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This article explores the unlikely collaboration between a Swedish art and dance historian, a Russian amateur historian, and a Russian-Swedish doctoral student to seek out the early career of migrating dancer Anna Robenne (one of her names). The article looks into the activist ways in which the explorers interacted with Russian, Swedish, and Finnish archives in order to both reveal and make accessible cross-border materials and knowledge pertaining to Robenne. To explore the relationship between the Robenne materials, the archival institutions, and the group of collaborating historians, the authors draw on Caswell and Cifor’s notion of “radical empathy”. The article thus brings new archival theory into the performing arts domain and makes a dance contribution to the broader field of critical archival and heritage studies. To cross borders to account for Robenne’s Russian legacy counters previous historiography’s disinterest in following the careers of non-canonized migrating artists in the Nordic-Baltic region.

Background. The last researches offer to conduct the study of Alzheimer disease (AD) mechanisms using diverse experimental models. However, it was not investigated the behavioral and cognitive impairment in rats at the different stages of vascular model of dementia of Alzheimer’s type developed by us. Subjects and methods. The experiment was performed on 32 male WAG rats weighing 180-250 g which were divided into 4 groups. Rats from group 1 and 3 were injected by aqueous solution of sodium nitrite at a dose of 50 mg/kg of body mass intraperitoneally during 14 and 28 days respectively. Groups 2 and 4 were received 500,000 mesenchymal stem cells in suspension intravenously against the background of experimental nitrite-induced AD. To estimate the behavioral reactions and cognitive functions the Open Field Test (OFT) and Passive Avoidance test (PAT) were used. Results. In all experimental groups in most cases it was found the significant decrease in vertical and horizontal activity (p

Despite numerous studies on the spatial distribution of pollutants in urban environments, little is known about their vertical profile. The presented research aims to analyze loadings and concentration of elements sedimented on exterior glass window surfaces of a high-rise building in Moscow city. The roadside part of a typical 17-level building was examined. Window sediments were collected along vertical from the 2nd to the 17th level using the novel instrumental approach of wet vacuum dust collection. A clear declining trend of sediment weight distribution was observed along the height increase. The values decreased by six times within 40-m vertical, from the 3rd (386 mg m

This article explores the creative practice of an independent Russian theatre collective, Soso Daughters. The collective was founded in Moscow by theatre director and playwright Zhenya Berkovich in 2018 and recently premiered their fourth production. The subjects of the productions the collective performs often delve into girls' and women's lives and traumatic experiences that are constantly undermined, dismissed or ignored. Driven by the feminist ethic of care, this article aims to situate Soso Daughters' creative work in the context of scan-aesthetic across the Nordic and Baltic regions, and to turn scholars and spectators' attention to the issue of an independent theatre practice in Russia. The article is based on interviews with the collective's director and scenographer as well as first-hand observations of their productions. The article further explores the possibility of representation of trauma and traumatic experience in The Rhyme, the collective's first production, grounding the analysis in that the holistic approach to scenography.

The article substantiates the relevance of detailing the existing network of state monitoring of water objects by forming an additional observation network for water objects being in municipal ownership. A brief description of the existing observation network of surface water monitoring carried out by Roshydromet is given. The main documents regulating the order of surface water monitoring are listed. The insufficient coverage of water bodies in municipal ownership by the observation program was noted. The layout of surface water monitoring stations in the Novosibirsk Region, confirming this fact, is given. The main types of water bodies that are the municipal ownership are listed. The list of the water objects that are the municipal property of Novosibirsk is resulted. The fact of active economic and recreational use of these objects that contradicts the municipal legislation and safety requirements is noted. A list of the possible use of these facilities for the benefit of the municipality is stated. Conclusions about the need for the formulation of water bodies in municipal ownership, cadastral registration for their effecient use in the economy are drawn. The main types of work planned to solve this problem are listed.

The article considers the issues of necessity of use of remote sensing data to obtain more information on the forest areas within the limits of forest lands. The causes of the lack of uniform methodology for the registration of forest land are identifield. The scheme of the forest plot registration with the use of remote sensing data is proposed.

