Focal adhesion alterations in G0-positive melanoma cells.

BACKGROUND: Melanoma is a highly heterogeneous malignant tumor that exhibits various forms of drug resistance. Recently, reversal transition of cancer cells to the G0 phase of the cell cycle under the influence of therapeutic drugs has been identified as an event associated with tumor dissemination. In the present study, we investigated the ability of chemotherapeutic agent dacarbazine to induce a transition of melanoma cells to the G0 phase as a mechanism of chemoresistance. METHODS: We used the flow cytometry to analyze cell distribution within cell cycle phases after dacarbazine treatment as well as to identifyG0 -positive cells population. Transcriptome profiling was provided to determine genes associated with dacarbazine resistance. We evaluated the activity of ß-galactosidase in cells treated with dacarbazine by substrate hydrolysis. Cell adhesion strength was measured by centrifugal assay application with subsequent staining of adhesive cells with Ki-67 monoclonal antibodies. Ability of melanoma cells to metabolize dacarbazine was determined by expressional analysis of CYP1A1, CYP1A2, CYP2E1 followed by CYP1A1 protein level evaluation by the ELISA method. RESULTS: The present study determined that dacarbazine treatment of melanoma cells could induce an increase in the percentage of cells in G0 phase without alterations of ß-galactosidase positive cells which corresponded to the fraction of the senescent cells. Transcriptomic profiling of cells under dacarbazine induction of G0 -positive cells percentage revealed that 'VEGFA-VEGFR2 signaling pathway' and 'Cell cycle' signaling were mostly enriched by dysregulated genes. 'Focal adhesion' signaling was also found to be triggered by dacarbazine. In melanoma cells treated with dacarbazine, an increase in G0 -positive cells among adherent cells was found. CONCLUSIONS: Dacarbazine induces the alteration in a percentage of melanoma cells residing in G0 phase of a cell cycle. The altered adhesive phenotype of cancer cells under transition in the G0 phase may refer to a specific intercellular communication pattern of quiescent/senescent cancer cells.

miR-204-5p in vivo inhibition cause diminished CD45RO cells rate in lungs of melanoma B16-bearing mice.

The treatment of melanoma remains a challenge, despite novel approaches recently becoming available for disseminated tumors. RNA targeting is being intensively studied in various types of disease. The aim of the present study was to explore whether the in vivo use of a microRNA (miR)-204-5p inhibitor affected melanoma progression, and whether its metastasis affects target organ remodeling. CD45RO+, CD3+, CD8+, forkhead box P3+, smooth muscle α-actin+ cells in the lungs of B16 melanoma-bearing mice were evaluated using immunohistochemistry following miR-204-5p inhibitor transfection. Next, CD45RO expression in peripheral blood mononuclear cells (PBMCs), as well as the apoptosis of these cells, were measured by flow cytometry. The results revealed that the number of CD45RO+ cells was decreased in the lungs of B16 melanoma-bearing mice and CD45RO+ PBMCs following the use of an miR-204-5p inhibitor, which was associated with increased levels of PBMC apoptosis. In conclusion, the findings of the present study suggested that targeting miR-204-5p in melanoma metastasis target organs could be used to develop novel approaches for the treatment of disseminated forms of the disease.

Temporal network embedding framework with causal anonymous walks representations.

Many tasks in graph machine learning, such as link prediction and node classification, are typically solved using representation learning. Each node or edge in the network is encoded via an embedding. Though there exists a lot of network embeddings for static graphs, the task becomes much more complicated when the dynamic (i.e., temporal) network is analyzed. In this paper, we propose a novel approach for dynamic network representation learning based on Temporal Graph Network by using a highly custom message generating function by extracting Causal Anonymous Walks. We provide a benchmark pipeline for the evaluation of temporal network embeddings. This work provides the first comprehensive comparison framework for temporal network representation learning for graph machine learning problems involving node classification and link prediction in every available setting. The proposed model outperforms state-of-the-art baseline models. The work also justifies their difference based on evaluation in various transductive/inductive edge/node classification tasks. In addition, we show the applicability and superior performance of our model in the real-world downstream graph machine learning task provided by one of the top European banks, involving credit scoring based on transaction data.

Transcriptomic Profiling Revealed Plexin A2 Downregulation With Migration and Invasion Alteration in Dacarbazine-Treated Primary Melanoma Cells.

