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Extreme environments, such as Antarctic habitats, present major challenges for many biological processes. Antarctic icefishes (Crynotothenioidea) represent a compelling system to investigate the molecular basis of adaptation to cold temperatures. Here, we explore how the sub-zero habitats of Antarctic icefishes have impacted rhodopsin (RH1) function, the temperature-sensitive dim-light visual pigment found in rod photoreceptors. Using likelihood models and ancestral reconstruction, we find that accelerated evolutionary rates in icefish RH1 underlie unique amino acid mutations absent from other deep-dwelling fishes, introduced before (S160A) and during (V259M) the onset of modern polar conditions. Functional assays reveal that these mutations red-shift rhodopsin spectral absorbance, consistent with spectral irradiance under sea ice. These mutations also lower the activation energy associated with retinal release of the light-activated RH1, and accelerate its return to the dark state, likely compensating for a cold-induced decrease in kinetic rates. These are adaptations in key properties of rhodopsin that mediate rod sensitivity and visual performance in the cold dark seas of the Antarctic. [ABSTRACT FROM AUTHOR]

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infects a broader range of mammalian species than previously predicted, binding a diversity of angiotensin converting enzyme 2 (ACE2) orthologs despite extensive sequence divergence. Within this sequence degeneracy, we identify a rare sequence combination capable of conferring SARS-CoV-2 resistance. We demonstrate that this sequence was likely unattainable during human evolution due to deleterious effects on ACE2 carboxypeptidase activity, which has vasodilatory and cardioprotective functions in vivo. Across the 25 ACE2 sites implicated in viral binding, we identify 6 amino acid substitutions unique to mouse—one of the only known mammalian species immune to SARS-CoV-2. Substituting human variants at these positions is sufficient to confer binding of the SARS-CoV-2 S protein to mouse ACE2, facilitating cellular infection. Conversely, substituting mouse variants into either human or dog ACE2 abolishes viral binding, diminishing cellular infection. However, these same substitutions decrease human ACE2 activity by 50% and are predicted as pathogenic, consistent with the extreme rarity of human polymorphisms at these sites. This trade-off can be avoided, however, depending on genetic background; if substituted simultaneously, these same mutations have no deleterious effect on dog ACE2 nor that of the rodent ancestor estimated to exist 70 million years ago. This genetic contingency (epistasis) may have therefore opened the road to resistance for some species, while making humans susceptible to viruses that use these ACE2 surfaces for binding, as does SARS-CoV-2. This study suggests that ancient events in mammalian cardiovascular evolution determined the host range of SARS-CoV-2 millions of years before the current pandemic. These physiological constraints are so inflexible that escape from SARS-CoV-2 susceptibility would likely have required significant alterations to the human cardiovascular system. [ABSTRACT FROM AUTHOR]

The ubiquitin-proteasome pathway (UPP) and autophagy play integral roles in cellular homeostasis. As part of their normal life cycle, most proteins undergo ubiquitination for some form of redistribution, localization and/or functional modulation. However, ubiquitination is also important to the UPP and several autophagic processes. The UPP is initiated after specific lysine residues of short-lived, damaged or misfolded proteins are conjugated to ubiquitin, which targets these proteins to proteasomes. Autophagy is the endosomal/lysosomal-dependent degradation of organelles, invading microbes, zymogen granules and macromolecules such as protein, carbohydrates and lipids. Autophagy can be broadly separated into three distinct subtypes termed microautophagy, chaperonemediated autophagy and macroautophagy. Although autophagy was once thought of as non-selective bulk degradation, advancements in the field have led to the discovery of several selective forms of autophagy. Here, we focus on the mechanisms of primary and selective mammalian autophagy pathways and highlight the current knowledge gaps in these molecular pathways. [ABSTRACT FROM AUTHOR]

Rhodopsin, the light-sensitive visual pigment expressed in rod photoreceptors, is specialized for vision in dim-light environments. Aquatic environments are particularly challenging for vision due to the spectrally dependent attenuation of light, which can differ greatly in marine and freshwater systems. Among fish lineages that have successfully colonized freshwater habitats from ancestrally marine environments, croakers are known as highly visual benthic predators. In this study, we isolate rhodopsins from a diversity of freshwater and marine croakers and find that strong positive selection in rhodopsin is associated with a marine to freshwater transition in South American croakers. In order to determine if this is accompanied by significant shifts in visual abilities, we resurrected ancestral rhodopsin sequences and tested the experimental properties of ancestral pigments bracketing this transition using in vitro spectroscopic assays. We found the ancestral freshwater croaker rhodopsin is redshifted relative to its marine ancestor, with mutations that recapitulate ancestral amino acid changes along this transitional branch resulting in faster kinetics that are likely to be associated with more rapid dark adaptation. This could be advantageous in freshwater due to the redshifted spectrum and relatively narrow interface and frequent transitions between bright and dim-light environments. This study is the first to experimentally demonstrate that positively selected substitutions in ancestral visual pigments alter protein function to freshwater visual environments following a transition from an ancestrally marine state and provides insight into the molecular mechanisms underlying some of the physiological changes associated with this major habitat transition. [ABSTRACT FROM AUTHOR]