The article discusses the types of agricultural land use; The main characteristics of the land fund of the Russian Federation governing land monitoring are given. At the same time, the problem of forming effective approaches for the rational use of land resources in a market economy requires an urgent solution in order to increase the efficiency of not only the use of land resources, but also the creation of conditions for obtaining environmentally safe products from each hectare of land. With this in mind, the article analyzes various methods of land resources assessment, reviewed the existing proposals for the integrated development of rural areasA brief description of the analysis of the state of land resources is given. The conclusions about the future direction of research.

Background: Monitoring of height and body mass index (BMI) is an important for patients with axial spondyloarthritis (axSpA). Reduction of height not only reflects spine remodeling, but affects various indexes, such as BMI (fig.1). However, BMI calculations assume that the adult patient‘s height is a constant. Therefore these calculations may be inaccurate in patients whose height is susceptible to decrease. Objectives: To evaluate changes in height and BMI over time in patients with axSpA, treated with tumor necrosis factor-α inhibitors (iTNF-α). Methods: The study included 51 patients with axSpA (ASAS criteria 2009). All the patients were treated with iTNF-α for one and > years. The patient’s height and weight measured before the beginning TNF-α inhibitors and during the treatment. Only patients with stable weight (variability ± 2.0 kg from baseline) were included in the study. Results: Mean age of axSpA patients at baseline was 40.22±13.51, 38 (74.5%) were male, disease duration 12.77±6.82, duration of TNF-α inhibitors treatment 5.64±2.21 years, all the patients had inactive disease. In 19 patients with axSpA diagnostic delay was 8 years. Patients’ height varied between 1.55 – 1.92 m (average height 1.73±0.07 m). Male’s mean height was 1.78±0.05 m, women’s – 1.63±0.05 m. Height decrease over time in patients with axSpa was 3.74±1.83 cm (from 1.0 to 15.0 cm). Maximal decrease in height was shown in patients with 10 years after the first symptoms emerged. Mean weight in patients who had height loss was 79.75±12.7 kg. Among patients who had decrease of height and stable body mass the difference between BMI at subsequent measurement and the time of treatment initiation (ΔBMI) was 1.15 kg/m2: BMI at baseline = 26.25±3.34 kg/m2, over time of observation BMI = 27.39±3.38 kg/m2). Conclusion: Height loss in axSpA is significant even in patients with inactive disease. Height loss affects BMI calculation that could lead to overestimation of cardiovascular risk, obesity and could change all of the BMI-dependent areas. Correction of BMI calculation for the patients with unstable height is needed. Disclosure of interests: Elizaveta Vasilenko: None declared, Olga Nikolaeva: None declared, anna Dadalova: None declared, Ekaterina Gaydukova: None declared, V Mazurov Grant/research support from: JSC BIOCAD, inna Gaydukova Grant/research support from: JSC BIOCAD, Speakers bureau: paiment from Pfizer, Novartis, abbvie, Biocad, Selgene, MSD, Sanofy not exceed 10 000 euros