Melanoma is highly heterogeneous type of malignant neoplasm that is responsible for the majority of deaths among other types of skin cancer. In the present study, we screened a list of differentially expressed genes in two primary, drug-naïve melanoma cell lines derived from patients with melanoma following treatment of the cells with the chemotherapeutic agent dacarbazine. The aim was to determine the transcriptomic profiles and associated alterations in the cell phenotype. We found the vascular endothelial growth factor A/vascular endothelial growth factor receptor 2, phosphoinositide 3-kinase/protein kinase B and focal adhesion signaling pathways to be top altered after dacarbazine treatment. In addition, we observed the expression levels of genes associated with tumor dissemination, integrin ß8 and matrix metalloproteinase-1, to be diminished in both cell lines studied, the results of which were confirmed by reverse transcription-quantitative polymerase chain reaction. By contrast, plexin A2 expression was found to be upregulated in K2303 cells, where reduced migration and invasion were also observed, following dacarbazine treatment. Plexin A2 downregulation was associated with the promotion of migrative and invasive capacities in B0404 melanoma cells. Since plexin A2 is semaphorin co-receptor that is involved in focal adhesion and cell migration regulation, the present study suggested that plexin A2 may be implicated in the dacarbazine-mediated phenotypic shift of melanoma cells. We propose that the signature of cancer cell invasiveness can be revealed by using a combination of transcriptomic and functional approaches, which should be applied in the development of personalized therapeutic strategies for each patient with melanoma.

Efficient video face recognition based on frame selection and quality assessment.

The article is considering the problem of increasing the performance and accuracy of video face identification. We examine the selection of the several best video frames using various techniques for assessing the quality of images. In contrast to traditional methods with estimation of image brightness/contrast, we propose to utilize the deep learning techniques that estimate the frame quality by using the lightweight convolutional neural network. In order to increase the effectiveness of the frame quality assessment step, we propose to distill knowledge of the cumbersome existing FaceQNet model for which there is no publicly available training dataset. The selected K-best frames are used to describe an input set of frames with a single average descriptor suitable for the nearest neighbor classifier. The proposed algorithm is compared with the traditional face feature extraction for each frame, as well as with the known clustering methods for a set of video frames.

[The activity of NAD- and NAD(P)-dependent dehydrogenases in lymphocytes of peripheral blood in Graves' disease].

BACKGROUND: The regulatory effect of thyroid hormones on the metabolism of the immune system cells (activation of oxidative processes, separation of oxidative phosphorylation and increased protein synthesis) depends on their number. Changes in the activity of intracellular enzymes in Graves’ disease (GD) can determine the mechanisms of maintaining autoimmune inflammation in relapse of the disease. The exact role of NAD(P)-dependent dehydrogenases in the development and maintenance of immune response in GD is still poorly investigated. AIMS: To study the activity of NAD(P)-dependent dehydrogenases in lymphocytes of peripheral blood in patients with manifestation and relapse of GD to clarify the mechanisms of development and progression of the autoimmune process. METHODS: A single-center, cohort, prospective, continuous, observational, open-label, controlled trial was conducted to evaluate the lymphocytes NAD(P)-dependent activity in 151 women with GD and hyperthyroidism, mean age 40.7±13.2, 52 (37.14%), who were on follow-up at the endocrinology center of Krasnoyarsk Regional clinical hospital from 2016 to 2019. The NAD(P)-dependent dehydrogenases activity measured using biochemiluminescence method. RESULTS: In patients with newly diagnosed of GD, relative to the control values and levels detected in relapse group we observe the increase of G6PDH and decrease of NADH-LDH. In GD relapse group compare to the control range in blood lymphocytes decreases the activity of LDH and NAD(P)-ICDH. In patients with newly diagnosed GD, two positive сorrelation were found: between fT3 level and MDG activity (r=0.90, p=0.037), and between fT4 level and NAD(P)-ICDH activity (r=0.82, p=0.007). In patients with relapse of GD positive relationships between the concentration of TSH and the activity of LDH (r=0.73, p=0.039), and MDH (r=0.93, p=0.002), as well as in a pair of fT4 and NADGDH (r=0.70, p=0.036) were revealed. CONCLUSION: The established differences in the activity of NAD(P)-dependent dehydrogenases in peripheral blood lymphocytes in patients with manifestation and relapse of GD can reflect in the first case the response of immune cells to a functional-regulatory signal with the development of hyperthyroidism, and in the second case, adaptive changes with the progression of autoimmune process.

