Characterization by photoaffinity labeling of a steroid binding protein in rat liver plasma membrane

Summary The mechanism of steroid uptake by the cell remains controversial. [³H]R5020 was utilized to characterize by photoaffinity labeling the steroid binding site in plasma membrane. This binding was saturable, reversible and had one type of binding site (K _d = 33 ± 4 nm, B _max = 32 ± 2 pmol/mg). [³H]R5020 could be prevented from binding by a variety of steroids (cortisol, progesterone, deoxycorticosterone, and levonorgestrel); estradiol did not have affinity for this binding site. The kinetics of R5020 photoactivation was time dependent and saturable. SDS-PAGE showed a specific band which corresponded to a 53-kDa peptide. The sucrose density gradient analysis has revealed the existence of a protein with a sedimentation coefficient of 3.6 ± 0.2 S. This polypeptide shows different characteristics than cytosolic steroid receptor or serum steroid binding proteins. This binding protein could correspond to the steroid binding site previously found in the plasma membrane.This work was supported by grants PB85-0461 from the Comisión Asesora de Investigatión Científica y Técnica and PGV-8612 from the Departamento de Educatión, Universidades e Investigation del Gobierno Vasco. We thank Roussel-Uclaf (France) for the nonradioactive RU-steroids kindly provided.     

Cell suspension cultures ofOnonis natrix L. subspeciesramosissima: Establishment and culture conditions

Summary Explants from petioles, folioles or hypocotyls ofOnonis natrix have been used for calli initiation. Hypocotyls inoculated on MS medium supplemented with 2% sucrose and 0.5 mg.1^–1 2,4-D / 1 mg.1^–1 Kin showed to be the best primary explant. Cell suspension cultures were established in MS basal medium supplemented with 2% sucrose, 0.5 mg.1^–1 NAA or 2,4-D and 1 mg.1^–1 Kin. Different subculturing periods, inoculum density, hormonal supplementation and sucrose concentration were assayed in order to obtain the best culture growth conditions. The optimal conditions were achieved with cultures initiated with 40 g.1^–1 of initial inoculum, growing in MS basal medium supplemented with 4% sucrose, 0.5 mg.1^–1 NAA and 1 mg.1^–1 Kin subcultured every twelve days. Under these experimental conditions, the cultures showed a doubling time of 36.3 hours.     

Anoxic survival of the isolated cerebellum of the turtle Pseudemis scripta elegans

Intact turtle brain provides a useful model for the study of anoxia and potential survival strategies, since this tissue maintains transmembrane ion gradients and ATP levels during prolonged anoxia and recovers functional activity afterwards. Since isolated tissues offer experimental advantages, the present study sought to determine effects of anoxia on the isolated turtle cerebellum and to define relationships between anoxia survival and glucose supply. In normoxia, the extracellular potassium ([K+]o) activity and evoked potentials were maintained with 5 mM glucose, but 20 mM glucose was required to maintain adenosine triphosphate (ATP) levels and prevent significant increases in [K+]o during anoxia. Inhibition of glycolysis by iodoacetic acid (IAA) during anoxia provoked large increases in [K+]o at all glucose levels. These results demonstrate the usefulness of the isolated turtle cerebellum for studies of anoxic survival since this tissue can maintain ATP levels and [K+]o during prolonged anoxia with 20 mM glucose in the artificial cerebrospinal fluid medium. They also suggest the presence of a Pasteur effect at least during the transition to a hypometabolic state.     

Some characteristics of Ca2+ uptake by yeast cells

Summary Experiments were performed to obtain information on: (i) the specific properties of Ca²⁺ binding and transport in yeast (ii) the relationship between both parameters; (iii) similarities to or differences from other biological systems as measured by the effects of inhibitors; and (iv) the effects of mono and divalent cations, in order to get some insight on the specificity and some characteristics of the mechanism of the transport system for divalent cations in yeast.The results obtained gave some kinetic parameters for a high affinity system involved in the transport of Ca²⁺ in yeast. These were obtained mainly by considering actual concentrations of Ca²⁺ in the medium after substracting the amounts bound to the cell. Ak _m of 1.9 m and aV _max of 1.2 nmol (100 mg·3 min)^–1 were calculated.The effects of some inhibitors and other cations on Ca²⁺ uptake allow one to postulate some independence between binding and transport for this divalent cation.Of the inhibitors tested, only lanthanum seems to be a potent inhibitor of Ca²⁺ uptake in yeast.The effects of Mg²⁺ on the uptake of Ca²⁺ agree with the existence of a single transport system for both divalent cations.The actions of Na⁺ and K⁺ on the transport of Ca²⁺ offer interesting possibilities to study further some of the mechanistic properties of this transport system for divalent cations.     