Background: Patients (pts) with axial spondyloarthritis (axSpA) have increased cardiovascular morbidity and mortality, compared to the general population. However, assessment of cardiovascular risk (CVR) in axSpA is complicated, as in age Objectives: To measure CVR in young pts with low activity of axSpA, and to evaluate the interrelation between CVR, measured with QRISK and gene predisposition. Methods: The study included 46 pts 25-40 years old with axSpA (ASAS criteria 2009), achieved inactive disease on TNF-α inhibitors. AxSpA activity was measured with ASDAS and BASDAI. CVR was calculated with the modified QRISK (QRISK3) (https://qrisk.org/three/). All parameters of QRISK3 were collected: smoking and diabetes statuses, angina or heart attack in a 1st degree relative Results: Mean age ± SD of included pts was 32.5±3.87 years (26% of pts aged 25 – 30, 28.3% – 30-35, 28.3% – 35-40 years), 36 (78.3%) patient were male, symptoms duration 8.71±4.72 years, duration of TNF-α inhibitor treatment 3.1±2.4 years, ASDAS 1.95±1.23, BASDAI 1.4±0.8. All three age groups were comparable (p≥0.05). Out of the 46 pts 25 – 40 years old 34 (75.6%) had a low CVR and 11 (23,9%) had increased CVR (fig.1). Average QRISK3 score of all the patients was 1.18±1.64%. In the 25-30 year old age QRISK3 was 1.51±0.25%, in pts 30-35years old – 1.09±0.6%, in 35-40 years old–2.31±1.0% (p All patients with increased CVR had a direct associations with homozygous in CC allele IL17F-11139 gene, heterozygous in GA allele of TNF-308 and heterozygous in CG allele IL6-174. Negative associations were found between CVR and heterozygosity in His/Arg and homozygous in His/His of IL17F7, homozygosity in GG allele TNF-308, homozygosity in CC IL6-174, table 1. Interrelation of CVR with another gene polymorphisms was not significant (data are not present). Conclusion: A quarter of young axSpA pts had an increased 10-year probability of cardiovascular events. Gene polymorphisms in IL-6, IL17F, and TNF-α locuses are strongly associated with increased CVR. QRISK3 and gene polymorphisms could be promising tools in CVR assessment in axSpA. Further larger studies are needed for evaluation of CVR factors in axSpA. Reference [1] Julia Hippisley-Cox, et al. BMJ2017; 357 doi: https://doi.org/10.1136/bmj.j2099. Disclosure of Interests: Elizaveta Vasilenko: None declared, Olga Nikolaeva: None declared, Anna Dadalova: None declared, Maxim Korolev: None declared, V Mazurov Grant/research support from: JSC BIOCAD, Inna Gaydukova Grant/research support from: JSC BIOCAD, Speakers bureau: paiment from Pfizer, Novartis, Abbvie, Biocad, Selgene, MSD, Sanofy does not exceed 10 000 euros

Antibodies are indispensable tools for therapy and diagnostics. Here, we present a novel technique that allows for an accelerated synthesis of functionally active antibodies. This is achieved by using a cell-free translation system based on mammalian cells.

The efficient recycling of the chromophore of visual pigments, 11-cis-retinal, through the retinoid visual cycle is an essential process for maintaining normal vision. RPE65 is the isomerohydrolase in retinal pigment epithelium and generates predominantly 11-cis-retinol (11cROL) and a minor amount of 13-cis-retinol (13cROL), from all-trans-retinyl ester (atRE). We recently identified and characterized novel homologues of RPE65, RPE65c, and 13-cis-isomerohydrolase (13cIMH), which are expressed in the zebrafish inner retina and brain, respectively. Although these two homologues have 97% identical amino acid sequences, they exhibit distinct product specificities. Under the same assay conditions, RPE65c generated predominantly 11cROL, similar to RPE65, while 13cIMH generated exclusively 13cROL from atRE substrate. To study the impacts of the key residues determining the isomerization product specificity of RPE65, we replaced candidate residues by site-directed mutagenesis in RPE65c and 13cIMH. Point mutations at residues Tyr58, Phe103, and Leu133 in RPE65c resulted in significantly altered isomerization product specificities. In particular, our results showed that residue 58 is a primary determinant of isomerization specificity, because the Y58N mutation in RPE65c and its reciprocal N58Y mutation in 13cIMH completely reversed the respective enzyme isomerization product specificities. These findings will contribute to the elucidation of molecular mechanisms underlying the isomerization reaction catalyzed by RPE65.

α-Phenyl-N-tert-butylnitrone (PBN), a free radical spin trap, has been shown previously to protect retinas against light-induced neurodegeneration, but the mechanism of protection is not known. Here we report that PBN-mediated retinal protection probably occurs by slowing down the rate of rhodopsin regeneration by inhibiting RPE65 activity. PBN (50 mg/kg) protected albino Sprague-Dawley rat retinas when injected 0.5-12 h before exposure to damaging light at 2,700 lux intensity for 6 h but had no effect when administered after the exposure. PBN injection significantly inhibited in vivo recovery of rod photoresponses and the rate of recovery of functional rhodopsin photopigment. Assays for visual cycle enzyme activities indicated that PBN inhibited one of the key enzymes of the visual cycle, RPE65, with an IC(50) = 0.1 mm. The inhibition type for RPE65 was found to be uncompetitive with K(i) = 53 μm. PBN had no effect on the activity of other visual cycle enzymes, lecithin retinol acyltransferase and retinol dehydrogenases. Interestingly, a more soluble form of PBN, N-tert-butyl-α-(2-sulfophenyl) nitrone, which has similar free radical trapping activity, did not protect the retina or inhibit RPE65 activity, providing some insight into the mechanism of PBN specificity and action. Slowing down the visual cycle is considered a treatment strategy for retinal diseases, such as Stargardt disease and dry age-related macular degeneration, in which toxic byproducts of the visual cycle accumulate in retinal cells. Thus, PBN inhibition of RPE65 catalytic action may provide therapeutic benefit for such retinal diseases.

