modified short service use questionnaire will inform the health and social resource costs for any future economic evaluation.Young people recruited into the trial will also complete a 28-day timeline follow back questionnaire at 12-month follow-up. A qualitative evaluation (with young people, school staff, learning mentors, and parents) will examine facilitators and barriers to the use of screening and brief intervention approaches in the school setting in this age group.Trial registrationTrial reference number ISRCTN07073105.     

Purinergic junctional transmission and propagation of calcium waves in cultured spinal cord microglial networks

In order to elucidate the mechanisms of purinergic transmission of calcium (Ca^2 +) waves between microglial cells, we have employed micro-photolithographic methods to form discrete patterns of microglia that allow quantitative measurements of Ca^2 + wave propagation. Microglia were confined to lanes 20–100 wide and Ca^2 + waves propagated from a point of mechanical stimulation, with a diminution in amplitude, for about 120 . The number of cells participating in propagation also decreased over this distance. Ca^2 + waves could propagate across a cell-free lane from one microglia lane to another if this distance of separation was less than about 60 , indicating that propagation involved diffusion of a chemical transmitter. This transmitter was identified as ATP since all Ca^2 + wave propagation was blocked by the purinoceptor antagonist suramin, which blocks P2Y₂ and P2Y₁₂ at relatively low concentrations. Antibodies to P2Y₁₂ showed these at very high density compared with P2Y₂, indicating a role for P2Y₁₂ receptors. These observations were quantitatively accounted for by a model in which the main determinants are the diffusion of ATP released from a stimulated microglial cell and differences in the dissociation constant of the purinoceptors on the microglial cells.