Birds (Aves) display high metabolic rates and oxygen consumption relative to mammals, increasing reactive oxygen species (ROS) formation. Although excess ROS reduces lifespan by causing extensive cellular dysfunction and damage, birds are remarkably long-lived. We address this paradox by identifying the constitutive activation of the NRF2 master antioxidant response in Neoaves (~95% of bird species), providing an adaptive mechanism capable of counterbalancing high ROS levels. We demonstrate that a KEAP1 mutation in the Neoavian ancestor disrupted the repression of NRF2 by KEAP1, leading to constitutive NRF2 activity and decreased oxidative stress in wild Neoaves tissues and cells. Our evidence suggests this ancient mutation induced a compensatory program in NRF2-target genes with functions beyond redox regulation—including feather development—while enabling significant metabolic rate increases that avoid trade-offs with lifespan. The strategy of NRF2 activation sought by intense clinical investigation therefore appears to have also unlocked a massively successful evolutionary trajectory. [ABSTRACT FROM AUTHOR]

This research aims at evaluating the effects of the dietary addition of a supplement containing 75% of chestnut tannins (CT) on growth performance, slaughter traits (Experiment 1) and on nutrient utilisation (Experiment 2) of Italian heavy pigs. Exp. 1 compared a control (C) diet with diets containing 0.15 or 0.30% of CT supplement. Forty-two barrows (91 ± 6 kg of BW) were divided into pairs and kept in 21 partially-slatted pens equipped with individual feeding (7 pens/diet). Animals were slaughtered at a BW of 174 ± 6 kg. The CT inclusion did not modify the performance and the slaughter traits but lowered the intensity of red colour (p <.05) and brightness (p <.05) of the gastric mucosa. Exp. 2 measured digestibility of nutrients, nitrogen (N) balance and energy utilisation of a C diet compared with low protein diets containing 0 or 0.53% of CT supplement (low protein, LP and low protein plus tannins, LPT, respectively). Eighteen barrows (BW 153 ± 4 kg) were housed in 6 metabolic cages in 3 periods of 14 days, with 6 animals per diet. LPT pigs produced less urine than LP (2132 vs. 2561 g/d, p <.05) and both were lower than C (2978 g/d, p <.05). LP and LPT diets had similar digestibility (87.6 vs. 87.8% for DM, and 85.1 vs. 83.9% for CP). N and energy balance were similar among all diets (34.8, 39.0, 39.2% retained N, and 37.6, 35.7, 37.3% retained energy, for C, LP and LPT, respectively). In conclusion, tannins do not exert anti-nutritional effect at the concentrations applied. Dietary chestnut tannin (CT) addition (at 1.5–5.3 g/kg) does not modify digestibility, metabolic use of nutrients and performance of heavy pigs. Dietary CT addition reduces urine volumes of animals with potential impact on farm slurry management. Changes of colour and hypertrophy of gastric mucosa of pigs fed CT are proxy indicators of metabolic reactions to be further investigated. [ABSTRACT FROM AUTHOR]

Bats are excellent models for studying the molecular basis of sensory adaptation. In Chiroptera, a sensory trade-off has been proposed between the visual and auditory systems, though the extent of this association has yet to be fully examined. To investigate whether variation in visual performance is associated with echolocation, we experimentally assayed the dim-light visual pigment rhodopsin from bat species with differing echolocation abilities. While spectral tuning properties were similar among bats, we found that the rate of decay of their light-activated state was significantly slower in a nonecholocating bat relative to species that use distinct echolocation strategies, consistent with a sensory trade-off hypothesis. We also found that these rates of decay were remarkably slower compared with those of other mammals, likely indicating an adaptation to dim light. To examine whether functional changes in rhodopsin are associated with shifts in selection intensity upon bat Rh1 sequences, we implemented selection analyses using codon-based likelihood clade models. While no shifts in selection were identified in response to diverse echolocation abilities of bats, we detected a significant increase in the intensity of evolutionary constraint accompanying the diversification of Chiroptera. Taken together, this suggests that substitutions that modulate the stability of the light-activated rhodopsin state were likely maintained through intensified constraint after bats diversified, being finely tuned in response to novel sensory specializations. Our study demonstrates the power of combining experimental and computational approaches for investigating functional mechanisms underlying the evolution of complex sensory adaptations. [ABSTRACT FROM AUTHOR]

Abstract: Stress‐inducible phosphoprotein 1 (STI1) acts as a neuroprotective factor in the ischemic brain and its levels are increased following ischemia. Previous work has suggested that some of these STI1 actions in a stroke model depend on the recruitment of bone marrow‐derived stem cells to improve outcomes after ischemic insult. However, STI1 can directly increase neuroprotective signaling in neurons by engaging with the cellular prion protein (PrPC) and activating α7 nicotinic acetylcholine receptors (α7nAChR). Given that α7nAChR activation has also been involved in neuroprotection in stroke, it is possible that STI1 can have direct actions on neurons to prevent deleterious consequences of ischemic insults. Here, we tested this hypothesis by exposing primary neuronal cultures to 1‐h oxygen‐glucose deprivation (OGD) and reperfusion and assessing signaling pathways activated by STI1/PrPC. Our results demonstrated that STI1 treatment significantly decreased apoptosis and cell death in mouse neurons submitted to OGD in a manner that was dependent on PrPC and α7nAChR, but also on the activin A receptor 1 (ALK2), which has emerged as a signaling partner of STI1. Interestingly, pharmacological inhibition of the ALK2 receptor prevented neuroprotection by STI1, while activation of ALK2 receptors by bone morphogenetic protein 4 (BMP4) either before or after OGD was effective in decreasing neuronal death induced by ischemia. We conclude that PrPC/STI1 engagement and its subsequent downstream signaling cascades involving α7nAChR as well as the ALK2 receptor may be activated in neurons by increased levels of STI1. This signaling pathway protects neurons from ischemic insults. [ABSTRACT FROM AUTHOR]

