Identification of the 11-cis-specific retinyl-ester synthase in retinal Müller cells as multifunctional O-acyltransferase (MFAT)

Absorption of a photon by a rhodopsin or cone-opsin pigment isomerizes its 11-cis-retinaldehyde (11-cis-RAL) chromophore to all-trans-retinaldehyde (all-trans-RAL), which dissociates after a brief period of activation. Light sensitivity is restored to the resulting apo-opsin when it recombines with another 11-cis-RAL. Conversion of all-trans-RAL to 11-cis-RAL is carried out by an enzyme pathway called the visual cycle in cells of the retinal pigment epithelium. A second visual cycle is present in Müller cells of the retina. The retinol isomerase for this noncanonical pathway is dihydroceramide desaturase (DES1), which catalyzes equilibrium isomerization of retinol. Because 11-cis-retinol (11-cis-ROL) constitutes only a small fraction of total retinols in an equilibrium mixture, a subsequent step involving selective removal of 11-cis-ROL is required to drive synthesis of 11-cis-retinoids for production of visual chromophore. Selective esterification of 11-cis-ROL is one possibility. Crude homogenates of chicken retinas rapidly convert all-trans-ROL to 11-cis-retinyl esters (11-cis-REs) with minimal formation of other retinyl-ester isomers. This enzymatic activity implies the existence of an 11-cis-specific retinyl-ester synthase in Müller cells. Here, we evaluated multifunctional O-acyltransferase (MFAT) as a candidate for this 11-cis-RE-synthase. MFAT exhibited much higher catalytic efficiency as a synthase of 11-cis-REs versus other retinyl-ester isomers. Further, we show that MFAT is expressed in Müller cells. Finally, homogenates of cells coexpressing DES1 and MFAT catalyzed the conversion of all-trans-ROL to 11-cis-RP, similar to what we observed with chicken-retina homogenates. MFAT is therefore an excellent candidate for the retinyl-ester synthase that cooperates with DES1 to drive synthesis of 11-cis-retinoids by mass action.Light perception begins with the absorption of a photon by an opsin pigment in the membranous outer segment (OS) of a rod or cone photoreceptor cell. The light-absorbing chromophore in most vertebrate opsins is 11-cis-retinaldehyde (11-cis-RAL). Photon capture isomerizes the 11-cis-RAL to all-trans-retinaldehyde (all-trans-RAL), inducing conformational changes in the protein that lead to its active meta-II state. After a brief period of signaling through the transduction cascade, meta II decays to yield apo-opsin and free all-trans-RAL. Light sensitivity is restored to the apo-opsin when it combines with 11-cis-RAL to regenerate the pigment. Conversion of all-trans-RAL to 11-cis-RAL is carried out by a multistep enzyme pathway called the visual cycle, located in cells of the retinal pigment epithelium (RPE) (1, 2). The retinoid isomerase in this pathway is Rpe65, which converts an all-trans-retinyl ester (all-trans-RE), such as all-trans-retinyl palmitate (all-trans-RP), to 11-cis-retinol (11-cis-ROL) and a free fatty acid (3–5). Retinyl esters are synthesized in RPE cells by lecithin:retinol acyl transferase (LRAT), which transfers a fatty acid from phosphatidylcholine to retinol (6, 7). LRAT converts both all-trans-ROL and 11-cis-ROL to their cognate esters with similar catalytic efficiency (8).A second visual cycle is present in Müller cells of the retina, providing 11-cis-ROL to cones (9–11). Cones, but not rods, can use 11-cis-ROL as a chromophore precursor to regenerate bleached opsin pigments (10, 12, 13). The isomerase in the noncanonical pathway is dihydroceramide desaturase (DES1) (11). DES1 catalyzes rapid equilibrium isomerization of retinol (11). At equilibrium, 11-cis-ROL is much less abundant than all-trans-ROL, due to the 4.1 kcal/mole difference in free energy between these isomers (14). Accordingly, a secondary source of energy is required to drive the conversion of all-trans-ROL to 11-cis-ROL by DES1. Retinas from cone-dominant species contain 11-cis-retinyl esters (11-cis-REs), whereas retinyl esters are much less abundant in retinas from rod-dominant species (11, 13, 15). Homogenates from cone-dominant chicken and ground-squirrel retinas convert all-trans-ROL predominantly to 11-cis-REs in the presence of palmitoyl CoA (palm CoA) (13, 16, 17). These observations suggest that selective esterification of 11-cis-ROL may be the driving force for 11-cis-retinoid formation. In the current work, we sought to identify the protein responsible for the 11-cis-RE-synthase activity in Müller cells. We evaluated multifunctional O-acyltransferase (MFAT) as a candidate for this synthase. MFAT, also called acyl-CoA wax-alcohol acyltransferase-2 (AWAT2), catalyzes palm CoA-dependent synthesis of triglycerides, wax monoesters, and retinyl esters (18). It is present in the endoplasmic reticulum and predominantly expressed in skin (18). The retinol-isomer specificity of MFAT, and its expression in ocular tissues, has not been studied.     