Cone photoreceptors have faster light responses than rods and a higher demand for 11-cis retinal (11cRAL), the chromophore of visual pigments. RPE65 is the isomerohydrolase in the retinal pigment epithelium (RPE) that converts all-trans retinyl ester to 11-cis retinol, a key step in the visual cycle for regenerating 11cRAL. Accumulating evidence suggests that cone-dominant species express an alternative isomerase, likely in retinal Muller cells, to meet the high demand for the chromophore by cones. In the present study, we describe the identification and characterization of a novel isomerohydrolase, RPE65c, from the cone-dominant zebrafish retina. RPE65c shares 78% amino acid sequence identity with RPE-specific zebrafish RPE65a (orthologue of human RPE65) and retains all of the known key residues for the enzymatic activity of RPE65. Similar to the other RPE-specific RPE65, RPE65c was present in both the membrane and cytosolic fractions, used all-trans retinyl ester as its substrate and required iron for its enzymatic activity. However, immunohistochemistry detected RPE65c in the inner retina, including Muller cells, but not in the RPE. Furthermore, double-immunostaining of dissociated retinal cells using antibodies for RPE65c and glutamine synthetase (a Muller cell marker), showed that RPE65c co-localized with the Muller cell marker. These results suggest that RPE65c is the alternative isomerohydrolase in the intra-retinal visual cycle, providing 11cRAL to cone photoreceptors in cone-dominant species. Identification of an alternative visual cycle will contribute to the understanding of the functional differences of rod and cone photoreceptors. Structured digital abstract • RPE65c colocalizes with Calnexin by cosedimentation (View interaction)

The visual cycle is a multi-step pathway to recycle 11-cis retinal, the chromophore for both rod and cone visual pigments. The isomerohydrolase RPE65, a membrane-associated enzyme, converts atRE (all-trans-retinyl ester) to 11-cis-retinol, a key step in the visual cycle. Previously, it has been shown that membrane association of RPE65 is essential for its catalytic activity. Using purified recombinant chicken RPE65 and an in vitro liposome-based floatation assay, we present evidence that the RPE65 membrane-binding affinity was significantly facilitated by incorporation of atRE, the substrate of RPE65, into liposomal membrane. Using tryptophan emission fluorescence quenching and CD spectroscopy, we showed that, upon membrane binding, RPE65 undergoes conformational changes at both the tertiary and secondary structural levels. Specifically, tryptophan fluorescence quenching showed that the tertiary RPE65 structure became more open towards the hydrophilic environment upon its association with the membrane. Simultaneously, a decrease in the α-helix content of RPE65 was revealed upon binding with the lipid membrane containing atRE. These results demonstrated that RPE65's functional activity depends on its conformational changes caused by its association with the membrane.

13-cis Retinoic acid (13cRA), a stereoisomeric form of retinoic acid, is naturally generated in the body and is also used clinically to treat acute promyelocytic leukaemia, some skin diseases, and cancer. Furthermore, it has been suggested that 13cRA modulates brain neurochemical systems, as increased 13cRA levels are correlated with depression and increased suicidal tendencies. However, the mechanism for the generation of endogenous 13cRA is not well understood. This study identified and characterized a novel enzyme in zebrafish brain, 13-cis isomerohydrolase (13cIMH), that exclusively generated 13-cis retinol, which can be oxidized to 13cRA. 13cIMH shares 74% amino acid sequence identity with human RPE65, an 11-cis isomerohydrolase in the visual cycle, and retains the key residues essential for RPE65’s isomerohydrolase activity. Similar to RPE65, 13cIMH is a membrane-associated protein, requires all-trans retinyl ester as its intrinsic substrate, and its enzymatic activity is dependent on iron. The purified 13cIMH converted atRE exclusively to 13-cis retinol with Km = 2.6 μM and kcat = 4.4 × 10−4s−1. RT-PCR, Western blot analysis and immunohistochemistry detected 13cIMH expression in the brain. These results suggest that 13cIMH may play a key role in the generation of 13cRA and in the modulation of neuronal functions in the brain.