Convergent evolution in response to similar selective pressures is a well-known phenomenon in evolutionary biology. Less well understood is how selection drives convergence in protein function, and the underlying mechanisms by which this can be achieved. Here, we investigate functional convergence in the visual system of two distantly related lineages of high-altitude adapted Andean and Himalayan catfishes. Statistical analyses revealed in the two high-altitude lineages, a parallel acceleration of evolutionary rates in rhodopsin, the dim-light visual pigment. However, the elevated rates were found to be accompanied by substitutions at different sites in the protein. Experiments substituting Andean- or Himalayan-specific residues significantly accelerated the kinetic rates of rhodopsin, destabilizing the ligand-bound forms. As found in cold-adapted enzymes, this phenotype likely compensates for a cold-induced decrease in kinetic rates, properties of rhodopsin mediating rod sensitivity and visual performance. Our study suggests that molecular convergence in protein function can be driven by parallel shifts in evolutionary rates but via nonparallel molecular mechanisms. Signatures of natural selection may therefore be a powerful guide for identifying complex instances of functional convergence across a wider range of protein systems. [ABSTRACT FROM AUTHOR]

Cichlids encompass one of the most diverse groups of fishes in South and Central America, and show extensive variation in life history, morphology, and colouration. While studies of visual system evolution in cichlids have focussed largely on the African rift lake species flocks, Neotropical cichlids offer a unique opportunity to investigate visual system evolution at broader temporal and geographic scales. South American cichlid colonization of Central America has likely promoted accelerated rates of morphological evolution in Central American lineages as they encountered reduced competition, renewed ecological opportunity, and novel aquatic habitats. To investigate whether such transitions have influenced molecular evolution of vision in Central American cichlids, we sequenced the dim-light rhodopsin gene in 101 Neotropical cichlid species, spanning the diversity of the clade. We find strong evidence for increased rates of evolution in Central American cichlid rhodopsin relative to South American lineages, and identify several sites under positive selection in rhodopsin that likely contribute to adaptation to different photic environments. We expressed a Neotropical cichlid rhodopsin protein in vitro for the first time, and found that while its spectral tuning properties were characteristic of typical vertebrate rhodopsin pigments, the rate of decay of its active signalling form was much slower, consistent with dim light adaptation in other vertebrate rhodopsins. Using site-directed mutagenesis combined with spectroscopic assays, we found that a key amino acid substitution present in some Central American cichlids accelerates the rate of decay of active rhodopsin, which may mediate adaptation to clear water habitats. [ABSTRACT FROM AUTHOR]

High-altitude environments present a range of biochemical and physiological challenges for organisms through decreases in oxygen, pressure, and temperature relative to lowland habitats. Proteinlevel adaptations to hypoxic high-altitude conditions have been identified in multiple terrestrial endotherms; however, comparable adaptations in aquatic ectotherms, such as fishes, have not been as extensively characterized. In enzyme proteins, cold adaptation is attained through functional trade-offs between stability and activity, often mediated by substitutions outside the active site. Little is known whether signaling proteins [e.g., G protein-coupled receptors (GPCRs)] exhibit natural variation in response to cold temperatures. Rhodopsin (RH1), the temperature-sensitive visual pigment mediating dim-light vision, offers an opportunity to enhance our understanding of thermal adaptation in a model GPCR. Here, we investigate the evolution of rhodopsin function in an Andean mountain catfish system spanning a range of elevations. Using molecular evolutionary analyses and site-directed mutagenesis experiments, we provide evidence for cold adaptation in RH1. We find that unique amino acid substitutions occur at sites under positive selection in high-altitude catfishes, located at opposite ends of the RH1 intramolecular hydrogen-bonding network. Natural high-altitude variants introduced into these sites via mutagenesis have limited effects on spectral tuning, yet decrease the stability of dark-state and light-activated rhodopsin, accelerating the decay of ligand-bound forms. As found in cold-adapted enzymes, this phenotype likely compensates for a cold-induced decrease in kinetic rates--properties of rhodopsin that mediate rod sensitivity and visual performance. Our results support a role for natural variation in enhancing the performance of GPCRs in response to cold temperatures. [ABSTRACT FROM AUTHOR]

Rhodopsin is the visual pigment that mediates dim-light vision in vertebrates and is a model system for the study of retinal disease. The majority of rhodopsin experiments are performed using bovine rhodopsin; however, recent evidence suggests that significant functional differences exist among mammalian rhodopsins. In this study, we identify differences in both thermal decay and light-activated retinal release rates between bovine and human rhodopsin and perform mutagenesis studies to highlight two clusters of substitutions that contribute to these differences. We also demonstrate that the retinitis pigmentosa-associated mutation G51A behaves differently in human rhodopsin compared to bovine rhodopsin and determine that the thermal decay rate of an ancestrally reconstructed mammalian rhodopsin displays an intermediate phenotype compared to the two extant pigments. [ABSTRACT FROM AUTHOR]

All-trans retinoic acid (ATRA) is the only clinically useful differentiating agent, being used in the treatment of acute promyelocytic leukemia (APL). The use of ATRA in other types of acute myelogenous leukemia (AML) calls for the identification of novel strategies aimed at increasing its therapeutic activity. Here, we provide evidence that pharmacological inhibition of the mitogen-activated protein kinase, p38α, or silencing of the corresponding gene sensitizes APL and AML cell lines, as well as primary cultures of AML blasts to the anti-proliferative and cyto-differentiating activity of ATRA and synthetic retinoids. P38α inhibits ligand-dependent transactivation of the nuclear retinoic acid receptor, RARα, and the derived chimeric protein expressed in the majority of APL cases, PML-RARα. Inhibition is the consequence of ligand-independent binding of p38α, which results in stabilization of RARα and PML-RARα via blockade of their constitutive degradation by the proteasome. The inhibitory effect requires a catalytically active p38α and direct physical interaction with RARα and PML-RARα. Ser-369 in the E-region of RARα is essential for the binding of p38α and the ensuing functional effects on the activity of the receptor. [ABSTRACT FROM AUTHOR]