Peculiarities of Platelet Metabolism in Patients with Acute Coronary Syndrome with Anxiety-Depressive Disorders and Informativity of Enzymes in the Forecast of Development of Cardiovascular Complications.

Anxiety-depressive disorders (ADD) are a risk factor of cardiovascular mortality in patients with coronary artery disease (CAD). Acute coronary syndrome (ACS) is the main clinical manifestation of a progressing CAD. Metabolic processes disorder in platelets can be one of the causes of cardiovascular complications in patients with ACS and concomitant ADD. We studied platelets metabolism and prognostic informativity of NAD(P)-dependent dehydrogenases of platelets in ACS patients with ADD in terms of forecasting cardiovascular complications development over a year of observation. The levels of NAD- and NADP-dependent dehydrogenases of platelets were determined by means of a bioluminescent method during the first 24 h after admission to hospital and in dynamics in 10 days. Among 315 examined patients, ADD was found in 161 (51.1%). ACS patients with concomitant ADD had both cytoplasmic and mitochondrial processes impairment in platelets that consisted in a decrease of energy metabolism intensity, inhibition of anaerobic glycolysis reactions and lipid catabolism. After 12 months of follow-up, 41 (25.5%) cardiovascular complications were detected in the group of ACS patients with ADD and 20 (13.0%) in the group of ACS patients without ADD. According to the results of the analysis of the neural network based on NAD(P)-dependent dehydrogenases of platelets activity in ACS patients with ADD, indicators were obtained that are informative for predicting the development of recurrent cardiovascular complications.

Probabilistic Neural Network With Complex Exponential Activation Functions in Image Recognition.

If the training data set in image recognition task is not very large, the feature extraction with a convolutional neural network is usually applied. Here, we focus on the nonparametric classification of extracted feature vectors using the probabilistic neural network (PNN). The latter is characterized by the high runtime and memory space complexity. We propose to overcome these drawbacks by replacing the exponential activation function in the Gaussian kernel to the complex exponential functions. Such complex nonlinearities make it possible to accurately approximate the unknown density function using the network with the number of neurons proportional to only cubic root of the database size. As a result, the proposed approach decreases the runtime and memory complexities of the PNN without losing its main advantages, namely, fast training and convergence to the Bayesian decision. In the experimental study, we describe a protocol for comparing recognition methods using the well-known visual object category data sets in the context of the small sample size problem. It has been experimentally shown that our approach rapidly obtains accurate decisions when compared to the known classifiers including the baseline PNN.

Semaphorin-5A downregulation is associated with enhanced migration and invasion of BRAF-positive melanoma cells under vemurafenib treatment in melanomas with heterogeneous BRAF status.

Tumor heterogeneity affects the efficacy of anticancer treatment as tumor subclones with distinct molecular patterns may be present within one tumor, leading to differing sensitivities to chemotherapeutic agents. In the present study, six melanoma tissue fragments were obtained from different parts of tumor of four patients and then the effect of vemurafenib treatment on biological characteristics and molecular processes of cell cultures was estimated by using MTT-test, apoptosis, migration and invasion assays, PCR real time. There was different BRAF status determined between cells derived from the central and peripheral regions of primary melanoma tumors. BRAF-positive melanoma cells showed an increased apoptotic rate under vemurafenib treatment, as well as increased migration and invasion rates, whereas BRAF-negative melanoma cells did not exhibit such tendency. Furthermore, semaphorin-5A levels were diminished in BRAF-positive cells, but not in BRAF-negative ones, which could be related to increased migration and invasion. Melanoma cells derived from different regions of the same tumor may differ by mutations status, molecular processes and biological response to target therapy. The downregulation of semaphorin-5A may be involved in divergent effects of anticancer agents on tumor cell biology.

Efficient facial representations for age, gender and identity recognition in organizing photo albums using multi-output ConvNet.