Stargardt disease (STGD) is the major form of inherited juvenile macular degeneration. Pyridinium bis-retinoid A2E is a major component of lipofuscin which accumulates in retinal pigment epithelium (RPE) cells in STGD and contributes to the disease pathogenesis. However, the precise role of A2E in vision loss is unclear. Here we report that A2E efficiently inhibits RPE65 isomerohydrolase, a key enzyme in the visual cycle. Subretinal injection of A2E significantly inhibited retinoid isomerohydrolase activity in mice. Likewise, A2E also inhibited isomerohydrolase activity in cells coexpressing RPE65, lecithin retinol acyltransferase (LRAT), and cellular retinaldehyde-binding protein. In vitro isomerohydrolase activity assays confirmed that A2E inhibited enzymatic activity of recombinant RPE65 in a concentration-dependent manner, but did not inhibit LRAT activity. The inhibition type for isomerohydrolase was found to be reversible and competitive with K i = 13.6 μM. To determine the direct interaction of A2E with RPE65 protein, fluorescence binding assays were performed. As shown by fluorimetric titration, binding of purified RPE65 with A2E enhanced the bis-retinoid fluorescence. Consistently, the fluorescence of RPE65 decreased upon incubation with A2E. Both of these experiments suggest a direct, specific binding of A2E to RPE65. The binding constant for A2E and purified RPE65 was calculated to be 250 nM. These results demonstrate that A2E inhibits the regeneration of 11- cis retinal, the chromophore of visual pigments, which represents a unique mechanism by which A2E may impair vision in STGD.

Recently, it was shown that replication of bovine viral diarrhea virus (BVDV) is sensitive to inhibitors of host ER glucosidases. Consistent with these findings, we report that incubation of BVDV-infected MDBK cells with the glucosidase inhibitor n-butyl-deoxynojirimycin (nB-DNJ) reduced BVDV yields by 70- to 100-fold (n = 27), while having no effect on MDBK cell viability. However, the 70- to 100-fold reduction in infectious virus was associated with only a 2-fold reduction in genomic RNA synthesis and secretion of enveloped virus particles. Analysis of secreted virions showed that in the absence of glucosidase inhibitor, approximately 50% of the virion-associated BVDV E2 glycoprotein was resistant to endoglycosidase H (endo H) digestion, whereas intracellular E2 was completely sensitive to endo H digestion. In the presence of glucosidase inhibitor, virion-associated E2 and intracellular E2 were completely sensitive to endo H digestion. Taken together, these results suggest that BVDV is secreted through a Golgi-mediated pathway and that host ER glucosidase activity is required for production of infectious virions and Golgi processing of envelope E2 protein during virus egress.

Der Artikel „Neue Systeme zur Antikörperherstellung“, von Marlitt Stech, Olga Nikolaeva und Stefan Kubick, wurde ursprünglich am 30. September 2017 ohne Open Access online auf SpringerLink publiziert. Die Autoren haben sich jedoch nachträglich für eine Open Access-Veröffentlichung entschieden. Das Copyright des Artikels wurde deshalb im November 2017 in © Der/die Autor(en) 2017 geändert und der Artikel wird nun unter der Creative Commons Namensnennung 4.0 Interna tional Lizenz (http://creativecommons.org/ licenses/by/4.0/deed.de) veröffentlicht, welche die Nutzung, Vervielfältigung, Bearbeitung, Verbreitung und Wiedergabe in jeglichem Medium und Format erlaubt, sofern Sie den/die ursprünglichen Autor(en) und die Quelle ordnungsgemäß nennen, einen Link zur Creative Commons Lizenz beifügen und angeben, ob Änderungen vorgenommen wurden.

