Calcium content and distribution as a function of growth and transformation in the mouse 3T3 cell

Total Ca content and that fraction of Ca sensitive to removal by the chelator ethylene glycol-bis(β-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA) have been investigated in the mouse 3T3 cell as a function of growth stage, transformation with SV40 virus, and serum levels of the media. Cells were allowed to grow through several doublings in media containing (45)Ca. The cellular content of (45)Ca was used to access total cell Ca. That fraction of (45)Ca removed by EGTA was presumed to represent primarily surface-localized Ca. The data are expressed on a per cell volume basis to compensate for size differences as a function of growth stage and transformation. During exponential growth phase, the 3T3 cell contains 525pmol Ca/μl cell volume. Of this, approx. 457 pmol/μl is not removable by EGTA and, presumably, is cytoplasmically located. This value is in close agreement with previous studies on the HeLa cell (470 pmol Ca/μl cell water after the removal of the surface Ca). The low level of EGTA- removable Ca present in the 3T3 cell during early exponential growth (68 pmol Ca/μl cell volume) increases progressively with increasing cell density, and upon quiescence it is sevenfold greater. In contrast, SV40- transformed 3T3 cells growing exponentially possess total levels of Ca which are approximately two-thirds the levels of the normal 3T3 cell. However, their EGTA-sensitive Ca is not significantly different from that of exponentially growing, normal 3T3 cells. As the transformed cells continue to grow at high density, their total ca and their sensitivity to EGTA do not change, in contrast to the normal 3T3 cell. Thus, an increase in Ca associated with the cell surface appears to be correlated with growth inhibition. This has been investigated further by regulating growth of the normal and transformed cell with alterations in the serum level of the media. In 4 percent calf serum the normal cell is stopped from continued proliferation. Growth stoppage under these conditions is characterized by a nearly fourfold increase in EGTA-removable Ca, similar to the increase observed upon quiescence in depleted 10 percent serum. Similar treatment of the transformed cell does not reduce its growth rate, nor does it significantly alter Ca distribution. However, at 0.5 percent medium serum levels, the SV40 3T3 growth rate is substantially reduced and, under these conditions, EGTA-removable Ca increases twofold.     

Ecological role of <Emphasis Type="Italic">Phyllophora antarctica</Emphasis> drift accumulations in coastal soft-sediment communities of McMurdo Sound,Antarctica

At Cape Evans on Ross Island, Antarctica, the rhodophyte Phyllophora antarctica is the dominant primary producer in terms of biomass from 10 to >30 m depth. The vast majority of Phyllophora occurs as accumulations of unattached plants. Whilst decomposition and incorporation of macroalgal drift material into the food web is rapid in temperate ecosystems, we predicted these processes to be slow in Antarctica. We address the functional role of macroalgal detritus in fuelling the biodiversity of benthic communities at Cape Evans during the summers of 2001 and 2002. Specifically we (a) describe the distribution and biomass of attached and drift algae, (b) assess the photosynthetic capacity and degradation of drift accumulations using in situ fluorometry, (c) assess the effect of patches of drift Phyllophora on underlying macrofaunal communities, and, (d) use stable isotopes to investigate the possible uptake of Phyllophora by macrofauna. We found drift Phyllophora accumulations throughout the depth range investigated (3–31 m), with peak biomasses of 140±30 g dwt m^–2 in the 15–25 m depth strata. At this depth stratum Phyllophora was a conspicuous habitat element with the % cover on the seafloor averaging 30%. While initially the drift algal accumulations appeared in good health we measured significant declines in photosynthetic capacity between years suggesting ongoing, albeit slow, degradation of the drift algal accumulations. Our results demonstrate that Phyllophora drift accumulations have a structuring role on soft-sediment communities, which increases in strength with the gradual degradation of the algae. The longevity of Phyllophora is enhanced by secondary metabolites, which serve as protection against grazers, and their extreme shade adaptation. However, our carbon and nitrogen stable isotope data of polychaetes and amphipods associated with Phyllophora suggest that macroalgal detritus enters the food web, and although this process is slow, Phyllophora accumulations might serve to dampen the seasonality in food supply providing higher trophic levels with a more constant food source.     

Recombinant adeno-associated virus type 2-mediated gene delivery into the <Emphasis Type="Italic">Rpe65</Emphasis><Superscript>-/-</Superscript> knockout mouse eye results in limited rescue

Background  

Leber's congenital amaurosis (LCA) is a severe form of retinal dystrophy. Mutations in the RPE65 gene, which is abundantly expressed in retinal pigment epithelial (RPE) cells, account for approximately 10–15% of LCA cases. In this study we used the high turnover, and rapid breeding and maturation time of the Rpe65 ^-/- knockout mice to assess the efficacy of using rAAV-mediated gene therapy to replace the disrupted RPE65 gene. The potential for rAAV-mediated gene treatment of LCA was then analyzed by determining the pattern of RPE65 expression, the physiological and histological effects that it produced, and any improvement in visual function.     

Skeletal muscle mitochondrial FAT/CD36 content and palmitate oxidation are not decreased in obese women

A reduction in fatty acid oxidation has been associated with lipid accumulation and insulin resistance in the skeletal muscle of obese individuals. We examined whether this decrease in fatty acid oxidation was attributable to a reduction in muscle mitochondrial content and/or a dysfunction in fatty acid oxidation within mitochondria obtained from skeletal muscle of age-matched, lean [body mass index (BMI) = 23.3 +/- 0.7 kg/m2] and obese women (BMI = 37.6 +/- 2.2 kg/m2). The mitochondrial marker enzymes citrate synthase (-34%), beta-hydroxyacyl-CoA dehydrogenase (-17%), and cytochrome c oxidase (-32%) were reduced (P < 0.05) in obese participants, indicating that mitochondrial content was diminished. Obesity did not alter the ability of isolated mitochondria to oxidize palmitate; however, fatty acid oxidation was reduced at the whole muscle level by 28% (P < 0.05) in the obese. Mitochondrial fatty acid translocase (FAT/CD36) did not differ in lean and obese individuals, but mitochondrial FAT/CD36 was correlated with mitochondrial fatty acid oxidation (r = 0.67, P < 0.05). We conclude that the reduction in fatty acid oxidation in obese individuals is attributable to a decrease in mitochondrial content, not to an intrinsic defect in the mitochondria obtained from skeletal muscle of obese individuals. In addition, it appears that mitochondrial FAT/CD36 may be involved in regulating fatty acid oxidation in human skeletal muscle.     

