Bonamia exitiosa in farmed native oysters Ostrea angasi in Australia: optimal epidemiological qPCR cut-point and clinical disease risk factors.

Bonamiosis has developed as a problem in Australian native oysters Ostrea angasi since the parasite Bonamia spp. was first detected in Port Phillip Bay, Victoria, in the early 1990s. At that time, large-scale mortalities in both farmed and wild oysters saw the demise of the pilot native oyster culture industry. More recent attempts to farm the species resulted in subclinical infections that progressed over time to clinical disease. The aim of this work was to establish what environmental factors result in the clinical manifestation of disease; determine the diagnostic sensitivity and diagnostic specificity of histopathological examination and a quantitative polymerase chain reaction (qPCR) test for the diagnosis of B. exitiosa infection in clinically diseased farmed native oysters; and calculate the optimal qPCR threshold cycle (CT) epidemiological cut-point for classification of positive and negative cases. After applying a range of stressors to tank-held oysters, results indicated a 58% increased risk (95% CI: 16%, 99%) of a Bonamia-infected oyster dying if the oyster was held at a higher temperature (p = 0.048). Starving and tumbling oysters, in isolation, was not significantly associated with clinical bonamiosis, but a Bonamia-infected oyster was at the greatest risk of death when increased water temperature was combined with both starvation and increased motion (p = 0.02; odds ratio = 3.47). The diagnostic sensitivity and specificity of the World Organisation for Animal Health qPCR protocol were calculated for increasing CT value cut-points from ≤25 to ≤40, with an optimal cut-point identified at ≤34.5 (specificity: 92.2; 95% posterior credible intervals [PCI]: 76.2, 99.8; Sensitivity: 93.5; 95% PCI: 84.7, 99.1).

Muscle activity during running in water and on dry land: matched physiology.

We investigated muscle activity during deep water running (DWR) and treadmill running on dry land (TMR) at similar physiological responses. 9 subjects (30.7±10.4 years) participated in this study. The baseline conditions consisted of TMR at 3 ratings of perceived exertion (RPE) level (RPE 11, 13, and 15) with heart rate (HR) recorded during each condition. The target HR for each level of DWR condition was determined by the HR recorded during the TMR. Muscle activity from the rectus femoris (RF), biceps femoris (BF), tibialis anterior (TA), and gastrocnemius (GA) were measured. As originally planned, HR was not different between modes (P>0.05) and was different between exercise intensities (P<0.001). Only TA muscle activity was influenced by the interaction of mode and intensity (P<0.05). Muscle activity from the GA during DWR was significantly lower than that of TMR (a 34-48% decrease; P<0.05), although muscle activity from the remaining tested muscles were not influenced by modes of exercise (P>0.05). These observations suggest that matching HR can be recommended to produce similar magnitude of lower extremity muscle activity during DWR to that of TMR, with the exception of the GA.

Determination of muscle activity during running at reduced body weight.

The aim of this study was to investigate how lower extremity muscles are influenced by body weight support during running at different speeds. Nine participants (age 24 ± 2 years, height 1.75 ± 0.12 m, mass 73.5 ± 15.7 kg) ran at 100%, 115%, and 125% of preferred speed at 100%, 90%, 80%, 70%, and 60% of body weight on a treadmill that provided body weight support. Preferred speed was self-selected by each participant and represented a speed that he or she could sustain if going for a 30 min run. Electromyography (EMG) data were recorded (1000 Hz, 1 min) from the bicep femoris, rectus femoris, tibialis anterior, and gastrocnemius for each condition together with knee angle (electrogoniometer). Average and root mean square EMG were calculated across 30 s. Muscle patterns were determined by smoothing (low-pass filter, 4 Hz) and extracting patterns for 49 cycles defined by consecutive maximum knee flexion angles. Repeated-measures analyses of variance were used to compare average and root mean square across body weight and speeds. Correlations were computed between the 100% speed/100% body weight condition and all other conditions per muscle. There was no interaction between body weight and speed (P > 0.05). Average and root mean square decreased as body weight decreased for all muscles (P < 0.05) and increased across speeds for all muscles (P < 0.05). Correlations for all muscles between conditions were high (range: 0.921-0.999). Although a percent reduction in body weight did not lead to the same reduction in muscle activity, it was clear that reducing body weight leads to a reduction in muscle activity with no changes in muscle activity patterns.