Epidermal growth factor (EGF) and insulin receptor tyrosine kinases (RTKs) exemplify how receptor location is coupled to signal transduction. Extracellular binding of ligands to these RTKs triggers their concentration into vesicles that bud off from the cell surface to generate intracellular signaling endosomes. On the exposed cytosolic surface of these endosomes, RTK autophosphorylation selects the downstream signaling proteins and lipids to effect growth factor and polypeptide hormone action. This selection is followed by the recruitment of protein tyrosine phosphatases that inactivate the RTKs and deliver them by membrane fusion and fission to late endosomes. Coincidentally, proteinases inside the endosome cleave the EGF and insulin ligands. Subsequent inward budding of the endosomal membrane generates multivesicular endosomes. Fusion with lysosomes then results in RTK degradation and downregulation. Through the spatial positioning of RTKs in target cells for EGF and insulin action, the temporal extent of signaling, attenuation, and downregulation is regulated. [ABSTRACT FROM AUTHOR]

The aim was to evaluate the effect of lowprotein (LP) or low-amino acid diets on digestibility, energy and nitrogen (N) utilisation in 2 genetic groups (GG) of pigs (129±11 kg BW). Duroc Large White (A) pigs were chosen to represent a traditional GG for ham production, and Danbred Duroc (D) pigs to represent a GG with fast growing rate and high carcass lean yield. Dietary treatments: a conventional diet (CONV) containing 13.2% CP, and two LP diets, one with LP (10.4%) and low essential AA (LP1), the second with LP (9.7%) and high essential AA (LP2). Compared to CONV, LP2 had the same essential AA content per unit feed, while LP1 the same essential AA content per unit CP. Feed was restricted (DMI=6.8% BW0.75). Four consecutive digestibility/balances periods were conducted with 24 barrows, 12 A and 12 D. Metabolic cages and respiration chambers were used. No significant difference between diets was registered for digestibility. Nitrogen excreted: 41.3, 33.4 and 29.0 g/d (P=0.009), for CONV, LP1 and LP2 diets, respectively. Nitrogen retention was similar between the diets. Heat production (HP) was the lowest for LP diets. There was a tendency (P=0.079) for a lower energy digestibility in D group. The D pigs also had a higher HP and hence a lower retained energy in comparison with the A pigs. In conclusion: it is possible to reduce N excretion using very LP diets and LP low AA diets; Danbred GG have a higher heat production and a lower energy retention than A pigs. [ABSTRACT FROM AUTHOR]

Transforming growth factor ß (TGFß) signaling controls many cellular responses including proliferation, epithelial to mesenchymal transition and apoptosis, through the activation of canonical (Smad) as well as non-canonical (e.g. Par6) pathways. Previous studies from our lab have demonstrated that aPKC inhibition regulates TGFß receptor trafficking and signaling. Here, we report that downstream TGFß-dependent transcriptional responses in aPKC-silenced NSCLC cells were reduced compared with those of control cells, despite a temporal extension of Smad2 phosphorylation. We assessed SARA-Smad2-Smad4 association and observed that knockdown of aPKC increased SARA (also known as ZFYVE9) levels and SARA-Smad2 complex formation, increased cytoplasmic retention of Smad2 and reduced Smad2-Smad4 complex formation, which correlated with reduced Smad2 nuclear translocation. Interestingly, we also detected an increase in p38 MAPK phosphorylation and apoptosis in aPKC-silenced cells, which were found to be TRAF6-dependent. Taken together, our results suggest that aPKC isoforms regulate Smad and non-Smad TGFß pathways and that aPKC inhibition sensitizes NSCLC cells to undergo TGFß-dependent apoptosis. [ABSTRACT FROM AUTHOR]

This paper describes our experience in designing, developing and deploying systems for supporting human–robot teams during disaster response. It is based on R&D performed in the EU-funded project NIFTi. NIFTi aimed at building intelligent, collaborative robots that could work together with humans in exploring a disaster site, to make a situational assessment. To achieve this aim, NIFTi addressed key scientificdesignaspects in building up situation awareness in a human–robot team,developingsystems using a user-centric methodology involving end users throughout the entire R&D cycle, and regularlydeployingimplemented systems under real-life circumstances for experimentation and testing. This has yielded substantial scientific advances in the state-of-the-art in robot mapping, robot autonomy for operating in harsh terrain, collaborative planning, and human–robot interaction. NIFTi deployed its system in actual disaster response activities in Northern Italy, in July 2012, aiding in structure damage assessment. [ABSTRACT FROM AUTHOR]

Epithelial-to-mesenchymal transition (EMT) is controlled by cellular signaling pathways that trigger the loss of cell-cell adhesion and lead to the restructuring of the cell cytoskeleton. Transforming growth factor β (TGF-β) has been shown to regulate cell plasticity through the phosphorylation of Par6 on a conserved serine residue ($345) by the type II TGF-β receptor. We show here that atypical protein kinase C (aPKC) is an essential component to this signaling pathway in non-small-cell lung cancer (NSCLC) cells. We show that the aPKC, PKCh interacts with TGF-β receptors through Par6 and that these proteins localize to the leading edge of migrating cells. Furthermore, Par6 phosphorylation on serine 345 by TGF-β receptors is enhanced in the presence of aPKC. aPKC kinase activity, as well as an association with Par6, were found to be important for Par6 phosphorylation. In effect, small interfering RNA-targeting aPKC reduces TGF-β-induced RhoA and E-cadherin loss, cell morphology changes, stress fiber production, and the migration of NSCLC cells. Interestingly, reintroduction of a phosphomimetic Par6 (Par6-S345E) into aPKC-silenced cells rescues both RhoA and E-cadherin loss with TGF-β stimulation. In conclusion, our results suggest that aPKCs cooperate with TGF-β receptors to regulate phospho-Par6-dependent EMT and cell migration. [ABSTRACT FROM AUTHOR]