This paper is focused on the automatic extraction of persons and their attributes (gender, year of born) from album of photos and videos. A two-stage approach is proposed in which, firstly, the convolutional neural network simultaneously predicts age/gender from all photos and additionally extracts facial representations suitable for face identification. Here the MobileNet is modified and is preliminarily trained to perform face recognition in order to additionally recognize age and gender. The age is estimated as the expected value of top predictions in the neural network. In the second stage of the proposed approach, extracted faces are grouped using hierarchical agglomerative clustering techniques. The birth year and gender of a person in each cluster are estimated using aggregation of predictions for individual photos. The proposed approach is implemented in an Android mobile application. It is experimentally demonstrated that the quality of facial clustering for the developed network is competitive with the state-of-the-art results achieved by deep neural networks, though implementation of the proposed approach is much computationally cheaper. Moreover, this approach is characterized by more accurate age/gender recognition when compared to the publicly available models.

The Prognostic Value of Combinations of Genetic Polymorphisms in the ITGB3, ITGA2, and CYP2C19*2 Genes in Predicting Cardiovascular Outcomes After Coronary Bypass Grafting.

AIM: To determine if there are any associations between the single nucleotide polymorphisms (SNPs): rs2046934, rs1126643, rs5918, rs6065, rs4244285; rs4986893 and the occurrence of cardiovascular events (CVE) in patients following coronary artery bypass grafting (CABG) surgery. MATERIALS AND METHODS: The study included 130 CABG patients with stable angina grades II-IV. After CABG 69 of the patients were treated with acetylsalicylic acid (ASA) alone, and 61 received dual antiplatelet therapy (ASA+clopidogrel). Platelet function was assessed by light transmission aggregometry with adenosinediphosphate and arachidonic acid. The SNPs were identified by real-time polymerase chain reaction (PCR) with electrophoretic detection. The mean follow-up period was equal to 10.9 ± 5.2 months. The primary end point included the composite of all-cause mortality, myocardial infarction (MI), and ischemic stroke. RESULTS: During the follow-up period 12 CVE were registered: 3 deaths, 6 MI, 3 strokes. Patients with composite mutant alleles of ITGB3+CYP2C19*2 or CYP2C19*2 + ITGA2, and with the mutant allele (*2) of CYP2C19, met end points more often than patients with other gene combinations (wild-type homozygotes, presence of one mutant allele of ITGB3 or ITGA2, the composite of mutant alleles of ITGB3+ITGA2 or ITGB3+ITGA2+CYP2C19*2; hazard ratio = 4, 95% confidence interval: 2.19-7.29, p = 0.008). CONCLUSION: Carriage of a combination of mutant alleles in multiple genes including ITGB3+CYP2C19*2 or CYP2C19*2 + ITGA2 or CYP2C19*2 are possible predictors of CVE in patients after CABG.

Aptamers Selected to Postoperative Lung Adenocarcinoma Detect Circulating Tumor Cells in Human Blood.

Circulating tumor cells (CTCs) are rare cells and valuable clinical markers of prognosis of metastasis formation and prediction of patient survival. Most CTC analyses are based on the antibody-based detection of a few epithelial markers; therefore miss an important portion of mesenchymal cancer cells circulating in blood. In this work, we selected and identified DNA aptamers as specific affinity probes that bind to lung adenocarcinoma cells derived from postoperative tissues. The unique feature of our selection strategy is that aptamers are produced for lung cancer cell biomarkers in their native state and conformation without previous knowledge of the biomarkers. The aptamers did not bind to normal lung cells and lymphocytes, and had very low affinity to A549 lung adenocarcinoma culture. We applied these aptamers to detect CTCs, apoptotic bodies, and microemboli in clinical samples of peripheral blood of lung cancer and metastatic lung cancer patients. We identified aptamer-associated protein biomarkers for lung cancer such as vimentin, annexin A2, annexin A5, histone 2B, neutrophil defensin, and clusterin. Tumor-specific aptamers can be produced for individual patients and synthesized many times during anticancer therapy, thereby opening up the possibility of personalized diagnostics.

Enzymatic relay mechanism stimulates cyclic GMP synthesis in rod photoresponse: biochemical and physiological study in guanylyl cyclase activating protein 1 knockout mice.