Subsurface cisterns in paraventricular nuclei of the hypothalamus of the rat

Summary Structures identified as subsurface cisterns (SSC's) were found in neurons of the paraventricular nuclei of the rat hypothalamus. They appeared as cytoplasmic organelles consisting most often of stacks of parallel cisterns apposed to the neuronal plasmalemma. These SSC's were located in the interneurons of the parvocellular system, but not in neurosecretory cells and glial cells. SSC's were seen at zones of cytoplasm apposed to neuronal or glial cell processes, showing in some instances specific relationships with synaptic areas.The morphological features of these SSC's are described, and their possible functional significance is briefly discussed.     

Effect of maternal diet on fetal hepatic lipogenesis.

The effects of: a, maternal diet; b, cyclic-3',5'-adenosinemonophosphate (cyclic AMP) and c, clofibrate on hepatic lipogenesis in fetal rats were studied. The experimental diets contained 22% protein, 40--50% carbohydrate, adequate vitamins, and minerals. In addition, the fat-containing diets were supplemented with either 15% corn oil, 25% corn oil, or 5% cholesterol + 10% oleic acid. In the clofibrate feeding studies, 0.3% (w/v) of the ethyl ester was added to a stock ration or to fat-free diet. Lipogenesis was measured in liver slices incubated with [2-14C]pyruvate, [1-14C]acetate, or 3H2O. In addition, activities of lipogenic enzymes were measured in cytosol fractions from liver homogenates. The effec-s of the experimental diets on liver composition were also examined. Lipogenic activity was higher in fetal than in maternal liver. When 15% corn oil was added to the maternal diet, fatty acid synthesis in fetal liver did not decrease as it did in maternal liver. Maternal fasting decreased fetal fatty acid synthesys by 50% when measured with 14C and less than 10% when measured with 3H2O. Although the addition of cholesterol to the maternal diet decreased cholesterol synthesis in maternal liver, no such decrease was observed in fetal liver. Changes in enzyme activities paralleled alterations in lipogenesis in maternal but not in fetal liver. Corn oil feeding or fasting increased the rate of transfer of linoleate from the dam to the fetus. However, accumulation of linoleate in fetal liver did not correlate with a decreased rate of fatty acid synthesis as it did in maternal liver. Maternal hepatic glycogen stores were depleted by fasting, but glycogen levels in fetal liver remained high under these conditions.     

Comparing sources of mobility for modelling the epidemic spread of Zika virus in Colombia

Timely, accurate, and comparative data on human mobility is of paramount importance for epidemic preparedness and response, but generally not available or easily accessible. Mobile phone metadata, typically in the form of Call Detail Records (CDRs), represents a powerful source of information on human movements at an unprecedented scale. In this work, we investigate the potential benefits of harnessing aggregated CDR-derived mobility to predict the 2015-2016 Zika virus (ZIKV) outbreak in Colombia, when compared to other traditional data sources. To simulate the spread of ZIKV at sub-national level in Colombia, we employ a stochastic metapopulation epidemic model for vector-borne diseases. Our model integrates detailed data on the key drivers of ZIKV spread, including the spatial heterogeneity of the mosquito abundance, and the exposure of the population to the virus due to environmental and socio-economic factors. Given the same modelling settings (i.e. initial conditions and epidemiological parameters), we perform in-silico simulations for each mobility network and assess their ability in reproducing the local outbreak as reported by the official surveillance data. We assess the performance of our epidemic modelling approach in capturing the ZIKV outbreak both nationally and sub-nationally. Our model estimates are strongly correlated with the surveillance data at the country level (Pearson’s r = 0.92 for the CDR-informed network). Moreover, we found strong performance of the model estimates generated by the CDR-informed mobility networks in reproducing the local outbreak observed at the sub-national level. Compared to the CDR-informed networks, the performance of the other mobility networks is either comparatively similar or substantially lower, with no added value in predicting the local epidemic. This suggests that mobile phone data captures a better picture of human mobility patterns. This work contributes to the ongoing discussion on the value of aggregated mobility estimates from CDRs data that, with appropriate data protection and privacy safeguards, can be used for social impact applications and humanitarian action.     

