Altered Lipid Droplet Dynamics in Hepatocytes Lacking Triacylglycerol Hydrolase Expression

Lipid droplets (LDs) form from the endoplasmic reticulum (ER) and grow in size by obtaining triacylglycerols (TG). Triacylglycerol hydrolase (TGH), a lipase residing in the ER, is involved in the mobilization of TG stored in LDs for the secretion of very-low-density lipoproteins. In this study, we investigated TGH-mediated changes in cytosolic LD dynamics. We have found that TGH deficiency resulted in decreased size and increased number of LDs in hepatocytes. Using fluorescent fatty acid analogues to trace LD formation, we observed that TGH deficiency did not affect the formation of nascent LDs on the ER. However, the rate of lipid transfer into preformed LDs was significantly slower in the absence of TGH. Absence of TGH expression resulted in increased levels of membrane diacylglycerol and augmented phospholipid synthesis, which may be responsible for the delayed lipid transfer. Therefore, altered maturation (growth) rather than nascent formation (de novo synthesis) may be responsible for the observed morphological changes of LDs in TGH-deficient hepatocytes.     

Intracellular sodium sensing: SIK1 network,hormone action and high blood pressure

Sodium is the main determinant of body fluid distribution. Sodium accumulation causes water retention and, often, high blood pressure. At the cellular level, the concentration and active transport of sodium is handled by the enzyme Na⁺,K⁺-ATPase, whose appearance enabled evolving primitive cells to cope with osmotic stress and contributed to the complexity of mammalian organisms. Na⁺,K⁺-ATPase is a platform at the hub of many cellular signaling pathways related to sensing intracellular sodium and dealing with its detrimental excess. One of these pathways relies on an intracellular sodium-sensor network with the salt-inducible kinase 1 (SIK1) at its core. When intracellular sodium levels rise, and after the activation of calcium-related signals, this network activates the Na⁺,K⁺-ATPase and expel the excess of sodium from the cytosol. The SIK1 network also mediates sodium-independent signals that modulate the activity of the Na⁺,K⁺-ATPase, like dopamine and angiotensin, which are relevant per se in the development of high blood pressure. Animal models of high blood pressure, with identified mutations in components of multiple pathways, also have alterations in the SIK1 network. The introduction of some of these mutants into normal cells causes changes in SIK1 activity as well. Some cellular processes related to the metabolic syndrome, such as insulin effects on the kidney and other tissues, also appear to involve the SIK1. Therefore, it is likely that this protein, by modulating active sodium transport and numerous hormonal responses, represents a “crossroad” in the development and adaptation to high blood pressure and associated diseases.     

The nucleotide sugar transporters AtUTr1 and AtUTr3 are required for the incorporation of UDP‐glucose into the endoplasmic reticulum,are essential for pollen development and are needed for embryo sac progress in Arabidopsis thaliana

Uridine 5′‐diphosphate (UDP)‐glucose is transported into the lumen of the endoplasmic reticulum (ER), and the Arabidopsis nucleotide sugar transporter AtUTr1 has been proposed to play a role in this process; however, different lines of evidence suggest that another transporter(s) may also be involved. Here we show that AtUTr3 is involved in the transport of UDP‐glucose and is located at the ER but also at the Golgi. Insertional mutants in AtUTr3 showed no obvious phenotype. Biochemical analysis in both AtUTr1 and AtUTr3 mutants indicates that uptake of UDP‐glucose into the ER is mostly driven by these two transporters. Interestingly, the expression of AtUTr3 is induced by stimuli that trigger the unfolded protein response (UPR), a phenomenon also observed for AtUTr1, suggesting that both AtUTr1 and AtUTr3 are involved in supplying UDP‐glucose into the ER lumen when misfolded proteins are accumulated. Disruption of both AtUTr1 and AtUTr3 causes lethality. Genetic analysis showed that the atutr1 atutr3 combination was not transmitted by pollen and was poorly transmitted by the ovules. Cell biology analysis indicates that knocking out both genes leads to abnormalities in both male and female germ line development. These results show that the nucleotide sugar transporters AtUTr1 and AtUTr3 are required for the incorporation of UDP‐glucose into the ER, are essential for pollen development and are needed for embryo sac progress in Arabidopsis thaliana.     