EMG sensor location: Does it influence the ability to detect differences in muscle contraction conditions?

Even though it is well known that electromyography (EMG) characteristics are influenced by electrode placement it is common to use a single pair of sensors per muscle for EMG. This study was designed to determine if the ability to distinguish between contraction conditions was influenced by sensor location. Subjects (n = 10; 27+/-5.3 years; 82+/-13.4 kg; 178+/-7.1 cm) completed six elbow flexor conditions: three isometric contraction intensities (100% maximum effort, 80%, 50%) and three isotonic contraction intensities (heavy weight, 80% and 50% of the weight). Three pairs of electrodes were placed centrally, medially and laterally on the biceps brachii belly in line with the muscle fibers. Isometric contractions were held for 5s, with the middle 3 s analyzed. Isotonic exercises included five repetitions of elbow flexion-extension, with the middle three repetitions analyzed. Average EMG (EMG(AVG)), root mean square EMG (EMG(RMS)) and mean power frequency (MPF) were calculated for each extracted data set. Dependent variables were analyzed using 2 (contraction type) x 3 (intensity) repeated measures ANOVAs per sensor. EMG(AVG) was influenced by the interaction between contraction type and intensity for all sensors (p < 0.05). EMG(RMS) as well as MPF were influenced by the interaction between contraction type and intensity for the lateral and central leads (p < 0.05) but not the medial leads (p > 0.05). Different conclusions could have been reached from the same experiment due to different sensor locations. These differences were primarily related to comparing contraction types (i.e., isotonic vs. isometric).

Variation in stakeholder opinion on countermeasures across Europe.

A compendium of agricultural countermeasures and rural waste disposal options has been compiled as part of the EC STRATEGY (Sustainable Restoration and Long-Term Management of Contaminated Rural, Urban and Industrial Ecosystems) project. The compendium was discussed by the FARMING (Food and Agriculture Restoration Management Involving Networked Groups) network of stakeholders during meetings of national panels in the UK, Finland, Belgium, Greece and France in 2002. Their preliminary feedback has been summarised in terms of whether an option is generally acceptable, unacceptable or only acceptable under specific circumstances. A considerable divergence of opinion between national panels was apparent for many of the options considered. This could be attributed to differences in geomorphology, climate, land management, infrastructure, consumer confidence, sociopolitical context and culture. Where consensus was reached between stakeholders it was generally for those countermeasures that provide public reassurance, sustain farming practices and minimise environmental impact. Furthermore, whilst there was general agreement that contaminated food should not enter the food chain, many of the options proposed for its subsequent management were not generally acceptable to stakeholders.

Achievements, difficulties and future challenges for the FARMING network.

The EC FARMING network (Food and Agriculture Restoration Management Involving Networked Groups) was set up to bring together the many and diverse stakeholders who would be involved in intervention following wide scale radioactive contamination of the food chain, so that acceptable strategies can be developed for maintaining agricultural production and safe food supply. The network comprises stakeholder panels in the UK, Finland, Belgium, France and Greece that have met regularly since 2001 to debate, discuss and exchange opinion on the acceptability, constraints and impact of various countermeasure options and strategies. The objectives of this paper are to consolidate the main achievements of the FARMING project over the period 2000-2004, to highlight the various difficulties that were encountered and to discuss the challenges for engaging stakeholders in off-site emergency management and long-term rehabilitation in the future.

Statistics and vertical directionality of low-frequency ambient noise at the North Pacific Acoustic Laboratory site.

We examine statistical and directional properties of the ambient noise in the 10-100 Hz frequency band from the NPAL array. Marginal probability densities are estimated as well as mean square levels, skewness and kurtoses in third octave bands. The kurotoses are markedly different from Gaussian except when only distant shipping is present. Extremal levels reached approximately 150 dB re 1 micro Pa, suggesting levels 60dB greater than the mean ambient were common in the NPAL data sets. Generally, these were passing ships. We select four examples: i) quiescent noise, ii) nearby shipping, iii) whale vocalizations and iv) a micro earthquake for the vertical directional properties of the NPAL noise since they are representative of the phenomena encountered. We find there is modest broadband coherence for most of these cases in their occupancy band across the NPAL aperture. Narrowband coherence analysis from VLA to VLA was not successful due to ambiguities. Examples of localizing sources based upon this coherence are included. kw diagrams allow us to use data above the vertical aliasing frequency. Ducted propagation for both the quiescent and micro earthquake (T phase) are identified and the arrival angles of nearby shipping and whale vocalizations. MFP localizations were modestly successful for nearby sources, but long range ones could not be identified, most likely because of signal mismatch in the MFP replica.