β-arrestins play critical roles in chemotaxis and cytoskeletal reorganization downstream of several receptor types, including G protein-coupled receptors (GPCRs), which are targets for greater than 50% of all pharmaceuticals. Among them, receptors for lysophosphatidic acid (LPA), namely LPA1 are overexpressed in breast cancer and promote metastatic spread. We have recently reported that β-arrestin2 regulates LPA1-mediated breast cancer cell migration and invasion, although the underlying molecular mechanisms are not clearly understood. We show here that LPA induces activity of the small G protein, Rap1 in breast cancer cells in a β-arrestin2-dependent manner, but fails to activate Rap1 in non-malignant mammary epithelial cells. We found that Rap1A mRNA levels are higher in human breast tumors compared to healthy patient samples and Rap1A is robustly expressed in human ductal carcinoma in situ and invasive tumors, in contrast to the normal mammary ducts. Rap1A protein expression is also higher in aggressive breast cancer cells (MDA-MB-231 and Hs578t) relative to the weakly invasive MCF-7 cells or non-malignant MCF10A mammary cells. Depletion of Rap1A expression significantly impaired LPA-stimulated migration of breast cancer cells and invasiveness in three-dimensional Matrigel cultures. Furthermore, we found that β-arrestin2 associates with the actin binding protein IQGAP1 in breast cancer cells, and is necessary for the recruitment of IQGAP1 to the leading edge of migratory cells. Depletion of IQGAP1 blocked LPA-stimulated breast cancer cell invasion. Finally, we have identified that LPA enhances the binding of endogenous Rap1A to β-arrestin2, and also stimulates Rap1A and IQGAP1 to associate with LPA1. Thus our data establish novel roles for Rap1A and IQGAP1 as critical regulators of LPA-induced breast cancer cell migration and invasion. [ABSTRACT FROM AUTHOR]

IL-1β-induced anorexia may depend on interactions of the cytokine with neuropeptides and neurotransmitters of the central nervous system; control of energy balance and serotonin is likely to be one catabolic mediator targeted by IL-1β. In the complex interplay involved in feeding modulation, nitric oxide has been ascribed a stimulatory action, which could be of significance in counteracting IL-1β effects. The present study aims to explore the participation of the nitric oxide and the serotonin systems on the central mechanisms induced by IL-1β and the relevance of their putative interactions to IL-1β hypophagia in normal rats. Serotonin levels were determined in microdialysates of the ventromedial hypothalamus after a single intracerebroventricular injection of 10 ng of IL-1β , with or without the pre-injection of 20 μg of the nitric oxide precursor L-arginine. IL-1β significantly stimulated hypothalamic serotonin extracellular levels, with a peak variation of 130 ±37% above baseline. IL- 1β also reduced the 4-h and the 24-h food intakes (by 23% and 58%, respectively). The IL-1β- induced serotonergic activation was abolished by the pre-injection of L-arginine while the hypophagic effect was unaffected. The data showed that one central effect of IL-1β is serotonergic stimulation in the ventromedial hypothalamus, an action inhibited by nitric oxide activity. It is suggested that, although serotonin participates in IL-1β anorexia, other mechanisms recruited by IL-1β in normal rats are able to override the absence of the serotonergic hypophagic influence. [ABSTRACT FROM AUTHOR]

The high cardiovascular risk of HIV infected (HIV+) patients is still partly unexplained. We aimed to evaluate if HIV infection and highly active antiretroviral therapy (HAART) are linked per se to left ventricular (LV) remodelling, independently of blood pressure (BP) values. We enrolled 4 groups of patients matched by gender, age, body mass index and smoking habit: 30 HIV+ hypertensives, 30 HIV+ normotensives, 30 not-infected (HIV−) hypertensives and 30 HIV− normotensives. HIV+ patients were on chronic HAART. Hypertension was newly diagnosed (6 months) and never treated. Each patient underwent blood tests, 24-h BP monitoring and LV echocardiogram. The 4 groups had similar fasting glucose and cholesterol; triglycerides, HOMA index and prevalence of metabolic syndrome were higher in the HIV+ groups. Despite similar 24-h BP values, HIV+ hypertensives had greater LV mass and higher prevalence of preclinical diastolic dysfunction than HIV− hypertensives. Compared to HIV− normotensives, HIV+ normotensives had similar 24-h BP values, but greater LV mass and lower LV diastolic indices, similar to HIV− hypertensives, whose 24-h BP values were higher. Asymptomatic HIV infection and chronic HAART are associated with myocardial hypertrophy and preclinical diastolic dysfunction, independently of BP values. [ABSTRACT FROM AUTHOR]