In vivo anticancer activity of rhomboidal Pt(II) metallacycles

The development of novel antitumor agents that have high efficacy in suppressing tumor growth, have low toxicity to nontumor tissues, and exhibit rapid localization in the targeted tumor sites is an ongoing avenue of research at the interface of chemistry, cancer biology, and pharmacology. Supramolecular metal-based coordination complexes (SCCs) have well-defined shapes and geometries, and upon their internalization, SCCs could affect multiple oncogenic signaling pathways in cells and tissues. We investigated the uptake, intracellular localization, and antitumor activity of two rhomboidal Pt(II)-based SCCs. Laser-scanning confocal microscopy in A549 and HeLa cells was used to determine the uptake and localization of the assemblies within cells and their effect on tumor growth was investigated in mouse s.c. tumor xenograft models. The SCCs are soluble in cell culture media within the entire range of studied concentrations (1 nM–5 µM), are nontoxic, and showed efficacy in reducing the rate of tumor growth in s.c. mouse tumor xenografts. These properties reveal the potential of Pt(II)-based SCCs for future biomedical applications as therapeutic agents.Molecular assemblies of nanoscale-size and well-defined geometries have recently emerged as an interesting new paradigm in drug design and drug delivery. To date, liposomes, the self-assembled lipid nanoparticles held together by weak interactions, are among the most widely studied and clinically successful nanoparticle-based drug carriers. Their use allows the drug to achieve sustained plasma levels while encapsulated, with the size preventing the fast clearance by the kidneys that often occurs with the free drug. However, liposomes themselves do not produce a therapeutic effect and their application as drug carriers for medical purposes has often been hindered by poor loading capacity (<5 wt %) and the inability to pass through biological barriers (1, 2). Inorganic and hybrid porous materials, such as molecular organic frameworks (MOFs), have also shown promise due to their higher loading capacities (>25 wt %) (3–5), but MOFs have poor hydrolytic stability (6, 7). Recent studies on materials from Institut Lavoisier (MIL)-100(Cr) and MIL-100(Fe), however, suggest that MOFs can persist in biologically relevant environments and can act as vehicles for some anticancer and antiviral agents (8–10). These early findings have prompted further investigations into the biomedical applications of supramolecular coordination complexes (SCCs) (11–24). SCCs preserve the attractive properties of MOFs, such as building block modularity (22, 23, 25), yet afford an increased solubility in the biological milieu and lend themselves to small-molecule characterization techniques due to their well-defined structure.Although development of SCCs for biomedical applications is in its infancy, some SCCs, such as trigonal prisms self-assembled from p-cymene and ruthenium-based metal fragments with pyridyl donors, have shown the ability to act as effective carriers of some chemotherapeutic agents (26–28). Moreover, a library of cytotoxic to cancer cells p-cymene ruthenium-based polygons and cages has also been developed (11). For biomedical applications, the information about the cellular uptake, delivery of a guest, and metabolism of the drug delivery vehicle is critical, although currently the fate of SCCs in biological environments is not well understood. In a rare report, a systematic investigation of the structural stability of a water-soluble, hexacationic ruthenium-based trigonal prism was performed; however, it was determined that the ruthenium-based trigonal prisms decompose in the presence of amino acids histidine, lysine, and arginine (29).An intriguing approach is the design of tumor-targeted modalities that combine detection and treatment through the self-assembly of emissive, metal-based coordination complexes. Such modalities can be especially valuable as they often do not require photoexcitation to elicit cytotoxicity. Recently Gray, Gross, and Medina-Kauwe and coworkers reported HerGa, a self-assembled tumor-targeted particle that bears the Ga(III)-metalated derivative of the sulfonated corrole (30, 31). The particle, which contained Ga(III)-corrole noncovalently bound to the tumor-targeting cell penetration protein HerPBK10, provided both tumor detection and elimination. Systemic injection of this protein–corrole complex resulted in tumor accumulation, which can be visualized in vivo due to the red corrole fluorescence. Cytotoxic and cytostatic properties of these targeted Ga(III) corroles were found to be cell-line dependent, with the ability to induce late M-phase arrest in several cancer cell lines (32).Despite the well-known cytotoxic properties of mono- and multinuclear platinum complexes (33–35), studies of the antitumor properties of platinum-based SCCs are rare (17, 36). Moreover, recent reports have demonstrated that platinum-based SCCs can act as effective hosts for guests and have interesting photophysical properties (37–42). In particular, highly emissive rhomboids based on aniline-containing donors and Pt-based metal acceptors have been developed that display different photophysical properties from those of their constituent subunits (40). These assemblies are interesting targets to investigate the cytotoxicity of organoplatinum SCCs, whereas their emission spectra could be used for interrogating the structural integrity in vitro. Here, for the first time to our knowledge, we report the uptake of SCCs in vitro in cell-based assays, determined by using laser-scanning confocal microscopy, and an in vivo assessment of the anticancer activity of SCCs in mouse s.c. tumor xenograft models.     

