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.     

Uptake, transport and metabolism of cytokinin in moss protonema

Uptake, transport and metabolism of cytokinin in the protonemaof Funaria hygrometrica were studied using labelled kinetin(6-furfurylamino [8-¹⁴C]-purine). All cells of the protonema,chloronema and caulonema, were able to take up kinetin, whichwas carried in the symplastic transport system from cell tocell. Radioactivity was especially accumulated in growing cellsof the protonema. Kinetin was metabolized immediately afteruptake. While only very little kinetin (less than 1%) remainedas free kinetin and one part was immobilized in chromatographicseparation [e.g. attached to proteins and incorporated intonucleic acids (17)], most of the remaining kinetin was metabolizedto adenine derivatives. Exogenously supplied adenosine changedthe metabolism of kinetin. In the caulonema, adenosine reducedthe turnover of kinetin to other adenine derivatives and enhancedthe content of labelling in the start fraction. Thus adenosinecan stimulate cytokinin-dependent bud formation in moss protonema. (Received November 24, 1977; )     

[18F]FLT and [18F]FDG PET for Non-invasive Treatment Monitoring of the Nicotinamide Phosphoribosyltransferase Inhibitor APO866 in Human Xenografts

Introduction

APO866 is a new anti-tumor compound inhibiting nicotinamide phosphoribosyltransferase (NAMPT). APO866 has an anti-tumor effect in several pre-clinical tumor models and is currently in several clinical phase II studies. 3′-deoxy-3′-[18F]fluorothymidine ([18F]FLT) is a tracer used to assess cell proliferation in vivo. The aim of this study was non-invasively to study effect of APO866 treatment on [18F]FLT and 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) uptake.

Methods

In vivo uptake of [18F]FLT and [18F]FDG in human ovary cancer xenografts in mice (A2780) was studied at various time points after APO866 treatment. Baseline [18F]FLT or [18F]FDG scans were made before treatment and repeated after 24 hours, 48 hours and 7 days. Tumor volume was followed with computed tomography (CT). Tracer uptake was quantified using small animal PET/CT. One hour after iv injection of tracer, static PET scans were performed. Imaging results were compared with Ki67 immunohistochemistry.

Results

Tumors treated with APO866 had volumes that were 114% (24 h), 128% (48 h) and 130% (Day 7) relative to baseline volumes at Day 0. In the control group tumor volumes were 118% (24 h), 145% (48 h) and 339% (Day 7) relative to baseline volumes Day 0. Tumor volume between the treatment and control group was significantly different at Day 7 (P = 0.001). Compared to baseline, [18F]FLT SUVmax was significantly different at 24 h (P<0.001), 48 h (P<0.001) and Day 7 (P<0.001) in the APO866 group. Compared to baseline, [18F]FDG SUVmax was significantly different at Day 7 (P = 0.005) in the APO866 group.

Conclusions

APO866 treatment caused a significant decrease in [18F]FLT uptake 24 and 48 hours after treatment initiation. The early reductions in tumor cell proliferation preceded decrease in tumor volume. The results show the possibility to use [18F]FLT and [18F]FDG to image treatment effect early following treatment with APO866 in future clinical studies.     

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.     

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.     