Overview of the wisdom stakeholder workshop on restoration management.

A Workshop to extend the Involvement of Stakeholders in Decisions On restoration Management (WISDOM) was held at New College, Oxford from 15 to 17 September 2003. The aim was to promote awareness and interest in the wider application of stakeholder involvement in the formulation of strategies for the management of contaminated agricultural land and produce following a nuclear accident. The workshop, through 25 plenary papers and a set of two facilitated discussion sessions, provided valuable feedback on a wide range of issues including technical and social factors affecting countermeasure selection, acceptability of intervention levels, the challenges of rural waste disposal and crisis management. The workshop achieved its aim and the findings will be disseminated widely. Stakeholder groups are already active in the UK, Finland, Belgium, France and Greece; there was commitment from participants to establish further, similar groups in other member states within the European Union.

Characteristics of shock attenuation during fatigued running.

The aim of this study was to examine shock attenuation before and after completing a maximal effort graded exercise test while running on a treadmill. Ten individuals ran before and after a maximal graded exercise test with running speed controlled between conditions. Transfer functions were calculated using surface-mounted accelerometers to represent shock attenuation. An accelerometer was mounted on the distal aspect of the tibia and another on the anterior aspect of the forehead. Ten strides were analysed in each condition for all participants. Paired t-tests were used to compare each dependent variable (shock attenuation, stride length, rate of oxygen consumption) between conditions (running before vs after the exercise test). Oxygen consumption was 16% greater when running after the graded exercise test (47.9 +/- 5.0 ml x kg(-1) x min(-1); mean+/-s) than when running before it (41.1 +/- 2.7 ml x kg(-1) x min(-1)) (P < 0.05). Stride length was similar during running before (2.71 +/- 0.15 m) and after (2.75 +/- 0.17 m) the graded exercise test (P > 0.05). Shock attenuation was, on average, 12% lower during running after (-9.8 +/- 2.6 dB) than before (-11.3 +/- 2.7 dB) the graded exercise test (P < 0.05). We conclude that less shock was attenuated during fatigued than non-fatigued running and that only subtle changes in stride length were made while fatigued.

Myosin-I nomenclature.

We suggest that the vertebrate myosin-I field adopt a common nomenclature system based on the names adopted by the Human Genome Organization (HUGO). At present, the myosin-I nomenclature is very confusing; not only are several systems in use, but several different genes have been given the same name. Despite their faults, we believe that the names adopted by the HUGO nomenclature group for genome annotation are the best compromise, and we recommend universal adoption.

Myosin vb is associated with plasma membrane recycling systems.

Myosin Va is associated with discrete vesicle populations in a number of cell types, but little is known of the function of myosin Vb. Yeast two-hybrid screening of a rabbit parietal cell cDNA library with dominant active Rab11a (Rab11aS20V) identified myosin Vb as an interacting protein for Rab11a, a marker for plasma membrane recycling systems. The isolated clone, corresponding to the carboxyl terminal 60 kDa of the myosin Vb tail, interacted with all members of the Rab11 family (Rab11a, Rab11b, and Rab25). GFP-myosin Vb and endogenous myosin Vb immunoreactivity codistributed with Rab11a in HeLa and Madin-Darby canine kidney (MDCK) cells. As with Rab11a in MDCK cells, the myosin Vb immunoreactivity was dispersed with nocodazole treatment and relocated to the apical corners of cells with taxol treatment. A green fluorescent protein (GFP)-myosin Vb tail chimera overexpressed in HeLa cells retarded transferrin recycling and caused accumulation of transferrin and the transferrin receptor in pericentrosomal vesicles. Expression of the myosin Vb tail chimera in polarized MDCK cells stably expressing the polymeric IgA receptor caused accumulation of basolaterally endocytosed polymeric IgA and the polymeric IgA receptor in the pericentrosomal region. The myosin Vb tail had no effects on transferrin trafficking in polarized MDCK cells. The GFP-myosin Va tail did not colocalize with Rab11a and had no effects on recycling system vesicle distribution in either HeLa or MDCK cells. The results indicate myosin Vb is associated with the plasma membrane recycling system in nonpolarized cells and the apical recycling system in polarized cells. The dominant negative effects of the myosin Vb tail chimera indicate that this unconventional myosin is required for transit out of plasma membrane recycling systems.