Licensed human natural killer cells aid dendritic cell maturation via TNFSF14/LIGHT

Interactions between natural killer (NK) cells and dendritic cells (DCs) aid DC maturation and promote T-cell responses. Here, we have analyzed the response of human NK cells to tumor cells, and we identify a pathway by which NK–DC interactions occur. Gene expression profiling of tumor-responsive NK cells identified the very rapid induction of TNF superfamily member 14 [TNFSF14; also known as homologous to lymphotoxins, exhibits inducible expression, and competes with HSV glycoprotein D for HVEM, a receptor expressed by T lymphocytes (LIGHT)], a cytokine implicated in the enhancement of antitumor responses. TNFSF14 protein expression was induced by three primary mechanisms of NK cell activation, namely, via the engagement of CD16, by the synergistic activity of multiple target cell-sensing NK-cell activation receptors, and by the cytokines IL-2 and IL-15. For antitumor responses, TNFSF14 was preferentially produced by the licensed NK-cell population, defined by the expression of inhibitory receptors specific for self-MHC class I molecules. In contrast, IL-2 and IL-15 treatment induced TNFSF14 production by both licensed and unlicensed NK cells, reflecting the ability of proinflammatory conditions to override the licensing mechanism. Importantly, both tumor- and cytokine-activated NK cells induced DC maturation in a TNFSF14-dependent manner. The coupling of TNFSF14 production to tumor-sensing NK-cell activation receptors links the tumor immune surveillance function of NK cells to DC maturation and adaptive immunity. Furthermore, regulation by NK cell licensing helps to safeguard against TNFSF14 production in response to healthy tissues.Natural killer (NK) cells play an important role in protecting the host against viral infection and cancer. As well as having potent cytotoxic activity, NK cells are endowed with immunoregulatory activity (1, 2). For example, NK cell activation induces the production of chemokines, such as macrophage inflammatory protein-1α (MIP-1α) and IL-8, and proinflammatory cytokines, such as IFN-γ, GM-CSF, and TNF-α. These molecules regulate the recruitment and activity of numerous immune cell types (1, 2). Importantly, NK cells can promote development of T-cell responses via NK–dendritic cell (DC) interactions that favor both DC maturation and NK-cell activation (3–5), with NK cell-derived IFN-γ skewing T-cell differentiation toward the Th1 phenotype (6, 7).Cytotoxic activity and cytokine production are coupled to signaling pathways downstream of a repertoire of activating and inhibitory receptors; signals from activating receptors (including NKG2D, DNAM-1, and 2B4, as well as the natural cytotoxicity receptors NKp30, NKp44, and NKp46) compete with signals from inhibitory receptors such as the killer cell immunoglobulin-like receptors (KIRs) and CD94/NKG2A heterodimers to regulate activation. In addition, NK cells express CD16, the low-affinity receptor for IgG, conferring antibody-dependent cellular cytotoxicity (8–10). Activation thus coordinates the killing of target cells, the induction of inflammation, and the promotion of adaptive immunity. This potent cytotoxicity and proinflammatory activity must be strictly controlled to minimize damage to healthy tissue. Functional competency of unstimulated NK cells is achieved via a process termed “licensing” or “education” (11–14). Licensing ensures that only those NK cells expressing inhibitory receptors for self-MHC class I can respond to target cells and NK cells that lack inhibitory receptors for self-MHC class I molecules are rendered hyporesponsive, preventing them from attacking healthy cells expressing normal levels of MHC class I molecules.We have analyzed the consequences of human NK cell activation by tumor cells. Our results reveal induction of the TNF superfamily member 14 (TNFSF14), also known as homologous to lymphotoxins, exhibits inducible expression, and competes with HSV glycoprotein D for HVEM, a receptor expressed by T lymphocytes (LIGHT) (15). We show that activated NK cells produce TNFSF14 in response to different stimuli, that tumor cells induce TNFSF14 production by licensed NK cells, and that TNFSF14-producing NK cells aid DC maturation during NK–DC cross-talk.     