There is a forthcoming link between chronic alcohol consumption and proteins covalently modified by ethanol metabolites and their antibodies. To identify sensitive probes of protein–ethanol conjugates, we screened for the ethanol-altered protein domains with a phage-display combinatorial peptide library. In principle, recognition of the epitopes by the library peptides occurs through protein–protein interactions. A general screening, M13-based library with 10 9 random sequences of linear heptameric peptides was used. The peptides were displayed in five copies each, as fusion proteins with phage's minor coat protein III. They were located on one end of the surface of the phage particles. The targets were a model protein, streptavidin, and protein–ethanol conjugates (hydroxyethyl radical- or acetaldehyde-modified bovine serum albumin). They were either immobilized on a surface by direct coating or affinity captured on floating beads. An enriched library of phages with the tightest peptide binders for each target was selected and amplified in a multiple-cycle biopanning in vitro procedure. Binders were characterized by DNA sequencing of the corresponding phages and by counter-screening with positive and negative targets in either an enzyme-linked immunosorbent assay or plaque assay. We obtained the HPQ motif for streptavidin and two unique subsets of peptides that recognized each ethanol target with a selectivity of two orders of magnitude above the carrier protein and controls. The application of biopanning processes, coupled with phage-display peptide libraries on biological fluids and tissues, could provide a systematic mapping of protein–ethanol conjugates and supply a means for early diagnosis and prognosis of chronic alcohol consumption in human beings.

Retinoic acid (atRA) signaling is essential for regulating embryonic development, and atRA levels must be tightly controlled in order to prevent congenital abnormalities and fetal death which can result from both excessive and insufficient atRA signaling. Cellular enzymes synthesize atRA from Vitamin A, which is obtained from dietary sources. Embryos express multiple enzymes that are biochemically capable of catalyzing the initial step of Vitamin A oxidation, but the precise contribution of these enzymes to embryonic atRA synthesis remains unknown. Using Rdh10trex-mutant embryos, dietary supplementation of retinaldehyde, and retinol dehydrogenase (RDH) activity assays, we demonstrate that RDH10 is the primary RDH responsible for the first step of embryonic Vitamin A oxidation. Moreover, we show that this initial step of atRA synthesis occurs predominantly in a membrane-bound cellular compartment, which prevents inhibition by the cytosolic cellular retinol-binding protein (RBP1). These studies reveal that widely expressed cytosolic enzymes with RDH activity play a very limited role in embryonic atRA synthesis under normal dietary conditions. This provides a breakthrough in understanding the precise cellular mechanisms that regulate Vitamin A metabolism and the synthesis of the essential embryonic regulatory molecule atRA.

13-cis Retinoic acid (13cRA), a stereoisomeric form of retinoic acid, is naturally generated in the body and is also used clinically to treat acute promyelocytic leukemia, some skin diseases and cancer. Furthermore, it has been suggested that 13cRA modulates brain neurochemical systems because increased 13cRA levels are correlated with depression and increased suicidal tendencies. However, the mechanism for the generation of endogenous 13cRA is not well understood. The present study identified and characterized a novel enzyme in zebrafish brain, 13-cis isomerohydrolase (13cIMH) (EC 5.2.1.7), which exclusively generated 13-cis retinol and can be oxidized to 13cRA. 13cIMH shares 74% amino acid sequence identity with human retinal pigment epithelium specific 65 kDa protein (RPE65), an 11-cis isomerohydrolase in the visual cycle, and retains the key residues essential for the isomerohydrolase activity of RPE65. Similar to RPE65, 13cIMH is a membrane-associated protein, requires all-trans retinyl ester as its intrinsic substrate, and its enzymatic activity is dependent on iron. The purified 13cIMH converted all-trans retinyl ester exclusively to 13-cis retinol with K(m) = 2.6 μm and k(cat) = 4.4 × 10(-4) ·s(-1) . RT-PCR, western blot analysis and immunohistochemistry detected 13cIMH expression in the brain. These results suggest that 13cIMH may play a key role in the generation of 13cRA, as well as in the modulation of neuronal functions in the brain.