Intercellular junctions: downstream and upstream of Ras?

Most human tumors are of epithelial origin, and these tumors gradually lose their epithelial character in a process termed the epithelial-mesenchymal transition. Approximately 40% of human tumors have activating mutations in one of the three RAS genes. Given these statistics, it is critically important to understand the role of Ras signaling in the epithelial-mesenchymal transition. This review considers the mechanisms and effectors through which Ras may regulate intercellular junction formation in epithelial cells. Conversely, intercellular junction proteins themselves may play a role in regulating Ras activation and signaling.

Identification of proteins in the postsynaptic density fraction by mass spectrometry.

Our understanding of the organization of postsynaptic signaling systems at excitatory synapses has been aided by the identification of proteins in the postsynaptic density (PSD) fraction, a subcellular fraction enriched in structures with the morphology of PSDs. In this study, we have completed the identification of most major proteins in the PSD fraction with the use of an analytical method based on mass spectrometry coupled with searching of the protein sequence databases. At least one protein in each of 26 prominent protein bands from the PSD fraction has now been identified. We found 7 proteins not previously known to be constituents of the PSD fraction and 24 that had previously been associated with the PSD by other methods. The newly identified proteins include the heavy chain of myosin-Va (dilute myosin), a motor protein thought to be involved in vesicle trafficking, and the mammalian homolog of the yeast septin protein cdc10, which is important for bud formation in yeast. Both myosin-Va and cdc10 are threefold to fivefold enriched in the PSD fraction over brain homogenates. Immunocytochemical localization of myosin-Va in cultured hippocampal neurons shows that it partially colocalizes with PSD-95 at synapses and is also diffusely localized in cell bodies, dendrites, and axons. Cdc10 has a punctate distribution in cell bodies and dendrites, with some of the puncta colocalizing with PSD-95. The results support a role for myosin-Va in transport of materials into spines and for septins in the formation or maintenance of spines.

Engineering of the myosin-ibeta nucleotide-binding pocket to create selective sensitivity to N(6)-modified ADP analogs.

Distinguishing the cellular functions carried out by enzymes of highly similar structure would be simplified by the availability of isozyme-selective inhibitors. To determine roles played by individual members of the large myosin superfamily, we designed a mutation in myosin's nucleotide-binding pocket that permits binding of adenine nucleotides modified with bulky N(6) substituents. Introduction of this mutation, Y61G in rat myosin-Ibeta, did not alter the enzyme's affinity for ATP or actin and actually increased its ATPase activity and actin-translocation rate. We also synthesized several N(6)-modified ADP analogs that should bind to and inhibit mutant, but not wild-type, myosin molecules. Several of these N(6)-modified ADP analogs were more than 40-fold more potent at inhibiting ATP hydrolysis by Y61G than wild-type myosin-Ibeta; in doing so, these analogs locked Y61G myosin-Ibeta tightly to actin. N(6)-(2-methylbutyl) ADP abolished actin filament motility mediated by Y61G, but not wild-type, myosin-Ibeta. Furthermore, a small fraction of inhibited Y61G molecules was sufficient to block filament motility mediated by mixtures of wild-type and Y61G myosin-Ibeta. Introduction of Y61G myosin-Ibeta molecules into a cell should permit selective inhibition by N(6)-modified ADP analogs of cellular processes dependent on myosin-Ibeta.

Absence of the tight junctional protein AF-6 disrupts epithelial cell-cell junctions and cell polarity during mouse development.