The status of dwarf carnivores on Cozumel Island, Mexico

Cozumel Island in the Mexican Caribbean is inhabited by four carnivores, of which two, the Cozumel coati Nasua nelsoni and pygmy raccoon Procyon pygmaeus, are endemic species. The taxonomic status of a third carnivore, a dwarf gray fox Urocyon cinereoargenteus, is undetermined, but may deserve subspecific or species-level recognition. The fourth species, the kinkajou (Potos flavus), may be a recent introduction. We review the status of these carnivores, report our field observations and results of line transect and trapping efforts, discuss current threats to these taxa, and make recommendations for their conservation. A population density of 0.43 ± 0.27 coatis/km², and a total island population size of 150 ± 95 individuals, was estimated from 386 km of line transects in 1994–1995. Intensive trapping efforts (1479 trap-nights) in 2001 at multiple localities were unsuccessful. Pygmy raccoons were observed in the mangrove and coastal wetland areas of the island and in 2001 we captured 11 individuals with the same sampling efforts as for coatis (8.8 raccoons/1000 trap-nights). The gray fox is also apparently very rare on the island. While a few observations of the animals have been made (1984, 1994 and 2001), no animals were seen along transects and none were trapped. The primary threats to the persistence of these taxa include introduced congeners, introduced predators, parasite and disease spill-over from exotic animals, habitat fragmentation, hunting and collection as pets, and hurricanes. We suggest that the Cozumel coati, pygmy raccoon, and the Cozumel population of the gray fox be considered as Critically Endangered according to the IUCN classification system. Current conservation actions focusing on Cozumel carnivores are extremely limited. We recommend eradication of introduced species, maintenance of habitat connectivity, ex situ conservation programs, explicit public policies on land-use and sustainable development, public awareness campaigns, and continuous scientific research and monitoring.     

IL17A augments autophagy in Mycobacterium tuberculosis-infected monocytes from patients with active tuberculosis in association with the severity of the disease

During mycobacterial infection, macroautophagy/autophagy, a process modulated by cytokines, is essential for mounting successful host responses. Autophagy collaborates with human immune responses against Mycobacterium tuberculosis (Mt) in association with specific IFNG secreted against the pathogen. However, IFNG alone is not sufficient to the complete bacterial eradication, and other cytokines might be required. Actually, induction of Th1 and Th17 immune responses are required for protection against Mt. Accordingly, we showed that IL17A and IFNG expression in lymphocytes from tuberculosis patients correlates with disease severity. Here we investigate the role of IFNG and IL17A during autophagy in monocytes infected with Mt H37Rv or the mutant MtΔRD1. Patients with active disease were classified as high responder (HR) or low responder (LR) according to their T cell responses against Mt. IL17A augmented autophagy in infected monocytes from HR patients through a mechanism that activated MAPK1/ERK2-MAPK3/ERK1 but, during infection of monocytes from LR patients, IL17A had no effect on the autophagic response. In contrast, addition of IFNG to infected monocytes, increased autophagy by activating MAPK14/p38 α both in HR and LR patients. Interestingly, proteins codified in the RD1 region did not interfere with IFNG and IL17A autophagy induction. Therefore, in severe tuberculosis patients' monocytes, IL17A was unable to augment autophagy because of a defect in the MAPK1/3 signaling pathway. In contrast, both IFNG and IL17A increased autophagy levels in patients with strong immunity to Mt, promoting mycobacterial killing. Our findings might contribute to recognize new targets for the development of novel therapeutic tools to fight the pathogen.