Changes in Implementation of Two Evidence‐Based Psychotherapies for PTSD in VA Residential Treatment Programs: A National Investigation

There has been little investigation of the natural course of evidence‐based treatments (EBTs) over time following the draw‐down of initial implementation efforts. Thus, we undertook qualitative interviews with the providers at 38 U.S. Department of Veterans Affairs’ residential treatment programs for posttraumatic stress disorder (PTSD) to understand implementation and adaptation of 2 EBTs, prolonged exposure (PE), and cognitive processing therapy (CPT), at 2 time points over a 4‐year period. The number of providers trained in the therapies and level of training improved over time. At baseline, of the 179 providers eligible per VA training requirements, 65 (36.4%) had received VA training in PE and 111 (62.0%) in CPT with 17 (9.5%) completing case consultation or becoming national trainers in both PE and CPT. By follow‐up, of the increased number of 190 eligible providers, 87 (45.8%) had received VA training in PE and 135 (71.1%) in CPT, with 69 (36.3%) and 81 (42.6%) achieving certification, respectively. Twenty‐two programs (57.9%) reported no change in PE use between baseline and follow‐up, whereas 16 (42.1%) reported an increase. Twenty‐four (63.2%) programs reported no change in their use of CPT between baseline and follow‐up, 12 (31.6%) programs experienced an increase, and 2 (5.2%) programs experienced a decrease in use. A significant number of providers indicated that they made modifications to the manuals (e.g., tailoring, lengthening). Reasons for adaptations are discussed. The need to dedicate time and resources toward the implementation of EBTs is noted.     

Autonomic neural dysfunction in recently diagnosed diabetic subjects

Because onset of autonomic neural dysfunction in the diabetic syndrome has not been well established, sensitive and quantitative measures of autonomic nervous system (ANS) function were made in 19 non-insulin-dependent (NIDD) and 14 insulin-dependent (IDD) recent-onset diabetic subjects. The known duration of diabetes mellitus in the NIDD subjects was less than or equal to 12 mo. The duration in the IDD subjects was less than or equal to 24 mo. RR-variation during beta adrenergic blockade (an index of an ANS reflex involving the cardiac parasympathetic nervous system [PNS] pathway) was smaller than that of control subjects in both NIDD (P less than 0.001) and IDD subjects (P less than 0.01). This PNS abnormality was not likely to be due to volume depletion since acute volume depletion induced by furosemide in six normal subjects (1608 +/- 105 ml, mean +/- SEM) did not change RR-variation. Dark-adapted pupil size after topical PNS blockade (an index of iris sympathetic nervous system [SNS] activity) was also smaller in both groups of diabetic subjects (NIDD, P less than 0.01; IDD, P less than 0.05). Pupillary latency time (an index of an ANS reflex involving iris PNS pathway) was prolonged in the NIDD subjects (P less than 0.005) but was not significantly altered in the IDD subjects. Thus, it would appear that the ANS is impaired soon after the diagnosis of diabetes mellitus. We hypothesize that early impairment of the ANS is common in IDD and NIDD subjects. This finding is consistent with the hypothesis that abnormal carbohydrate metabolism is an important factor in the etiology of diabetic autonomic neuropathy.     

Effect of early use of noradrenaline on in-hospital mortality in haemorrhagic shock after major trauma: a propensity-score analysis

Background

The role of vasopressors in trauma-related haemorrhagic shock (HS) remains a matter of debate. They are part of the most recent European recommendations on the management of HS and are regularly used in France. We assessed the effect of early administration of noradrenaline in 24 h mortality of trauma patients in HS, using a propensity-score analysis.