All-trans retinoic acid (ATRA) is the only clinically useful differentiating agent, being used in the treatment of acute promyelocytic leukemia (APL). The use of ATRA in other types of acute myelogenous leukemia (AML) calls for the identification of novel strategies aimed at increasing its therapeutic activity. Here, we provide evidence that pharmacological inhibition of the mitogen-activated protein kinase, p38α, or silencing of the corresponding gene sensitizes APL and AML cell lines, as well as primary cultures of AML blasts to the anti-proliferative and cyto-differentiating activity of ATRA and synthetic retinoids. P38α inhibits ligand-dependent transactivation of the nuclear retinoic acid receptor, RARα, and the derived chimeric protein expressed in the majority of APL cases, PML-RARα. Inhibition is the consequence of ligand-independent binding of p38α, which results in stabilization of RARα and PML-RARα via blockade of their constitutive degradation by the proteasome. The inhibitory effect requires a catalytically active p38α and direct physical interaction with RARα and PML-RARα. Ser-369 in the E-region of RARα is essential for the binding of p38α and the ensuing functional effects on the activity of the receptor. [ABSTRACT FROM AUTHOR]

Kisspeptins (Kp), peptide products of the Kisspeptin-1 (KISS1) gene are endogenous ligands for a G protein-coupled receptor 54 (GPR54). Previous findings have shown that KISS1 acts as a metastasis suppressor in numerous cancers in humans. However, recent studies have demonstrated that an increase in KISS1 and GPR54 expression in human breast tumors correlates with higher tumor grade and metastatic potential. At present, whether or not Kp signaling promotes breast cancer cell invasiveness, required for metastasis and the underlying mechanisms, is unknown. We have found that kisspeptin-10 (Kp-10), the most potent Kp, stimulates the invasion of human breast cancer MDA-MB-231 and Hs578T cells using Matrigel-coated Transwell chamber assays and induces the formation of invasive stellate structures in threedimensional invasion assays. Furthermore, Kp-10 stimulated an increase in matrix metalloprotease (MMP)-9 activity. We also found that Kp-10 induced the transactivation of epidermal growth factor receptor (EGFR). Knockdown of the GPCR scaffolding protein, b-arrestin 2, inhibited Kp-10-induced EGFR transactivation as well as Kp-10 induced invasion of breast cancer cells via modulation of MMP-9 secretion and activity. Finally, we found that the two receptors associate with each other under basal conditions, and FRET analysis revealed that GPR54 interacts directly with EGFR. The stability of the receptor complex formation was increased upon treatment of cells by Kp-10. Taken together, our findings suggest a novel mechanism by which Kp signaling via GPR54 stimulates breast cancer cell invasiveness. [ABSTRACT FROM AUTHOR]

The TGFβ (transforming growth factor β) pathway is an essential cell signalling pathway that is implicated in both normal developmental processes, such as organogenesis, and pathological disorders, such as cancer and fibrosis. There are three prototypical TβRs (TGFβ receptors): TβRI (TβR type I), TGβRII (TβR type II) and TGFβRIII (TβR type III, also known as betaglycan). Whereas the role of TβRII and TβRI in TGFβ signal propagation has been established, the contribution of TβRIII to TGFβ signalling is less well understood. At the cell surface, TβRI and TβRII receptors can be internalized by clathrin-mediated endocytosis and clathrin-independent membrane-raft-dependent endocytosis. Interestingly, the endocytic route of the receptors plays a direct role in TGFβ-dependent Smad signal transduction; receptors endocytosed via clathrin-mediated endocytosis activate Smad signalling, whereas receptors endocytosed via membrane rafts are targeted for degradation. The objective of the present study was to evaluate the contribution of TβRIII to TβRII and TβRI membrane partitioning, receptor half-life and signalling. Using sucrose-density ultracentrifugation to isolate membrane-raft fractions, we show that TβRIII recruits both TβRII and TβRI to non-raft membrane fractions. Immunofluorescence microscopy analysis demonstrated that overexpression of TβRIII affects intracellular trafficking of TβRII by recruiting TβRII to EEA1 (early endosome antigen 1)- and Rab5-positive early endosomes. Using 125I-labelled TGFβ1 to follow cell-surface receptor degradation we show that overexpression of TβRIII also extends the receptor half-life of the TβRII–TβRI complex. Interestingly, we also show, using a luciferase reporter assay, that TβRIII increases basal TGFβ signalling. As numerous pathologies show aberrant activation of TGFβ signalling, the present study illustrates that TβRIII may represent a novel therapeutic target. [ABSTRACT FROM AUTHOR]

The aim of this study was to carry out two different dose estimation approaches in patients with non-Hodgkin’s lymphoma (NHL) treated with a myeloablative amount of 90Y-labelled ibritumomab tiuxetan (Zevalin®) in an open-label dose escalation study. Twenty-seven patients with relapsed/refractory or de novo high-risk NHL receiving one myeloablative dose of 90Y-ibritumomab tiuxetan followed by tandem stem cell reinfusion were evaluated for dose estimate. The injected activity was 30 MBq/kg in 12 patients and 45 MBq/kg in 15 patients. Dose estimation was performed 1 week prior to 90Y-ibritumomab tiuxetan by injection of 111In-ibritumomab tiuxetan (median activity: 200 MBq). The absorbed dose (D) and the biologically effective dose (BED) were calculated. The absorbed doses per unit activity (Gy/GBq) were [median (range)]: heart wall 4.6 (2.5–9.7), kidneys 5.1 (2.8–10.5), liver 6.1 (3.9–10.4), lungs 2.9 (1.5–6.8), red marrow 1.0 (0.5–1.7), spleen 7.0 (1.5–14.4) and testes 4.9 (2.9–16.7). The absorbed dose (Gy) for the 15 patients treated with 45 MBq/kg were: heart wall 17.0 (8.7–25.4), kidneys 17.1 (7.9–22.4), liver 20.8 (15.4–28.3), lungs 8.1 (5.4–11.4), red marrow 3.1 (2.0–4.0), spleen 26.2 (17.0–35.6) and testes 17.3 (9.0–28.4). At the highest activities the acute haematological toxicity was mild or moderate and of very short duration, and it was independent of the red marrow absorbed dose. No secondary malignancy or treatment-related myelodysplastic syndrome was observed. No non-haematological toxicity (liver, kidney, lung) was observed during a follow-up period of 24–48 months. The use of 45 MBq/kg of 90Y-ibritumomab tiuxetan in association with stem cell autografting resulted in patients being free of toxicity in non-haematological organs. These clinical findings were in complete agreement with our dose estimations, considering both organ doses and BED values. [ABSTRACT FROM AUTHOR]