Anticipating future conditions via trajectory sensitivity

Plants are known to be highly responsive to environmental heterogeneity and normally allocate more biomass to organs that grow in richer patches. However, recent evidence demonstrates that plants can discriminately allocate more resources to roots that develop in patches with increasing nutrient levels, even when their other roots develop in richer patches. Responsiveness to the direction and steepness of spatial and temporal trajectories of environmental variables might enable plants to increase their performance by improving their readiness to anticipated resource availabilities in their immediate proximity. Exploring the ecological implications and mechanisms of trajectory-sensitivity in plants is expected to shed new light on the ways plants learn their environment and anticipate its future challenges and opportunities.Key words: Gradient perception, phenotypic plasticity, anticipatory responses, plant behavior, plant learningNatural environments present organisms with myriad challenges of surviving and reproducing under changing conditions.¹ Depending on its extent, predictability and costs, environmental heterogeneity may select for various combinations of genetic differentiation and phenotypic plasticity.^2–6 However, phenotypic plasticity is both limited and costly.⁷ One of the main limitations of phenotypic plasticity is the lag between the perception of the environment and the time the products of the plastic responses are fully operational.⁷ For instance, the developmental time of leaves may significantly limit the adaptive value of their plastic modification due to mismatches between the radiation levels and temperatures prevailing during their development and when mature and fully functional.^8,9 Accordingly, selection is expected to promote responsiveness to cues that bear information regarding the probable future environment.^9,10Indeed, anticipatory responses are highly prevalent, if not universal, amongst living organisms. Whether through intricate cerebral processes, such as in vertebrates, nervous coordination, as in Echinoderms,¹¹ or by relatively rudimentary non-neural processes, such as in plants¹² and bacteria,¹³ accumulating examples suggest that virtually all known life forms are able to not only sense and plastically respond to their immediate environment but also anticipate probable future conditions via environmental correlations.¹⁰Perhaps the best known example of plants'' ability to anticipate future conditions is their responsiveness to spectral red/far-red cues, which is commonly tightly correlated with future probability of light competition.¹⁴ Among others, plants have been shown to respond to cues related to anticipated herbivory^15,16 and nitrogen availability.¹⁷ Imminent stress is commonly anticipated by the perception of a prevailing stress. For example, adaptation to anticipated severe stress was demonstrated to be inducted by early priming by sub-acute drought,¹⁸ root competition¹⁹ and salinity.²⁰Future conditions can also be anticipated by gradient perception: because resource and stress levels are often changing along predictable spatial and temporal trajectories, spatio-temporal dynamics of environmental variables might convey information regarding anticipated growth conditions (Fig. 1). For example, the order of changes in day length, rather than day length itself, are known to assist plants in differentiating fall from spring and thus avoid blooming in the wrong season.²¹ In addition, responsiveness to environmental gradients as such, i.e., sensitivity to the direction and steepness of environmental trajectories, independently from the stationary levels of the same factors, has been demonstrated in higher organisms, such as the perception of acceleration in contrast to velocity;²² and the dynamics of skin temperature in contrast to stationary skin temperature;²³ where the adaptive value of the second-order derivatives of environmental factors is paramount. Similar perception capabilities have also been demonstrated in rudimentary life forms such as bacteria (reviewed in refs. ¹³ and ²⁴) and plants.^25,26 Specifically, perception of environmental trajectories might assist organisms to both anticipate future conditions and better utilize the more promising patches in their immediate environment.^27,28Open in a separate windowFigure 1Trajectory sensitivity in plants. The hypothetical curves depict examples of spatio-temporal trajectories of resource availability, which might be utilized by plants to increase foraging efficiency in newly-encountered patches. When young or early-in-the-season (segment 1–2), plants are expected to allocate more resources to roots that experience the most promising (steepest increases or shallowest decreases) resource availabilities (e.g., allocating more resources to organs in INC-1 than INC-2). In addition, plants are predicted to avoid allocation to roots experiencing decreasing trajectories (DEC, segment 1–2); although temporarily more abundant with resources, such DEC patches are expected to become poorer than alternative patches in the longer run (segment 2–3).²⁹ However, responsiveness to environmental trajectories is only predicted where the expected period of resource uptake is relatively long, e.g., when plants are still active in segment 2–3, a stipulation which might not be fulfilled in e.g., short-living annuals with life span shorter than segment 1–2.In a recent study, Pisum plants have been demonstrated to be sensitive to temporal changes in nutrient availabilities. Specifically, plants allocated greater biomass to roots growing under dynamically-improving nutrient levels than to roots that grew under continuously higher, yet stationary or deteriorating, nutrient availabilities.²⁹ Allocation to roots in poorer patches might seem maladaptive if only stationary nutrient levels are accounted for, and indeed-almost invariably, plants are known to allocate more resources to organs that experience higher (non-toxic) resource levels (reviewed in ref. ³³). Accordingly, the new findings suggest that rather than merely responding to the prevailing nutrient availabilities, root growth and allocation are also responsive to trajectories of nutrient availabilities (Fig. 1).¹⁰Although Shemesh et al.²⁹ demonstrated trajectory-sensitivity of individual roots to temporal gradient of nutrient availabilities, it is likely that this sensitivity helps plants sense spatial gradients, whereby root tips perceive changes in growth conditions as they move through space.³⁴ Interestingly, because the trajectory-sensitivity was observed when whole roots were subjected to changing nutrient levels, it is likely that trajectory sensitivity in roots is based on the integration of sensory inputs perceived by yet-to-be-determined parts of the root over time, i.e., temporal sensitivity/memory (e.g. reviewed in ref. ³⁵), rather than on the integration of sensory inputs at different locations on the same individual roots (i.e., spatial sensitivity).Besides the direction of change, it is hypothesized that plants are also sensitive to the steepness of environmental trajectories (Fig. 1). This might be especially crucial in short-living annuals, which are expected to only be responsive to trajectories steep enough to be indicative of changes in growth conditions before the expected termination of the growth season (Fig. 1).Studying responsiveness to environmental variability is pivotal for understanding the ecology and evolution of any living organism. However, until recently most attention has been given to the study of responses to stationary spatial and temporal heterogeneities in growth conditions. Exploring the ecological implications and mechanisms of trajectory sensitivity in plants is expected to shed new light on the ways plants learn their immediate environment and anticipate its future challenges and opportunities.     

