Characterization of [3H]Dopamine Uptake Sites and [3H]Cocaine Recognition Sites in Primary Cultures of Mesencephalic Neurons During In Vitro Development

[3H]Dopamine uptake and [3H]cocaine binding sites were studied in primary cultures of ventral mesencephalon from 14-day-old rat embryos. Specific binding sites for [3H]cocaine and [3H]mazindol were detected only in intact cell cultures of ventral mesencephalon, and were absent in sonicated, washed membranes prepared from these cell cultures. [3H]Cocaine was not taken up by the cells through an active transport process because [3H]cocaine binding occurred also at 4 degrees C. Moreover, the possibility of [3H]cocaine entering the cells by passive diffusion and ion trapping was also excluded because extensive washing failed to remove [3H]cocaine from the cells. [3H]Cocaine binding was reduced to 6% of control when cells were permeabilized with streptolysin O (0.2 U/ml, 5 min). Taken together, these results suggest that in cultured mesencephalic neurons, [3H]cocaine may enter the cell by passive diffusion and then be sequestered by a cytosolic compartment that is lost in the process of permeabilization or sonication and washing of membrane preparations. Permeabilization of cultured neurons failed to alter the storage of [3H]dopamine. When cells were permeabilized with streptolysin O (0.2 U/ml; 5 min) after [3H]dopamine was taken up, [3H]dopamine was retained by the cells and did not leak into the incubation medium, indicating that [3H]dopamine was stored in sites that could not pass through the perforated membranes. In contrast, [3H]dopamine uptake into already permeabilized cells was reduced by 33%, suggesting that a cytosolic protein that had leaked out may play a functional role in the uptake process. In contrast to striatal membrane preparations of adult rats, [3H]cocaine binding in intact mesencephalic cell cultures was Na+ independent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Augmented Lung Inflammation Protects against Influenza A Pneumonia

Background

Influenza pneumonia causes high mortality every year, and pandemic episodes kill millions of people. Influenza-related mortality has been variously ascribed to an ineffective host response that fails to limit viral replication, an excessive host inflammatory response that results in lung injury and impairment of gas exchange, or to bacterial superinfection. We sought to determine whether lung inflammation promoted or impaired host survival in influenza pneumonia.

Methods and Findings

To distinguish among these possible causes of influenza-related death, we induced robust lung inflammation by exposing mice to an aerosolized bacterial lysate prior to challenge with live virus. The treatment induced expression of the inflammatory cytokines IL-6 and TNF in bronchoalveolar lavage fluid 8- and 40-fold greater, respectively, than that caused by lethal influenza infection. Yet, this augmented inflammation was associated with striking resistance to host mortality (0% vs 90% survival, p = 0.0001) and reduced viral titers (p = 0.004). Bacterial superinfection of virus infected lungs was not observed. When mice were repeatedly exposed to the bacterial lysate, as would be clinically desirable during an influenza epidemic, there was no tachyphylaxis of the induced viral resistance. When the bacterial lysate was administered after the viral challenge, there was still some mortality benefit, and when ribavirin was added to the aerosolized bacterial lysate, host survival was synergistically improved (0% vs 93.3% survival, p<0.0001).

Conclusions

Together, these data indicate that innate immune resistance to influenza can be effectively stimulated, and suggest that ineffective rather than excessive inflammation is the major cause of mortality in influenza pneumonia.     

Ecosystem restoration with teeth: what role for predators?

Recent advances highlight the potential for predators to restore ecosystems and confer resilience against globally threatening processes, including climate change and biological invasions. However, releasing the ecological benefits of predators entails significant challenges. Here, we discuss the economic, environmental and social considerations affecting predator-driven ecological restoration programmes, and suggest approaches for reducing the undesirable impacts of predators. Because the roles of predators are context dependent, we argue for increased emphasis on predator functionality in ecosystems and less on the identities and origins of species and genotypes. We emphasise that insufficient attention is currently given to the importance of variation in the social structures and behaviours of predators in influencing the dynamics of trophic interactions. Lastly, we outline experiments specifically designed to clarify the ecological roles of predators and their potential utility in ecosystem restoration.     

Gauging a hydrocarbon ruler by an intrinsic exciton probe

The structural basis of lipid acyl-chain selection by membrane-intrinsic enzymes is poorly understood because most integral membrane enzymes of lipid metabolism have proven refractory to structure determination; however, robust enzymes from the outer membranes of gram-negative bacteria are now providing a first glimpse at the underlying mechanisms. The methylene unit resolution of the phospholipid:lipid A palmitoyltransferase PagP is determined by the hydrocarbon ruler, a 16-carbon saturated acyl-chain-binding pocket buried within the transmembrane beta-barrel structure. Substitution of Gly88 lining the floor of the hydrocarbon ruler with Ala or Met makes the enzyme select specifically 15- or 12-carbon saturated acyl chains, respectively, indicating that hydrocarbon ruler depth determines acyl-chain selection. However, the Gly88Cys PagP resolution does not diminish linearly because it selects both 14- and 15-carbon saturated acyl chains. We discovered that an exciton, emanating from a buried Tyr26-Trp66 phenol-indole interaction, is extinguished by a local structural perturbation arising from the proximal Gly88Cys PagP sulfhydryl group. Site-specific S-methylation of the single Cys afforded Gly88Cys-S-methyl PagP, which reasserted both the exciton and methylene unit resolution by specifically selecting 13-carbon saturated acyl chains for transfer to lipid A. Unlike the other Gly88 substitutions, the Cys sulfhydryl group recedes from the hydrocarbon ruler floor and locally perturbs the subjacent Tyr26 and Trp66 aromatic rings. The resulting hydrocarbon ruler expansion thus occurs at the exciton's expense and accommodates an extra methylene unit in the selected acyl chain. The hydrocarbon ruler-exciton juxtaposition endows PagP with a molecular gauge for probing the structural basis of lipid acyl-chain selection in a membrane-intrinsic environment.     