Cell-surface TGFβ (transforming growth factor β) receptors partition into membrane rafts and the caveolin-positive endocytic compartment by an unknown mechanism. In the present study, we investigated the determinant in the TGFβ type II receptor (TβRII) that is necessary for membrane raft/caveolar targeting. Using subcellular fractionation and immunofluorescence microscopy techniques, we demonstrated that the extracellular domain of TβRII mediates receptor partitioning into raft and caveolin-positive membrane domains. Pharmacological perturbation of glycosylation using tunicamycin or the mutation of Mgat5 [mannosyl(α-1,6)-glycoprotein β-1,6-N-acetylglucosaminyltransferase V] activity interfered with the raft partitioning of TβRII. However, this was not due to the glycosylation state of TβRII, as a non-glycosylated TβRII mutant remained enriched in membrane rafts. This suggested that other cell-surface glycoproteins associate with the extracellular domain of TβRII and direct their partitioning in membrane raft domains. To test this we analysed a GMCSF (granulocyte/macrophage colony-stimulating factor)–TβRII chimaeric receptor, which contains a glycosylated GMCSF extracellular domain fused to the transmembrane and intracellular domains of TβRII. This chimaeric receptor was found to be largely excluded from membrane rafts and caveolin-positive structures. Our results indicate that the extracellular domain of TβRII mediates receptor partitioning into membrane rafts and efficient entrance into caveolin-positive endosomes. [ABSTRACT FROM AUTHOR]

Endocytosis of cell surface receptors is an important regulatory event in signal transduction. The transforming growth factor β (TGF-β) superfamily signals to the Smad pathway through heteromeric Ser-Thr kinase receptors that are rapidly internalized and then downregulated in a ubiquitin-dependent manner. Here we demonstrate that TGF-β receptors internalize into both caveolin- and EEA1-positive vesicles and reside in both lipid raft and non-raft membrane domains. Clathrin-dependent internalization into the EEA1-positive endosome, where the Smad2 anchor SARA is enriched, promotes TGF-β signalling. In contrast, the lipid raft-caveolar internalization pathway contains the Smad7-Smurf2 bound receptor and is required for rapid receptor turnover. Thus, segregation of TGF-β receptors into distinct endocytic compartments regulates Smad activation and receptor turnover. [ABSTRACT FROM AUTHOR]

In the NB4 model of acute promyelocytic leukemia (APL), ATRA, 9-cis retinoic acid (9-cis RA), the pan-RAR and RARαselective agonists, TTNPB and AM580, induce growth inhibition, granulocytic differentiation and apoptosis. By contrast, two RXR agonists, a RARβ agonist and an anti-AP1 retinoid have very limited activity, ATRA- and AM580dependent effects are completely inhibited by RAR antagonistic blockade, while 9-cis RA-induced cell-growth-inhibition and apoptosis are equally inhibited by RAR and RXR antagonists. ATRA, 9-cis RA and AM580 cause upregulation of the mRNAs coding for pro-caspase-1, -7, -8, and -9, which, however, results in increased synthesis of only pro-caspase-1 and -7 proteins. These phenomena are associated with activation of pro-caspase-6, -7 and -8, cytochrome c release from the mitochondria, inversion of Bcl-2/Bax ratio and degradation of PML-RARα. Caspase activation is fundamental for retinoid-induced apoptosis, which is suppressed by the caspase-inhibitor z-VAD. [ABSTRACT FROM AUTHOR]

To reduce the sequelae from CNS irradiation (RT), 16 children younger than 3 years with medulloblastoma-PNET (13 cases) and ependymoma (3 cases) were treated between 1987–1993 according to different postsurgical chemotherapy (CT) programs. None of these patients presented with metastases. Eleven patients were rendered disease-free by surgery, while 5 had residual tumor. Adjuvant therapy depended on patients’ age, postsurgical status and parents’ consent to radiotherapy (RT). Nine of the 16 infants remained alive in continuous complete remission from the first neoplasm (median follow-up 7 years). Three of them had been treated with CT alone and 6 with combined CT+RT (posterior fossa 4, whole CNS 2). Seven patients relapsed a median of 13 months after diagnosis, and all 7 of them died of their disease. Despite the omission of RT in 6 of the 16 patients and administration of only focal RT in 8 of the 16, the outcome of this series was satisfactory. Local failure (in 5/7 patients) was the major problem, despite the high dose of RT used in 2 of these 5. In 4 of 6 evaluable children school performance was satisfactory. One child in whom the entire CNS was irradiated developed glioblastoma multiforme 120 months after the first diagnosis of medulloblastoma. [ABSTRACT FROM AUTHOR]