BACKGROUND: The establishment, maintenance and rearrangement of junctions between epithelial cells are extremely important in many developmental, physiological and pathological processes. AF-6 is a putative Ras effector; it is also a component of tight and adherens junctions, and has been shown to bind both Ras and the tight-junction protein ZO-1. In the mouse, AF-6 is encoded by the Af6 gene. As cell-cell junctions are important in morphogenesis, we generated a null mutation in the murine Af6 locus to test the hypothesis that lack of AF-6 function would cause epithelial abnormalities. RESULTS: Although cell-cell junctions are thought to be important in early embryogenesis, homozygous mutant embryos were morphologically indistinguishable from wild-type embryos through 6.5 days post coitum (dpc) and were able to establish all three germ layers. The earliest morphological abnormalities were observed in the embryonic ectoderm of mutant embryos at 7.5 dpc. The length of the most apical cell-cell junctions was reduced, and basolateral surfaces of those cells were separated by multiple gaps. Cells of the embryonic ectoderm were less polarized as assessed by histological criteria and lateral localization of an apical marker. Mutant embryos died by 10 dpc, probably as a result of placental failure. CONCLUSIONS: AF-6 is a critical regulator of cell-cell junctions during mouse development. The loss of neuroepithelial polarity in mutants is consistent with a loss of efficacy of the cell-cell junctions that have a critical role in establishing apical/basolateral asymmetry.

Myosin-V: head to tail.

The myosin-V family is the most extensively studied of the unconventional myosin families. Most organisms examined have at least one member of the myosin-V family: many have multiple members. The wide range of species in which myosin-V has been identified suggests that myosin-V is a fundamental component of organelle transport in all higher eukaryotes. Possible cargoes for myosin-V range from melanosomes and synaptic vesicles in mammals to vacuoles and messenger RNA in yeast. In this review, we discuss the current state of research on the cellular function of myosin-V as described by the actions of the head, neck and tail domains.

Chromosomal location of three unconventional myosin heavy chain genes in the mouse.

We have used an interspecific backcross to map the chromosomal locations of the mouse homologs of the genes encoding myr 3 (unconventional myosin from rat), myr 4, and myr 5, members of the myosin super-family of motor proteins. The mouse loci have been named Myo1e, Myo1c, and Myo9b; they map to chromosomes 9 (MGD position 41), 11 (MGD position 46), and 8 (MGD position 31.5), respectively.

Cultured melanocytes from dilute mutant mice exhibit dendritic morphology and altered melanosome distribution.

Mutant alleles at the dilute unconventional myosin heavy chain locus cause diluted coat color, opisthotonic seizures, and death. The dilute coat color phenotype is caused by irregular clumping of pigment in the hair, but amounts of melanin are unchanged from wild-type controls. The melanocyte phenotype has been described as adendritic, since hair bulb and Harderian gland melanocytes appear to be rounded in tissue sections. These observations do not exclude the possibility that the processes lack pigment, since the melanocyte shape was judged by the distribution of melanin. We have tested this hypothesis by culturing primary melanocytes from dilute mutant and wild-type mice. The mutant melanocytes do not lack processes; instead, they exhibit a concentrated perinuclear distribution of melanosomes, while wild-type melanocytes have a very uniform cytoplasmic distribution of melanosomes. Electron micrographs show no detectable differences in melanosome morphology or maturation between dilute and wild-type melanocytes. Immunofluorescence experiments indicate that the dilute protein is concentrated in regions of the cytoplasm that contain melanosomes. These experiments show that the dilute myosin is necessary for the localization of melanosomes, either by active transport or tethering.

Cloning and characterization of myr 6, an unconventional myosin of the dilute/myosin-V family.

We have isolated cDNAs encoding a second member of the dilute (myosin-V) unconventional myosin family in vertebrates, myr 6 (myosin from rat 6). Expression of myr 6 transcripts in the brain is much more limited than is the expression of dilute, with highest levels observed in choroid plexus and components of the limbic system. We have mapped the myr 6 locus to mouse chromosome 18 using an interspecific backcross. The 3' portion of the myr 6 cDNA sequence from rat is nearly identical to that of a previously published putative glutamic acid decarboxylase from mouse [Huang, W.M., Reed-Fourquet, L., Wu, E. & Wu, J.Y. (1990) Proc. Natl. Acad. Sci. USA 87, 8491-8495].