Estimation of Primary Productivity by Chlorophyll a in vivo Fluorescence in Freshwater Phytoplankton

Primary productivity in marine waters is widely estimated by the measurements of ¹⁴C incorporation, the underwater light climate, and the absorption spectra of phytoplankton. In bio-optical models the quantum efficiency of carbon fixation derived from ¹⁴C incorporation rates, the photosynthetically absorbed radiation derived from the underwater light climate, and the phytoplankton absorption spectra are used to calculate time- and depth-integrated primary productivity. Due to the increased sensitivity of commercially available fluorometers, chlorophyll a in vivo fluorescence became a new tool to assess the photosynthetic activity of phytoplankton. Since fluorescence data yield only relative photosynthetic electron transport rates, a direct conversion into absolute carbon fixation rates is not possible. Here, we report a procedure how this problem can be adressed in freshwater phytoplankton. We adapted a marine bio-optical model to the freshwater situation and tested if this model yields realistic results when applied to a hypertrophic freshwater reservoir. Comparison of primary productivity derived from ¹⁴C incorporation to primary productivity derived from Chl a fluorescence showed that the conversion of fluorescence data into carbon fixation rates is still an unsolved problem. Absolute electron transport rates calculated from fluorescence data tend to overestimate primary production. We propose that the observed differences are caused mainly by neglecting the package effect of pigments in phytoplankton cells and by non-carbon related electron flow (e.g., nitrogen fixation). On the other hand, the ¹⁴C incorporation rates can be artificially influenced by "bottle effects", especially near the water surface, where photoinhibition, photorespiration, and Mehler reaction can play a major role.     

Conditioned Medium from Early-Outgrowth Bone Marrow Cells Is Retinal Protective in Experimental Model of Diabetes

Bone marrow-derived cells were demonstrated to improve organ function, but the lack of cell retention within injured organs suggests that the protective effects are due to factors released by the cells. Herein, we tested cell therapy using early outgrowth cells (EOCs) or their conditioned media (CM) to protect the retina of diabetic animal models (type 1 and type 2) and assessed the mechanisms by in vitro study. Control and diabetic (db/db) mice (8 weeks of age) were randomized to receive a unique intravenous injection of 5×10⁵EOCs or 0.25 ml thrice weekly tail-vein injections of 10x concentrated CM and Wystar Kyoto rats rendered diabetic were randomized to receive 0.50 ml thrice weekly tail-vein injections of 10x concentrated CM. Four weeks later, the animals were euthanized and the eyes were enucleated. Rat retinal Müller cells (rMCs) were exposed for 24 h to high glucose (HG), combined or not with EOC-conditioned medium (EOC-CM) from db/m EOC cultures. Diabetic animals showed increase in diabetic retinopathy (DR) and oxidative damage markers; the treatment with EOCs or CM infusions significantly reduced this damage and re-established the retinal function. In rMCs exposed to diabetic milieu conditions (HG), the presence of EOC-CM reduced reactive oxygen species production by modulating the NADPH-oxidase 4 system, thus upregulating SIRT1 activity and deacetylating Lys-310-p65-NFκB, decreasing GFAP and VEGF expressions. The antioxidant capacity of EOC-CM led to the prevention of carbonylation and nitrosylation posttranslational modifications on the SIRT1 molecule, preserving its activity. The pivotal role of SIRT1 on the mode of action of EOCs or their CM was also demonstrated on diabetic retina. These findings suggest that EOCs are effective as a form of systemic delivery for preventing the early molecular markers of DR and its conditioned medium is equally protective revealing a novel possibility for cell-free therapy for the treatment of DR.     

The in vitro synthesis and secretion of alpha-ecdysone by the ring glands of the fly, Sarcophaga bullata.

The in vitro secretory product of larval Sarcophage bullata ring glands has been identified as 2beta, 3beta, 14alpha, 22R, 25-pentahydroxy-5beta-cholest-7-en-6-one (alpha-ecdysone). Mid to late 3rd instar larval ecdysones were isolated and identified as 2beta, 3beta, 14alpha, 20R, 22R, 25-hexahydroxy-5beta-cholest-7-en-6-one (beta-ecdysone) and alpha-ecdysone at a ratio of 27:1. The low level of alpha-ecdysone in vivo, relative to its exclusive in vitro synthesis and secretion by the ring glands, is a function of the very active C20 hydroxylation mechanism in tissues peripheral to the ring gland. The role of alpha-ecdysone as a prohormone in dipteran metamorphosis is discussed.