A prelysosomal compartment sequesters membrane-impermeant fluorescent dyes from the cytoplasmic matrix of J774 macrophages

After the membrane impermeant dye Lucifer Yellow is introduced into the cytoplasmic matrix of J774 cells, the dye is sequestered within cytoplasmic vacuoles and secreted into the extracellular medium. In the present work we studied the intracellular transport of Lucifer Yellow in J774 macrophages and the nature of the cytoplasmic vacuoles into which this dye is sequestered. When the lysosomal system of J774 cells was prelabeled with a Texas red ovalbumin conjugate and Lucifer Yellow was then loaded into the cytoplasm of the cells by ATP-mediated permeabilization of the plasma membrane, the vacuoles that sequestered Lucifer Yellow 30 min later were distinct from the Texas red-stained lysosomes. After an additional 30 min Lucifer Yellow and Texas red colocalized in the same membrane bound compartments, indicating that the Lucifer Yellow had been delivered to lysosomes. We next prelabeled the plasma membrane of J774 cells with anti-macrophage antibody and Texas red protein A before Lucifer Yellow was loaded into the cells. The phase-lucent vacuoles that subsequently sequestered Lucifer Yellow also stained with Texas red, showing that they were part of the endocytic pathway. J774 cells were fractionated on percoll density gradients either 15 or 60 min after Lucifer Yellow was introduced into the cytoplasmic matrix of the cells. In cells fractionated after 15 min, Lucifer Yellow was contained within the fractions of light buoyant density that contain plasma membrane and endosomes; the dye later appeared in vesicles of higher density which contained lysosomes. Secretion of Lucifer Yellow from the cytoplasmic matrix of J774 cells is inhibited by the organic anion transport blocker probenecid. We found that probenecid also reversibly inhibited sequestration of dye, indicating that sequestration of dye within cytoplasmic vacuoles was also mediated by organic anion transporters. These studies show that the vacuoles that sequester Lucifer Yellow from the cytoplasmic matrix of J774 cells possess the attributes of endosomes. Thus, in addition to their role in sorting of membrane bound and soluble substances, macrophage endosomes may play a role in the accumulation and transport of molecules resident in the soluble cytoplasm

Macrophages possess probenecid-inhibitable organic anion transporters that remove fluorescent dyes from the cytoplasmic matrix

We introduced several membrane-impermeant fluorescent dyes, including Lucifer Yellow, carboxyfluorescein, and fura-2, into the cytoplasmic matrix of J774 cells and thioglycollate-elicited mouse peritoneal macrophages by ATP permeabilization of the plasma membrane and observed the subsequent fate of these dyes. The dyes did not remain within the cytoplasmic matrix; instead they were sequestered within phase-lucent cytoplasmic vacuoles and released into the extracellular medium. We used Lucifer Yellow to study these processes further. In cells incubated at 37 degrees C, 87% of Lucifer Yellow was released from the cells within 30 min after dye loading. The dye that remained within the cells at this time was predominantly within cytoplasmic vacuoles. Lucifer yellow transport was temperature dependent and occurred against a concentration gradient; therefore it appeared to be an energy-requiring process. The fluorescent dyes used in these studies are all organic anions. We therefore examined the ability of probenecid (p-[dipropylsulfamoyl]benzoic acid), which blocks organic anion transport across many epithelia, to inhibit efflux of Lucifer Yellow, and found that this drug inhibited this process in a dose-dependent and reversible manner. Efflux of Lucifer Yellow from the cells did not require Na+ co-transport or Cl- antiport; however, it was inhibited by lowering of the extracellular pH. These experiments indicate that macrophages possess probenecid-inhibitable transporters which are similar in their functional properties to organic anion transporters of epithelial cells. Such organic anion transporters have not been described previously in macrophages; they may mediate the release of naturally occurring organic anions such as prostaglandins, leukotrienes, glutathione, bilirubin, or lactate from macrophages

Firm finances, weather derivatives and geography

This paper considers some intellectual, practical and political dimensions of collaboration between human and physical geographers exploring how firms are using relatively new financial products – weather derivatives – to displace any costs of weather-related uncertainty and risk. The paper defines weather derivatives and indicates how they differ from weather insurance products before considering the geo-political, cultural and economic context for their creation. The paper concludes by reflecting on the challenges of research collaboration across the human–physical geography divide and suggests that while such initiatives may be undermined by a range of institutional and intellectual factors, conversations between physical and human geographers remain and are likely to become increasingly pertinent. The creation of a market in weather derivatives raises a host of urgent political and regulatory questions and the confluence of natural and social knowledges, co-existing within and through the geography academy, provides a constructive and creative basis from which to engage with this new market and wider discourses of uneven economic development and climate change