Mg2+ as an indicator of nutritional status in marine bacteria

Cells maintain an osmotic pressure essential for growth and division, using organic compatible solutes and inorganic ions. Mg²⁺, which is the most abundant divalent cation in living cells, has not been considered an osmotically important solute. Here we show that under carbon limitation or dormancy native marine bacterial communities have a high cellular concentration of Mg²⁺ (370–940 m) and a low cellular concentration of Na⁺ (50–170 m). With input of organic carbon, the average cellular concentration of Mg²⁺ decreased 6–12-fold, whereas that of Na⁺ increased ca 3–4-fold. The concentration of chlorine, which was in the range of 330–1200 m and was the only inorganic counterion of quantitative significance, balanced and followed changes in the concentration of Mg²⁺+Na⁺. In an osmotically stable environment, like seawater, any major shift in bacterial osmolyte composition should be related to shifts in growth conditions, and replacing organic compatible solutes with inorganic solutes is presumably a favorable strategy when growing in carbon-limited condition. A high concentration of Mg²⁺ in cells may also serve to protect and stabilize macromolecules during periods of non-growth and dormancy. Our results suggest that Mg²⁺ has a major role as osmolyte in marine bacteria, and that the [Mg²⁺]/[Na⁺] ratio is related to its physiological condition and nutritional status. Bacterial degradation is a main sink for dissolved organic carbon in the ocean, and understanding the mechanisms limiting bacterial activity is therefore essential for understanding the oceanic C-cycle. The [Mg²⁺]/[Na⁺]-ratio in cells may provide a physiological proxy for the transitions between C-limited and mineral nutrient-limited bacterial growth in the ocean''s surface layer.     

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).     

Salt tolerant sugarbeet progeny from tissue cultures challenged with multiple salts

Lines of sugarbeet (Beta vulgaris L.) tolerant of multiple salts was accomplished by an in vitro multiple salt challenge. Petioles were placed on RV medium amended with 5 different salts along with Murashige and Skoog base salts for one month. Surviving shoots were cultured on RV medium to obtain petioles for subsequent challenges. During the first, second and third challenges, organogenically regenerated shoots developed from 5%, 46%, and 80% of the petioles, respectively. After the third multiple salt challenge, tolerant shoots were rooted and transplanted in soil. Salt was added to this soil at 1.0% by weight and plants were observed for 2 months. The ten most salt tolerant plants were vernalized to obtain seed. The R₁ seed and controls were planted in soil containing 0%, 0.61% or 0.77% multiple salts per dry soil weight. Emergence of R₁ seedlings was significantly greater than the controls under salt stress. Multiple salt tolerant R₁ plants were maintained in salt amended soil to the 8–10 leaf stage and appeared as healthy and vigorous as the control growing in salt free soil.Contribution from Missouri Agricultural Experiment Station Journal Series No. 10948. University of Missouri, Columbia, MO, USA Mention of trade names does not constitute a guarantee or warranty of the product by University of Missouri of Holly Sugar Corporation and does not imply their approval to the exclusion of other products.     

Gastrointestinal Illness among Triathletes Swimming in Non-Polluted versus Polluted Seawater Affected by Heavy Rainfall,Denmark, 2010-2011

Recent years have seen an increase in the frequency of extreme rainfall and subsequent flooding across the world. Climate change models predict that such flooding will become more common, triggering sewer overflows, potentially with increased risks to human health. In August 2010, a triathlon sports competition was held in Copenhagen, Denmark, shortly after an extreme rainfall. The authors took advantage of this event to investigate disease risks in two comparable cohorts of physically fit, long distance swimmers competing in the sea next to a large urban area. An established model of bacterial concentration in the water was used to examine the level of pollution in a spatio-temporal manner. Symptoms and exposures among athletes were examined with a questionnaire using a retrospective cohort design and the questionnaire investigation was repeated after a triathlon competition held in non-polluted seawater in 2011. Diagnostic information was collected from microbiological laboratories. The results showed that the 3.8 kilometer open water swimming competition coincided with the peak of post-flooding bacterial contamination in 2010, with average concentrations of 1.5x10⁴ E. coli per 100 ml water. The attack rate of disease among 838 swimmers in 2010 was 42% compared to 8% among 931 swimmers in the 2011 competition (relative risk (RR) 5.0; 95% CI: 4.0-6.39). In 2010, illness was associated with having unintentionally swallowed contaminated water (RR 2.5; 95% CI: 1.8-3.4); and the risk increased with the number of mouthfuls of water swallowed. Confirmed aetiologies of infection included Campylobacter, Giardia lamblia and diarrhoeagenic E. coli. The study demonstrated a considerable risk of illness from water intake when swimming in contaminated seawater in 2010, and a small but measureable risk from non-polluted water in 2011. This suggests a significant risk of disease in people ingesting small amounts of flood water following extreme rainfall in urban areas.