Regulation of cGMP synthesis by retinal membrane guanylyl cyclase isozymes (RetGC1 and RetGC2) in rod and cone photoreceptors by calcium-sensitive guanylyl cyclase activating proteins (GCAP1 and GCAP2) is one of the key molecular mechanisms affecting the response to light and is involved in congenital retinal diseases. The objective of this study was to identify the physiological sequence of events underlying RetGC activation in vivo, by studying the electrophysiological and biochemical properties of mouse rods in a new genetic model lacking GCAP1. The GCAP1(-/-) retinas expressed normal levels of RetGC isozymes and other phototransduction proteins, with the exception of GCAP2, whose expression was elevated in a compensatory fashion. RetGC activity in GCAP1(-/-) retinas became more sensitive to Ca(2+) and slightly increased. The bright flash response in electroretinogram (ERG) recordings recovered quickly in GCAP1(-/-), as well as in RetGC1(-/-)GCAP1(-/-), and RetGC2(-/-)GCAP1(-/-) hybrid rods, indicating that GCAP2 activates both RetGC isozymes in vivo. Individual GCAP1(-/-) rod responses varied in size and shape, likely reflecting variable endogenous GCAP2 levels between different cells, but single-photon response (SPR) amplitude and time-to-peak were typically increased, while recovery kinetics remained faster than in wild type. Recovery from bright flashes in GCAP1(-/-) was prominently biphasic, because rare, aberrant SPRs producing the slower tail component were magnified. These data provide strong physiological evidence that rod photoresponse recovery is shaped by the sequential recruitment of RetGC isozyme activation by GCAPs according to the different GCAP sensitivities for Ca(2+) and specificities toward RetGC isozymes. GCAP1 is the 'first-response' sensor protein that stimulates RetGC1 early in the response and thus limits the SPR amplitude, followed by activation of GCAP2 that adds stimulation of both RetGC1 and RetGC2 to speed-up photoreceptor recovery.

Retinal guanylyl cyclase isozyme 1 is the preferential in vivo target for constitutively active GCAP1 mutants causing congenital degeneration of photoreceptors.

Two calcium-sensitive guanylyl cyclase activating proteins (GCAP1 and GCAP2) activate cGMP synthesis in photoreceptor by retinal membrane guanylyl cyclase isozymes (RetGC1 and RetGC2) to expedite recovery, but calcium-insensitive constitutively active GCAP1 mutants cause photoreceptor degeneration in human patients and transgenic mice. Although GCAP1 and GCAP2 can both activate RetGC1 and RetGC2 in vitro, we find that GCAP1 selectively regulates RetGC1 in vivo. Furthermore, elimination of RetGC1 but not RetGC2 isozyme reverses abnormal calcium sensitivity of cGMP synthesis and rescues mouse rods in transgenic mice expressing GCAP1 mutants causing photoreceptor disease. Rods expressing mutant GCAP1 not only survive in the absence of RetGC1 but also remain functional, albeit with reduced electroretinography (ERG) amplitudes typical of RetGC1-/- genotype. The rod ERG recovery from a strong flash, only slightly affected in both RetGC1-/- and RetGC2-/- mice, becomes very slow in RetGC1-/- but not RetGC2-/- mice when GCAP2 is not available to provide Ca²âº feedback to the remaining RetGC isozyme. The intrinsic biochemical properties of RetGC and GCAP determined in vitro do not explain the observed phenomena. Instead, our results argue that there must be a cellular mechanism that limits GCAP1 access to RetGC2 and makes RetGC1 isozyme a preferential target for the disease-causing GCAP1 mutants. A more general conclusion from our findings is that nondiscriminatory interactions between homologous effector enzymes and their regulatory proteins permitted by their intrinsic biochemical properties can be effectively restricted in a living photoreceptor.

Enzymatic properties and regulation of the native isozymes of retinal membrane guanylyl cyclase (RetGC) from mouse photoreceptors.

Mouse photoreceptor function and survival critically depend on Ca(2+)-regulated retinal membrane guanylyl cyclase (RetGC), comprised of two isozymes, RetGC1 and RetGC2. We characterized the content, catalytic constants, and regulation of native RetGC1 and RetGC2 isozymes using mice lacking guanylyl cyclase activating proteins GCAP1 and GCAP2 and deficient for either GUCY2F or GUCY2E genes, respectively. We found that the characteristics of both native RetGC isozymes were considerably different from other reported estimates made for mammalian RetGCs: the content of RetGC1 per mouse rod outer segments (ROS) was at least 3-fold lower, the molar ratio (RetGC2:RetGC1) 6-fold higher, and the catalytic constants of both GCAP-activated isozymes between 12- and 19-fold higher than previously measured in bovine ROS. The native RetGC isozymes had different basal activity and were accelerated 5-28-fold at physiological concentrations of GCAPs. RetGC2 alone was capable of contributing as much as 135-165 µM cGMP s(-1) or almost 23-28% to the maximal cGMP synthesis rate in mouse ROS. At the maximal level of activation by GCAP, this isozyme alone could provide a significantly high rate of cGMP synthesis compared to what is expected for normal recovery of a mouse rod, and this can help explain some of the unresolved paradoxes of rod physiology. GCAP-activated native RetGC1 and RetGC2 were less sensitive to inhibition by Ca(2+) in the presence of GCAP1 (EC(50Ca) ∼132-139 nM) than GCAP2 (EC(50Ca) ∼50-59 nM), thus arguing that Ca(2+) sensor properties of GCAP in a functional RetGC/GCAP complex are defined not by a particular target isozyme but the intrinsic properties of GCAPs themselves.