Explaining Myanmar's Regime Transition: The Periphery is Central

In 2010, Myanmar (Burma) held its first elections after 22 years of direct military rule. Few compelling explanations for this regime transition have emerged. This article critiques popular accounts and potential explanations generated by theories of authoritarian ‘regime breakdown’ and ‘regime maintenance’. It returns instead to the classical literature on military intervention and withdrawal. Military regimes, when not terminated by internal factionalism or external unrest, typically liberalise once they feel they have sufficiently addressed the crises that prompted their seizure of power. This was the case in Myanmar. The military intervened for fear that political unrest and ethnic-minority separatist insurgencies would destroy Myanmar’s always-fragile territorial integrity and sovereignty. Far from suddenly liberalising in 2010, the regime sought to create a ‘disciplined democracy’ to safeguard its preferred social and political order twice before, but was thwarted by societal opposition. Its success in 2010 stemmed from a strategy of coercive state-building and economic incorporation via ‘ceasefire capitalism’, which weakened and co-opted much of the opposition. Having altered the balance of forces in its favour, the regime felt sufficiently confident to impose its preferred settlement. However, the transition neither reflected total ‘victory’ for the military nor secured a genuine or lasting peace

Inhibition of adenosine monophosphate-activated protein kinase-3-hydroxy-3-methylglutaryl coenzyme a reductase signaling leads to hypercholesterolemia and promotes hepatic steatosis and insulin resistance

Adenosine monophosphate–activated protein kinase (AMPK) regulates multiple signaling pathways involved in glucose and lipid metabolism in response to changes in hormonal and nutrient status. Cell culture studies have shown that AMPK phosphorylation and inhibition of the rate‐limiting enzyme in the mevalonate pathway 3‐hydroxy‐3‐methylglutaryl (HMG) coenzyme A (CoA) reductase (HMGCR) at serine‐871 (Ser871; human HMGCR Ser872) suppresses cholesterol synthesis. In order to evaluate the role of AMPK‐HMGCR signaling in vivo, we generated mice with a Ser871‐alanine (Ala) knock‐in mutation (HMGCR KI). Cholesterol synthesis was significantly suppressed in wild‐type (WT) but not in HMGCR KI hepatocytes in response to AMPK activators. Liver cholesterol synthesis and cholesterol levels were significantly up‐regulated in HMGCR KI mice. When fed a high‐carbohydrate diet, HMGCR KI mice had enhanced triglyceride synthesis and liver steatosis, resulting in impaired glucose homeostasis. Conclusion: AMPK‐HMGCR signaling alone is sufficient to regulate both cholesterol and triglyceride synthesis under conditions of a high‐carbohydrate diet. Our findings highlight the tight coupling between the mevalonate and fatty acid synthesis pathways as well as revealing a role of AMPK in suppressing the deleterious effects of a high‐carbohydrate diet

Modelling environmental changes and effects on wild-caught species in Queensland. Environmental drivers.

We report on the findings of a collaborative research project that was designed to identify and measure the effects of environmental drivers on the abundance and population dynamics of key Queensland fishery species. The project was co-funded by the Commonwealth Government’s Fisheries Research and Development Corporation (FRDC) and carried out by a multi-disciplinary team of scientists from the University of Queensland (UQ), the Queensland Department of Agriculture and Fisheries (DAF) and the Australian Institute of Marine Science (AIMS). The research team applied modern statistical, data science and modelling techniques in combination with biological insights into the life cycles of the three target species. Background With increasing evidence that environmental conditions in the marine environment are changing rapidly, it is becoming ever more important to understand how these changes may impact on the population dynamics and abundance of important fish stocks. Understanding the influence of environmental conditions can provide greater certainty that the risk of overfishing (under adverse environmental conditions) or under harvesting (under favourable conditions) are accounted for by resource managers. This project aimed to identify the environmental factors which may be influencing the recruitment, catchability or productivity of Snapper, Pearl Perch, and Spanner Crab stocks in Queensland. Results from this work will support sustainable management of Queensland’s fisheries by directly informing the assessment and management of these key species within Queensland waters

The efficacy of a daily self-weighing weight loss intervention using smart scales and email

ObjectiveTo examine the impact of a weight loss intervention that focused on daily self-weighing for self-monitoring as compared to a delayed control group among 91 overweight adults.Design and MethodsThe 6-month intervention included a cellular-connected “smart” scale for daily weighing, web-based weight loss graph, and weekly emails with tailored feedback and lessons. An objective measure of self-weighing frequency was obtained. Weight was measured in clinic at 3 and 6 months. Caloric intake and expenditure, and perceptions of daily self-weighing were also measured.ResultsUsing intent-to-treat analyses, the intervention group lost significantly more weight compared to the control group [Mean (95%CI); 3 months: −4.41%(−5.5, −3.3) vs. −0.37%(−1.5, .76); 6 months: −6.55%(−7.7, −5.4) vs. −0.35%(−1.5, .79); group×time interaction: p<.001] and a greater percentage achieved 5% (42.6% vs. 6.8%; p<.0001) and 10% (27.7% vs. 0%; p<.0001) weight loss. On average, the intervention group self-weighed more days/week (6.1±1.1 vs. 1.1±1.5; p<.0001) and consumed fewer calories/day compared to the control group [Mean (95% CI); 6 months: 1509 (1291,1728) vs. 1856 (1637,2074); group×time interaction: p=.006]. Among intervention participants, daily self-weighing was perceived positively.ConclusionsThese results indicate that an intervention focusing on daily self-weighing can produce clinically significant weight loss

Daily Self-Weighing and Adverse Psychological Outcomes: A Randomized Controlled Trial

Despite evidence that daily weighing is an effective strategy for weight control, concerns remain regarding the potential for negative psychological consequences