Importance of Membrane Structural Integrity for RPE65 Retinoid Isomerization Activity

Regeneration of visual chromophore in the vertebrate visual cycle involves the retinal pigment epithelium-specific protein RPE65, the key enzyme catalyzing the cleavage and isomerization of all-trans-retinyl fatty acid esters to 11-cis-retinol. Although RPE65 has no predicted membrane spanning domains, this protein predominantly associates with microsomal fractions isolated from bovine retinal pigment epithelium (RPE). We have re-examined the nature of RPE65 interactions with native microsomal membranes by using extraction and phase separation experiments. We observe that hydrophobic interactions are the dominant forces that promote RPE65 association with these membranes. These results are consistent with the crystallographic model of RPE65, which features a large lipophilic surface that surrounds the entrance to the catalytic site of this enzyme and likely interacts with the hydrophobic core of the endoplasmic reticulum membrane. Moreover, we report a critical role for phospholipid membranes in preserving the retinoid isomerization activity and physical properties of RPE65. Isomerase activity measured in bovine RPE was highly sensitive to phospholipase A² treatment, but the observed decline in 11-cis-retinol production did not directly reflect inhibition by products of lipid hydrolysis. Instead, a direct correlation between the kinetics of phospholipid hydrolysis and retinoid isomerization suggests that the lipid membrane structure is critical for RPE65 enzymatic activity. We also provide evidence that RPE65 operates in a multiprotein complex with retinol dehydrogenase 5 and retinal G protein-coupled receptor in RPE microsomes. Modifications in the phospholipid environment affecting interactions with these protein components may be responsible for the alterations in retinoid metabolism observed in phospholipid-depleted RPE microsomes. Thus, our results indicate that the enzymatic activity of native RPE65 strongly depends on its membrane binding and phospholipid environment.     

Buoyancy under Control: Underwater Locomotor Performance in a Deep Diving Seabird Suggests Respiratory Strategies for Reducing Foraging Effort

Background

Because they have air stored in many body compartments, diving seabirds are expected to exhibit efficient behavioural strategies for reducing costs related to buoyancy control. We study the underwater locomotor activity of a deep-diving species from the Cormorant family (Kerguelen shag) and report locomotor adjustments to the change of buoyancy with depth.

Methodology/Principal Findings

Using accelerometers, we show that during both the descent and ascent phases of dives, shags modelled their acceleration and stroking activity on the natural variation of buoyancy with depth. For example, during the descent phase, birds increased swim speed with depth. But in parallel, and with a decay constant similar to the one in the equation explaining the decrease of buoyancy with depth, they decreased foot-stroke frequency exponentially, a behaviour that enables birds to reduce oxygen consumption. During ascent, birds also reduced locomotor cost by ascending passively. We considered the depth at which they started gliding as a proxy to their depth of neutral buoyancy. This depth increased with maximum dive depth. As an explanation for this, we propose that shags adjust their buoyancy to depth by varying the amount of respiratory air they dive with.

Conclusions/Significance

Calculations based on known values of stored body oxygen volumes and on deep-diving metabolic rates in avian divers suggest that the variations of volume of respiratory oxygen associated with a respiration mediated buoyancy control only influence aerobic dive duration moderately. Therefore, we propose that an advantage in cormorants - as in other families of diving seabirds - of respiratory air volume adjustment upon diving could be related less to increasing time of submergence, through an increased volume of body oxygen stores, than to reducing the locomotor costs of buoyancy control.     

Sleep Deprivation Influences Diurnal Variation of Human Time Perception with Prefrontal Activity Change: A Functional Near-Infrared Spectroscopy Study

Human short-time perception shows diurnal variation. In general, short-time perception fluctuates in parallel with circadian clock parameters, while diurnal variation seems to be modulated by sleep deprivation per se. Functional imaging studies have reported that short-time perception recruits a neural network that includes subcortical structures, as well as cortical areas involving the prefrontal cortex (PFC). It has also been reported that the PFC is vulnerable to sleep deprivation, which has an influence on various cognitive functions. The present study is aimed at elucidating the influence of PFC vulnerability to sleep deprivation on short-time perception, using the optical imaging technique of functional near-infrared spectroscopy. Eighteen participants performed 10-s time production tasks before (at 21:00) and after (at 09:00) experimental nights both in sleep-controlled and sleep-deprived conditions in a 4-day laboratory-based crossover study. Compared to the sleep-controlled condition, one-night sleep deprivation induced a significant reduction in the produced time simultaneous with an increased hemodynamic response in the left PFC at 09:00. These results suggest that activation of the left PFC, which possibly reflects functional compensation under a sleep-deprived condition, is associated with alteration of short-time perception.     