Immune status & enzymes activity in blood lymphocytes in adult patients at different stages of acute lymphoblastic leukaemia.

BACKGROUND & OBJECTIVES: Pathogenesis acute lymphoblastic leukaemia ( ALL ) in adults is not well understood, as it is more common in children. We examined the immunological status and the activity of certain enzymes in blood lymphocytes in adult patients of ALL at different stages. METHODS: ALL patients (n=71) admitted during 2000-2005 were included in this study. All patients had decreased T-lymphocytes content. At first attack, they had CD4 + -cells decreasing and increasing IgM and IgG concentration. In complete remission all examined parameters were low. The peculiarities of ALL recurrence were high NK-cells content and disbalances of the main immunoglobulin concentrations. RESULTS: In the first attack and recurrence the anaerobe glucose oxidation intensity and the reactions of macromolecular synthesis were lower in lymphocytes compared to control. In remission all these processes restored to normal. In all stages in lymphocytes GR had decreased activity. INTERPRETATION & CONCLUSIONS: Our results showed that most of changes in immune status of ALL patients were in a stage of complete remission when patients arrived on its maintenance through the small period from spent before therapy when the immune system of the patient has not been restored. Thus, probably cytostatic action causes immune failure in the future and starts disease again.

Constitutive excitation by Gly90Asp rhodopsin rescues rods from degeneration caused by elevated production of cGMP in the dark.

Previous experiments indicate that congenital human retinal degeneration caused by genetic mutations that change the Ca(2+) sensitivity of retinal guanylyl cyclase (retGC) can result from an increase in concentration of free intracellular cGMP and Ca(2+) in the photoreceptors. To rescue degeneration in transgenic mouse models having either the Y99C or E155G mutations of the retGC modulator guanylyl cyclase-activating protein 1 (GCAP-1), which produce elevated cGMP synthesis in the dark, we used the G90D rhodopsin mutation, which produces constitutive stimulation of cGMP hydrolysis. The effects of the G90D transgene were evaluated by measuring retGC activity biochemically, by recording single rod and electroretinogram (ERG) responses, by intracellular free Ca(2+) measurement, and by retinal morphological analysis. Although the G90D rhodopsin did not alter the abnormal Ca(2+) sensitivity of retGC in the double-mutant animals, the intracellular free cGMP and Ca(2+) concentrations returned close to normal levels, consistent with constitutive activation of the phosphodiesterase PDE6 cascade in darkness. G90D decreased the light sensitivity of rods but spared them from severe retinal degeneration in Y99C and E155G GCAP-1 mice. More than half of the photoreceptors remained alive, appeared morphologically normal, and produced electrical responses, at the time when their siblings lacking the G90D rhodopsin transgene lost the entire retinal outer nuclear layer and no longer responded to illumination. These experiments indicate that mutations that lead to increases in cGMP and Ca(2+) can trigger photoreceptor degeneration but that constitutive activation of the transduction cascade in these animals can greatly enhance cell survival.

Effects of low AIPL1 expression on phototransduction in rods.