RPE65 is the isomerohydrolase essential for regeneration of 11-cis retinal, the chromophore of visual pigments. Here we compared the impacts of two mutations in RPE65, E417Q identified in patients with Leber congenital amaurosis (LCA), and E417D on isomerohydrolase activity. Although both mutations decreased the stability of RPE65 and altered its sub-cellular localization, E417Q abolished isomerohydrolase activity whereas the E417D mutant retained partial enzymatic activity suggesting that the negative charge of E417 is important for RPE65 catalytic activity. Loss of charge at this position may represent a mechanism by which the E417Q mutation causes blindness in LCA patients.

Generation of 11-cis-retinol from all-trans-retinyl ester in the retinal pigment epithelium is a critical step in the visual cycle and is essential for perception of light. Recent findings from cell culture models suggest that protein RPE65 is the retinoid isomerohydrolase that catalyzes the reaction. However, previous attempts to detect the enzymatic activity of purified RPE65 were unsuccessful, and thus its enzymatic function remains controversial. Here, we developed a novel liposome-based assay for isomerohydrolase activity. The results showed that purified recombinant chicken RPE65 had a high affinity for all-trans-retinyl palmitate-containing liposomes and demonstrated a robust isomerohydrolase activity. Furthermore, we found that all-trans-retinyl ester must be incorporated into the phospholipid membrane to serve as a substrate for isomerohydrolase. This assay system using purified RPE65 enabled us to measure kinetic parameters for the enzymatic reaction catalyzed by RPE65. These results provide conclusive evidence that RPE65 is the isomerohydrolase of the visual cycle.

During initiation of apoptosis, Bcl-2 family proteins regulate the permeability of mitochondrial outer membrane. BH3-only protein, tBid, activates pro-apoptotic Bax to release cytochrome c from mitochondria. tBid also activates anti-apoptotic Bcl-2 in the mitochondrial outer membrane, changing it from a single-spanning to a multi-spanning conformation that binds the active Bax and inhibits cytochrome c release. However, it is not known whether other mitochondrial proteins are required to elicit the tBid-induced Bcl-2 conformational alteration. To define the minimal components that are required for the functionally important Bcl-2 conformational alteration, we reconstituted the reaction using purified proteins and liposomes. We found that purified tBid was sufficient to induce a conformational alteration in the liposome-tethered, but not cytosolic Bcl-2, resulting in a multi-spanning form that is similar to the one found in the mitochondrial outer membrane of drug treated cells. Mutations that abolished tBid/Bcl-2 interaction also abolished the conformational alteration, demonstrating that a direct tBid/Bcl-2 interaction at the membrane is both required and sufficient to elicit the conformational alteration. Furthermore, active Bax also elicited the Bcl-2 conformational alteration. Bcl-2 mutants that displayed increased or decreased activity in the conformational alteration assay, showed corresponding activities in inhibiting pore formation by Bax in vitro, and in preventing apoptosis in vivo. Thus, there is a strong correlation between the direct interaction of membrane-bound Bcl-2 and tBid with activation of Bcl-2 in vitro and in vivo.

Bcl-2 family proteins are critical regulators of apoptosis and function at the level of mitochondria and endoplasmic reticulum. Bcl-2 is a tail-anchored protein in the membranes, but its cytosolic ...

Golgi protein-73 (GP73) is up-regulated in hepatocellular carcinoma (HCC). The aims of this study were to determine if GP73 is detected in the serum, and to establish the sensitivity and specificity of serum GP73 for diagnosing HCC.Serum GP73 was detected by immunoblots and quantified by densitometric analysis.A total of 352 patients were studied. Serum GP73 levels were significantly higher in patients with HCC compared to those with cirrhosis (P0.001). GP73 had a sensitivity of 69% and a specificity of 75% at the optimal cutoff point of 10 relative units, with an area under the receiver operating curve of 0.79 vs. 0.61 for AFP (P = 0.001). GP73 levels had significantly higher sensitivity (62%) than AFP (25%) for diagnosing early HCC (P0.0001). Moreover, GP73 levels were elevated in the serum of 57% (32/56) of individuals with HCC who had serum AFP levels less than 20ng/ml.Higher levels of GP73 can be found in the serum of patients with HCC than of those without. GP73 was better than AFP for the diagnosis of early HCC. Further validation studies are needed to confirm the role of GP73 in the early detection of HCC.