Pair-level approximations to the spatio-temporal dynamics of epidemics on asymmetric contact networks

The process of infection during an epidemic can be envisaged as being transmitted via a network of routes represented by a contact network. Most differential equation models of epidemics are mean-field models. These contain none of the underlying spatial structure of the contact network. By extending the mean-field models to pair-level, some of the spatial structure can be contained in the model. Some networks of transmission such as river or transportation networks are clearly asymmetric, whereas others such as airborne infection can be regarded as symmetric. Pair-level models have been developed to describe symmetric contact networks. Here we report on work to develop a pair-level model that is also applicable to asymmetric contact networks. The procedure for closing the model at the level of pairs is discussed in detail. The model is compared against stochastic simulations of epidemics on asymmetric contact networks and against the predictions of the symmetric model on the same networks. DEFRA funded project FC1153     

Importance of factors determining the effective lifetime of a mass, long-lasting, insecticidal net distribution: a sensitivity analysis

Background

Long-lasting insecticidal nets (LLINs) reduce malaria transmission by protecting individuals from infectious bites, and by reducing mosquito survival. In recent years, millions of LLINs have been distributed across sub-Saharan Africa (SSA). Over time, LLINs decay physically and chemically and are destroyed, making repeated interventions necessary to prevent a resurgence of malaria. Because its effects on transmission are important (more so than the effects of individual protection), estimates of the lifetime of mass distribution rounds should be based on the effective length of epidemiological protection.

Methods

Simulation models, parameterised using available field data, were used to analyse how the distribution's effective lifetime depends on the transmission setting and on LLIN characteristics. Factors considered were the pre-intervention transmission level, initial coverage, net attrition, and both physical and chemical decay. An ensemble of 14 stochastic individual-based model variants for malaria in humans was used, combined with a deterministic model for malaria in mosquitoes.

Results

The effective lifetime was most sensitive to the pre-intervention transmission level, with a lifetime of almost 10 years at an entomological inoculation rate of two infectious bites per adult per annum (ibpapa), but of little more than 2 years at 256 ibpapa. The LLIN attrition rate and the insecticide decay rate were the next most important parameters. The lifetime was surprisingly insensitive to physical decay parameters, but this could change as physical integrity gains importance with the emergence and spread of pyrethroid resistance.

Conclusions

The strong dependency of the effective lifetime on the pre-intervention transmission level indicated that the required distribution frequency may vary more with the local entomological situation than with LLIN quality or the characteristics of the distribution system. This highlights the need for malaria monitoring both before and during intervention programmes, particularly since there are likely to be strong variations between years and over short distances. The majority of SSA's population falls into exposure categories where the lifetime is relatively long, but because exposure estimates are highly uncertain, it is necessary to consider subsequent interventions before the end of the expected effective lifetime based on an imprecise transmission measure.     

First detection, isolation and molecular characterization of infectious salmon anaemia virus associated with clinical disease in farmed Atlantic salmon (Salmo salar) in Chile

Background  

Several forms of progressive retinal atrophy (PRA) segregate in more than 100 breeds of dog with each PRA segregating in one or a few breeds. This breed specificity may be accounted for by founder effects and genetic drift, which have reduced the genetic heterogeneity of each breed, thereby facilitating the identification of causal mutations. We report here a new form of PRA segregating in the Border Collie breed. The clinical signs, including the loss of night vision and a progressive loss of day vision, resulting in complete blindness, occur at the age of three to four years and may be detected earlier through systematic ocular fundus examination and electroretinography (ERG).     

Changes in Ecosystem Function Across Sedimentary Gradients in Estuaries

The input of terrestrial silt and clay (hereafter mud) into coastal environments can alter sediment grain size distribution affecting the structure and functioning of benthic communities. The relationship between sediment mud content and macrofaunal community structure has been well documented, but not the effects on ecosystem function. In 143 plots from the mid-intertidal sites in 9 estuaries, we measured sediment properties, macrofaunal community composition and fluxes of O₂ and NH₄ ⁺ across the sediment–water interface to derive process-based measures of ecosystem function across the sand–mud gradient. We observed reductions in measures of macrofaunal diversity and decreases in the maximum density of key bioturbating bivalves (Austrovenus stutchburyi and Macomona liliana) with increased mud content. Concurrently, the maximum rates of sediment oxygen consumption (SOC), NH₄ ⁺ efflux (NH₄ ⁺) and biomass standardized gross primary production (GPP_Chl-a ) also decreased with increasing mud content. Environmental predictors explained 34–39% (P = 0.005–0.01) of the total variation in ecosystem function in distance-based linear models. After partitioning out the effect of mud, A. stutchburyi abundance was positively correlated and explained 25 and 23% (P = 0.0001) of the variation of SOC and NH₄ ⁺, respectively. Also, mud content (negatively correlated) and temperature (positively correlated) explained 26% of variability in GPP_Chl-a (P = 0.0001). Our results highlight the importance of increased mud content and the associated reduction in the abundance of strongly interacting key species on the loss of ecosystem function in intertidal sand flats.