PURPOSE: To investigate the impact of aryl hydrocarbon receptor-interacting protein-like (AIPL)-1 on photoreception in rods. METHODS: Photoresponses of mouse rods expressing lowered amounts of AIPL1 were studied by single-cell and electroretinogram (ERG) recordings. Phototransduction protein levels and enzymatic activities were determined in biochemical assays. Ca2+ dynamics were probed with a fluorescent dye. Comparisons were made to rods expressing mutant Y99C guanylate cyclase activating protein (GCAP)-1, to understand which effects arose from elevated dark levels of cGMP and Ca2+. RESULTS: Except for PDE, transduction protein levels were normal in low-AIPL1 retinas, as were guanylate cyclase (GC), rhodopsin kinase (RK), and normalized phosphodiesterase (PDE) activities. Y99C and low-AIPL1 rods were more sensitive to flashes than normal, but flash responses of low-AIPL1 rods showed an abnormal delay, reduced rate of increase, and longer recovery not present in Y99C rod responses. In addition, low-AIPL1 rods but not Y99C rods failed to reach the normal light-induced minimum in Ca2+ concentration. CONCLUSIONS: Reduced AIPL1 delayed the photoresponse, decreased its amplification constant, slowed a rate-limiting step in its recovery, and limited the light-induced decrease in Ca2+. Not all changes were attributable to decreased PDE or to elevated cGMP and Ca2+ in darkness. Therefore, AIPL1 directly or indirectly affects more than one component of phototransduction.

Mole quantity of RPE65 and its productivity in the generation of 11-cis-retinal from retinyl esters in the living mouse eye.

RPE65, a protein expressed in cells of the retinal pigment epithelium of the eye, is essential for the synthesis by isomerohydrolase of 11-cis-retinal, the chromophore of rod and cone opsins. Recent work has established that RPE65 is a retinyl ester binding protein, and as all-trans-retinyl esters are the substrate for isomerohydrolase activity, the hypothesis has emerged that RPE65 serves to deliver substrate to this enzyme or complex. We bred mice with five distinct combinations of the RPE65 Leu450/Met450 variants (Leu/Leu, Met/Met, Leu/Met, Leu/-, and Met/-), measured in mice of each genotype the mole quantity of RPE65 per eye, and measured the initial rate of rhodopsin regeneration after a nearly complete bleach of rhodopsin to estimate the maximum rate of 11-cis-retinal synthesis in vivo. The quantity of RPE65 per eye ranged from 5.7 pmol (Balb/c) to 0.32 pmol (C57BL/6N x Rpe65(-)(/)(-)); the initial rate of rhodopsin regeneration was a Michaelis function of RPE65, where V(max) = 18 pmol/min per eye and K(m) = 1.7 pmol, and not dependent on the Leu450/Met450 variant. At RPE65 levels well below the K(m), the rate of production of 11-cis-retinal per RPE65 molecule was approximately 10 min(-)(1). Thus, the results imply that as a chaperone each RPE65 molecule can deliver retinyl ester to the isomerohydrolase at a rate of 10 molecules/min; should RPE65 itself be identified as the isomerase, each copy must be able to produce at least 10 molecules of 11-cis-retinal per minute.

The Y99C mutation in guanylyl cyclase-activating protein 1 increases intracellular Ca2+ and causes photoreceptor degeneration in transgenic mice.

Guanylyl cyclase-activating proteins (GCAPs) are Ca2+-binding proteins that activate guanylyl cyclase when free Ca2+ concentrations in retinal rods and cones fall after illumination and inhibit the cyclase when free Ca2+ reaches its resting level in the dark. Several forms of retinal dystrophy are caused by mutations in GUCA1A, the gene coding for GCAP1. To investigate the cellular mechanisms affected by the diseased state, we created transgenic mice that express GCAP1 with a Tyr99Cys substitution (Y99C GCAP1) found in human patients with a late-onset retinal dystrophy (Payne et al., 1998). Y99C GCAP1 shifted the Ca2+ sensitivity of the guanylyl cyclase in photoreceptors, keeping it partially active at 250 nM free Ca2+, the normal resting Ca2+ concentration in darkness. The enhanced activity of the cyclase in the dark increased cyclic nucleotide-gated channel activity and elevated the rod outer segment Ca2+ concentration in darkness, measured by using fluo-5F and laser spot microscopy. In different lines of transgenic mice the magnitude of this effect rose with the Y99C GCAP1 expression. Surprisingly, there was little change in the rod photoresponse, indicating that dynamic Ca2+-dependent regulation of cGMP synthesis was preserved. However, the photoreceptors in these mice degenerated, and the rate of the cell loss increased with the level of the transgene expression, unlike in transgenic mice that overexpressed normal GCAP1. These results provide the first direct evidence that a mutation linked to congenital blindness increases Ca2+ in the outer segment, which may trigger the apoptotic process.