Potential Component Allee Effects and Their Impact on Wetland Management in the Conservation of Endangered Anurans

Effective management of wetland quantity and quality is crucial for effective conservation of declining amphibian populations. In particular, frogs and toads that employ aggregative breeding strategies may suffer negative population impacts in response to changes in availability of aquatic breeding habitat, including overabundance of suitable habitat, if density of conspecifics attending aggregations is positively correlated with reproductive success. Here we document such a positive relationship, potentially the first example of a component Allee effect in an anuran, in the critically endangered Houston toad (Bufo houstonensis). We assessed the relationship between mean yearly chorus size and reproductive success of males at the pond level using an information theoretic model selection approach and a two-sample t-test. The chosen model contained the single variable of mean yearly chorus size to predict probability of reproduction, as selected using the Akaike Information Criterion corrected for small sample size and Akaike weight. Mean chorus sizes were significantly higher among ponds exhibiting evidence of reproduction than in those that showed no evidence of reproduction. Our results suggest that chorusing alone is a poor proxy for inference of population stability and highlight a need for reassessment of widely-used amphibian monitoring protocols. Further, amphibian conservation efforts should account for potential Allee effects in order to optimize benefits and avoid underestimating critical population thresholds, particularly in species exhibiting rapid population declines.     

Extracellular BCL2 Proteins Are Danger-Associated Molecular Patterns That Reduce Tissue Damage in Murine Models of Ischemia-Reperfusion Injury

Background

Ischemia-reperfusion (I/R) injury contributes to organ dysfunction in a variety of clinical disorders, including myocardial infarction, stroke, organ transplantation, and hemorrhagic shock. Recent investigations have demonstrated that apoptosis as an important mechanism of cell death leading to organ dysfunction following I/R. Intracellular danger-associated molecular patterns (DAMPs) released during cell death can activate cytoprotective responses by engaging receptors of the innate immune system.

Methodology/Principal Findings

Ischemia was induced in the mouse hind limb by tourniquet or in the heart by coronary artery ligation. Reperfusion injury of skeletal or cardiac muscle was markedly reduced by intraperitoneal or subcutaneous injection of recombinant human (rh)BCL2 protein or rhBCL2-related protein A1 (BCL2A1) (50 ng/g) given prior to ischemia or at the time of reperfusion. The cytoprotective activity of extracellular rhBCL2 or rhBCL2A1 protein was mapped to the BH4 domain, as treatment with a mutant BCL2 protein lacking the BH4 domain was not protective, whereas peptides derived from the BH4 domain of BCL2 or the BH4-like domain of BCL2A1 were. Protection by extracellular rhBCL2 or rhBCL2A1 was associated with a reduction in apoptosis in skeletal and cardiac muscle following I/R, concomitant with increased expression of endogenous mouse BCL2 (mBCL2) protein. Notably, treatment with rhBCL2A1 protein did not protect mice deficient in toll-like receptor-2 (TLR2) or the adaptor protein, myeloid differentiation factor-88 (MyD88).

Conclusions/Significance

Treatment with cytokine-like doses of rhBCL2 or rhBCL2A1 protein or BH4-domain peptides reduces apoptosis and tissue injury following I/R by a TLR2-MyD88-dependent mechanism. These findings establish a novel extracellular cytoprotective activity of BCL2 BH4-domain proteins as potent cytoprotective DAMPs.     