A Unified Approach to Multi‐item Reliability

Summary The reliability of multi‐item scales has received a lot of attention in the psychometric literature, where a myriad of measures like the Cronbach's α or the Spearman–Brown formula have been proposed. Most of these measures, however, are based on very restrictive models that apply only to unidimensional instruments. In this article, we introduce two measures to quantify the reliability of multi‐item scales based on a more general model. We show that they capture two different aspects of the reliability problem and satisfy a minimum set of intuitive properties. The relevance and complementary value of the measures is studied and earlier approaches are placed in a broader theoretical framework. Finally, we apply them to investigate the reliability of the Positive and Negative Syndrome Scale, a rating scale for the assessment of the severity of schizophrenia.     

An abelisaurid humerus from the Upper Cretaceous of India

The Lameta Formation (Upper Cretaceous, Maastrichtian) of India has yielded abundant fossils of abelisaurid theropods, including bones from the cranium, vertebral column, pectoral and pelvic girdles, and hindlimb. However, the forelimbs of Indian abelisaurids remain unknown. Here we describe an abelisaurid humerus from exposure of the Lameta Formation near the village of Rahioli in northwestern India. This new material exhibits derived traits that are distinctive of Abelisauridae, for example an articular head that is hemispherical in proximal view, thus establishing the specimen as the first abelisaurid humerus from India.     

The kinetic parameters and energy cost of the Hsp70 chaperone as a polypeptide unfoldase

Hsp70-Hsp40-NEF and possibly Hsp100 are the only known molecular chaperones that can use the energy of ATP to convert stably pre-aggregated polypeptides into natively refolded proteins. However, the kinetic parameters and ATP costs have remained elusive because refolding reactions have only been successful with a molar excess of chaperones over their polypeptide substrates. Here we describe a stable, misfolded luciferase species that can be efficiently renatured by substoichiometric amounts of bacterial Hsp70-Hsp40-NEF. The reactivation rates increased with substrate concentration and followed saturation kinetics, thus allowing the determination of apparent V(max)' and K(m)' values for a chaperone-mediated renaturation reaction for the first time. Under the in vitro conditions used, one Hsp70 molecule consumed five ATPs to effectively unfold a single misfolded protein into an intermediate that, upon chaperone dissociation, spontaneously refolded to the native state, a process with an ATP cost a thousand times lower than expected for protein degradation and resynthesis.     

The plasma membrane Ca2+ pump isoform 4a differs from isoform 4b in the mechanism of calmodulin binding and activation kinetics: implications for Ca2+ signaling

The inhibition by the regulatory domain and the interaction with calmodulin (CaM) vary among plasma membrane calcium pump (PMCA) isoforms. To explore these differences, the kinetics of CaM effects on PMCA4a were investigated and compared with those of PMCA4b. The maximal apparent rate constant for CaM activation of PMCA4a was almost twice that for PMCA4b, whereas the rates of activation for both isoforms showed similar dependence on Ca2+. The inactivation of PMCA4a by CaM removal was also faster than for PMCA4b, and Ca2+ showed a much smaller effect (2- versus 30-fold modification). The rate constants of the individual steps that determine the overall rates were obtained from stopped-flow experiments in which binding of TA-CaM was observed by changes in its fluorescence. TA-CaM binds to two conformations of PMCA4a, an "open" conformation with high activity, and a "closed" one with lower activity. Compared with PMCA4b (Penheiter, A. R., Bajzer, Z., Filoteo, A. G., Thorogate, R., T?r?k, K., and Caride, A. J. (2003) Biochemistry 41, 12115-12124), the model for PMCA4a predicts less inhibition in the closed form and a much faster equilibrium between the open and closed forms. Based on the available kinetic parameters, we determined the constants to fit the shape of a Ca2+ signal in PMCA4b-overexpressing Chinese hamster ovary cells. Using the constants for PMCA4a, and allowing small variations in parameters of other systems contributing to a Ca2+ signal, we then simulated the effect of PMCA4a on the shape of a Ca2+ signal in Chinese hamster ovary cells. The results reproduce the published data (Brini, M., Coletto, L., Pierobon, N., Kraev, N., Guerini, D., and Carafoli, E. (2003) J. Biol. Chem. 278, 24500-24508), and thereby demonstrate the importance of altered regulatory kinetics for the different functional properties of PMCA isoforms.