The reovirus type 3 S1 gene product (type 3 hemagglutinin; HA3) is the viral protein responsible for binding to a mammalian cell-surface receptor. It has been shown that HA3 binding to its receptor inhibits cell growth, even in the continuous presence of serum mitogens. Here, receptor-mediated signal transduction leading to growth arrest was studied after binding with synthetic or recombinant ligands in the absence of viral infection. Receptor ligation caused rapid inactivation of p21ras, a decrease in Raf phosphorylation and in mitogenactivated protein kinase (MAPK) enzymatic activity, and G[sub 1] cell cycle arrest. Transfection and expression of constitutively active v-Has-ras prevented the G[sub 1] arrest, indicating that inactivation of p21ras is causative. Interestingly, v-Has-ras expression also decreased the efficiency of reoviridae replication, suggesting that inactivation of p21ras signals is required at some step of the viral cycle. This study may define new mechanisms regulating cell growth and support the approach of using viral proteins to identify and study cellular receptors. Synthetic receptor ligands with antiproliferative properties may be useful in drug development with the aim of blocking mitosis. [ABSTRACT FROM AUTHOR]

Acquired arachnoid cysts of the spinal cord are uncommon causes of spinal cord compression in the pediatric group. Meningitis, trauma and hemorrhage are considered to be causative or contributing factors. Interestingly, no spinal arachnoid cysts have been reported in patients after subarachnoid hemorrhage or meningitis, conditions expected to cause arachnoid scarring. We describe a child of 1 year and 10 months with thoracic spine trauma with crural paraplegia and anesthesia at level T5 submitted to serial magnetic resonance imagery at 5 days and 18 months, after trauma showing evolution from subarachnoid hemorrhage and adhesions of the arachnoid space to a posterior hyptertensive thoracic intradural arachnoid cyst. [ABSTRACT FROM AUTHOR]

The aim of this study was to establish a conversion factor for assessing the scattered radiation produced during intraoral radiography with a rectangular collimator in areas where the remnant beam is not present. The dose level in the remnant beam will be addressed in another study. A radiological phantom (Rando) in the upright position was exposed to radiation delivered by a Planmeca Intra X-ray unit over 9 exposure geometries (right side of a full mouth series). For each geometry, the scattered radiation dose was measured at 5 locations in the horizontal plane and 4 locations in the vertical plane in front of the patient. The measurements were corrected for background and leakage radiation. An inventory of the distributions of the different geometries with respect to their share of the total workload was created in order to assign the correct weightings of the total ambient scattered radiation for the different geometries. The weightings were used to construct a scattered radiation pattern for a representative mix of exposures. The maximum value found in this pattern was used to arrive at a safe conversion factor. The scattered ambient radiation dose was evenly distributed over the horizontal plane. The scattered ambient radiation dose ranged from 20 to 65% lower in the vertical plane. The conversion factor established for scattered ambient radiation was 0.05 µSv per mAs at 1 meter. This factor is 3-fold lower than the conversion factors reported previously. [ABSTRACT FROM AUTHOR]

Preterm infants are at increased risk of low bone mass. Quantitative ultrasound (GUS) is a non-invasive method for assessment of bone status. The application of QUS in a cohort of ex-preterm infants was investigated. This study suggests that GUS cannot be used as a surrogate for dual-energy x ray absorptiometry. [ABSTRACT FROM AUTHOR]

IL-1β-induced anorexia may depend on interactions of the cytokine with neuropeptides and neurotransmitters of the central nervous system control of energy balance and serotonin is likely to be one catabolic mediator targeted by IL-1β. In the complex interplay involved in feeding modulation, nitric oxide has been ascribed a stimulatory action, which could be of significance in counteracting IL-1β effects.The present study aims to explore the participation of the nitric oxide and the serotonin systems on the central mechanisms induced by IL-1β and the relevance of their putative interactions to IL-1β hypophagia in normal rats.Serotonin levels were determined in microdialysates of the ventromedial hypothalamus after a single intracerebroventricular injection of 10 ng of IL-1β , with or without the pre-injection of 20 μg of the nitric oxide precursor L-arginine. IL-1β significantly stimulated hypothalamic serotonin extracellular levels, with a peak variation of 130 ± 37% above baseline. IL- 1β also reduced the 4-h and the 24-h food intakes (by 23% and 58%, respectively). The IL-1β-induced serotonergic activation was abolished by the pre-injection of L-arginine while the hypophagic effect was unaffected.The data showed that one central effect of IL-1β is serotonergic stimulation in the ventromedial hypothalamus, an action inhibited by nitric oxide activity. It is suggested that, although serotonin participates in IL-1β anorexia, other mechanisms recruited by IL-1β in normal rats are able to override the absence of the serotonergic hypophagic influence. [ABSTRACT FROM AUTHOR]

Preterm infants are at increased risk of low bone mass. Quantitative ultrasound (QUS) is a non-invasive method for assessment of bone status. The application of QUS in a cohort of ex-preterm infants was investigated. This study suggests that QUS cannot be used as a surrogate for dual-energy x ray absorptiometry. [ABSTRACT FROM AUTHOR]

Dietary needs during adolescence lack specific definitions, and most evidence is derived from indirect indications. The data on dietary needs for energy and proteins are mainly extrapolated from subjects in other age-classes. Lipids and carbohydrates are being progressively considered for preventive purposes since the qualitative distribution of saturated and unsaturated fats and slowly and rapidly absorbed carbohydrates, respectively, seems to be associated with metabolic index predictors of degenerative disorders in later stages of life. The recent results of multicentric autoptic studies in young people from the US indicate that the lipoprotein status of the second decade of life is associated with the first raised arterial lesions in the third decade. The evidence of these links needs further confirmation from ongoing surveys. We must recognize that adolescence is a critical period of life, and food fads may deeply change the dietary habits acquired within the familiar group. As nutritionists, we may suggest that all adolescents should be supplied with nutritional support in terms of education, maybe at school, to improve their knowledge of nutrition. Descriptors: adolescence; dietary habits; dietary recommendations; prevention European Journal of Clinical Nutrition (2000) 54, Suppl 1, S7–S10. [ABSTRACT FROM AUTHOR]