Cathepsin L Inhibition Prevents Murine Autoimmune Diabetes via Suppression of CD8+ T Cell Activity

Background

Type 1 diabetes (T1D) is an autoimmune disease resulting from defects in central and peripheral tolerance and characterized by T cell-mediated destruction of islet β cells. To determine whether specific lysosomal proteases might influence the outcome of a T cell–mediated autoimmune response, we examined the functional significance of cathepsin inhibition on autoimmune T1D-prone non-obese diabetic (NOD) mice.

Methods and Findings

Here it was found that specific inhibition of cathepsin L affords strong protection from cyclophosphamide (CY)-induced insulitis and diabetes of NOD mice at the advanced stage of CD8⁺ T cell infiltration via inhibiting granzyme activity. It was discovered that cathepsin L inhibition prevents cytotoxic activity of CD8⁺ T cells in the pancreatic islets through controlling dipeptidyl peptidase I activity. Moreover, the gene targeting for cathepsin L with application of in vivo siRNA administration successfully prevented CY-induced diabetes of NOD mice. Finally, cathepsin L mRNA expression of peripheral CD8⁺ T cells from NOD mice developing spontaneous T1D was significantly increased compared with that from control mice.

Conclusions

Our results identified a novel function of cathepsin L as an enzyme whose activity is essential for the progression of CD8⁺ T cell-mediated autoimmune diabetes, and inhibition of cathepsin L as a powerful therapeutic strategy for autoimmune diabetes.     

Novel beta-1,3-, 1,6-oligoglucan elicitor from Alternaria alternata 102 for defense responses in tobacco

A novel elicitor that induces chitinases in tobacco BY-2 cells was isolated from Alternaria alternata 102. Six other fungi, including A. alternata IFO 6587, could not induce, or weakly induce chitinase activity. The purified elicitor was soluble in 75% methanol and showed the chitinase-inducing activity when applied at concentrations of as low as 25 ng x mL(-1). Structural determination by methylation analysis, reducing-end analysis, MALDI-TOF/MS, and NMR spectroscopy indicated that the elicitor was a mixture of beta-1,3-, 1,6-oligoglucans mostly with a degree of polymerization of between 8 and 17. Periodate oxidation of the elicitor suggested that the 1,6-linked and nonreducing terminal residues are essential for the elicitor activity. Further analysis of the elicitor responses in BY-2 cells indicated that the activity of this beta-1,3-, 1,6-glucan elicitor was about 1000 times more potent than that of laminarin, which is a known elicitor of defense responses in tobacco. Analyzing the expression of defense-related genes indicated that a phenylalanine ammonia-lyase gene and a coumaroyl-CoA O-methyltransferase gene were transiently expressed by this beta-1,3-, 1,6-glucan elicitor. The elicitor induced a weak oxidative burst but did not induce cell death in the BY-2 cells. In the tissue of tobacco plants, this beta-1,3-, 1,6-glucan elicitor induced the expression of basic PR-3 genes, the phenylpropanoid pathway genes, and the sesquiterpenoid pathway genes. In comparison with laminarin and laminarin sulfate, which are reported to be potent elicitors of defense responses in tobacco, the expression pattern of genes induced by the purified beta-1,3-, 1,6-glucan elicitor was more similar to that induced by laminarin than to that induced by laminarin sulfate.     

Determination of endogenous testosterone in rat tissues following fetal alcohol exposure using HPLC with UV detection

A novel method for quantitation of brain neurosteroid levels using HPLC with UV detection is described. In this simple and reliable method, testosterone from the brain and whole blood, and the internal standard, 17alpha-methyl testosterone, were extracted in 20% acetonitrile-phosphate buffer (pH 2.8), followed by solid phase extraction (SPE). The calibration curve was linear in concentration ranges from 0.1 to 10 ng from 0.2 g of tissue. We successfully applied this method to the analysis of endogenous testosterone in the male offspring of rats exposed to alcohol in utero. The concentration of testosterone at 21 post delivery in fetal alcohol exposure (FAE) group was significantly greater than the concentrations in either pair-fed or the ad libitum controls. These results support the usefulness of this method as a means of quantitating neurosteroids, and illustrate its applicability to fetal alcohol exposure.