Palmitoylation of Superoxide Dismutase 1 (SOD1) Is Increased for Familial Amyotrophic Lateral Sclerosis-linked SOD1 Mutants

Mutations in Cu,Zn-superoxide dismutase (mtSOD1) cause familial amyotrophic lateral sclerosis (FALS), a neurodegenerative disease resulting from motor neuron degeneration. Here, we demonstrate that wild type SOD1 (wtSOD1) undergoes palmitoylation, a reversible post-translational modification that can regulate protein structure, function, and localization. SOD1 palmitoylation was confirmed by multiple techniques, including acyl-biotin exchange, click chemistry, cysteine mutagenesis, and mass spectrometry. Mass spectrometry and cysteine mutagenesis demonstrated that cysteine residue 6 was the primary site of palmitoylation. The palmitoylation of FALS-linked mtSOD1s (A4V and G93A) was significantly increased relative to that of wtSOD1 expressed in HEK cells and a motor neuron cell line. The palmitoylation of FALS-linked mtSOD1s (G93A and G85R) was also increased relative to that of wtSOD1 when assayed from transgenic mouse spinal cords. We found that the level of SOD1 palmitoylation correlated with the level of membrane-associated SOD1, suggesting a role for palmitoylation in targeting SOD1 to membranes. We further observed that palmitoylation occurred predominantly on disulfide-reduced as opposed to disulfide-bonded SOD1, suggesting that immature SOD1 is the primarily palmitoylated species. Increases in SOD1 disulfide bonding and maturation with increased copper chaperone for SOD1 expression caused a decrease in wtSOD1 palmitoylation. Copper chaperone for SOD1 overexpression decreased A4V palmitoylation less than wtSOD1 and had little effect on G93A mtSOD1 palmitoylation. These findings suggest that SOD1 palmitoylation occurs prior to disulfide bonding during SOD1 maturation and that palmitoylation is increased when disulfide bonding is delayed or decreased as observed for several mtSOD1s.     

Investigation of genotoxic and antigenotoxic activities of chlorophylls and chlorophyllin in cultured V79 cells

Chlorophyll and its derivatives are examples of plant compounds (purified and/or extracted) which appear to protect DNA from damage caused by chemical or physical agents, although some studies have identified clastogenic activity of these compounds. This study was carried out to assess the genotoxic activity of chlorophyll-a (Chl-a), -b (Chl-b) and chlorophyllin (Chl) and their antigenotoxic activity against the DNA damage induced by methyl methanesulphonate (MMS) under conditions of simultaneous, pre-, post-treatment, and simultaneous treatment after pre-incubation of the chemical with MMS. The micronucleus (MN) test was used in binucleated cells (induced by cytochalasin-B) of a mammalian cell line (V79). The three concentrations of Chl-a, Chl-b or Chl (0.1375, 0.275, 0.55microM) were not genotoxic and the genotoxic action of MMS (400microM) decreased (74-117%) under all treatment conditions. The results showed that there was no significant difference among the treatment types, the concentration or the nature of chlorophyll used. The data obtained suggest that Chl-a, Chl-b and Chl when associated with the DNA damaging agent, MMS, may protect the DNA by desgenotoxic action and/or by bio-antigenotoxic mechanisms, with the similar efficiency.     

Enzymes and chelating agent in cotton pretreatment

Desized cotton fabric and cotton seed-coat fragments (impurities) have been treated with commercial cellulase (Celluclast 1.5 L), hemicellulase–pectinase (Viscozyme 120 L) and xylanase (Pulpzyme HC) enzymes. Seed-coat fragments hydrolyzed much faster than the cotton fabric itself. This relative difference in hydrolysis rates makes possible a direct enzymatic removal of seed-coat fragments from desized cotton fabric. Addition of chelating agents such as ethylenediamine-tetra-acetic acid (EDTA) markedly enhanced the directed enzyme action. Pretreatments carried out in acidic solution at pH 5 increased the lightness of seed-coat fragments, contrary to the samples treated in neutral medium at pH 7. Alkaline scouring resulted in darker seed-coat fragments except for the samples pretreated with Pulpzyme HC plus EDTA. This effect is similar to that observed in the biobleaching process in pulp and paper industry.     

Involvement of the metabolic hormones leptin, ghrelin, obestatin, IGF-I and of MAP kinase in control of porcine oocyte maturation

The general aim of our in vitro experiments was to study the role of the metabolic hormones leptin, ghrelin, obestatin and IGF-I and mitogen-activated protein kinase (MAPK)-dependent intracellular mechanisms in the control of nuclear maturation of porcine oocytes. For this purpose, porcine oocytes were isolated from the ovary and cultured in the presence of leptin, ghrelin, obestatin, IGF-I, MAPK blocker PD98059 and the combinations of hormones with PD98059. Proportions of matured oocytes (at metaphase II of meiosis, determined by DAPI staining) and of oocytes containing MAPK/ERK1-2 (determined by immunocytochemistry) were measured before and after culture. It was observed that the majority of oocytes isolated from the ovary before culture were immature and did not contain visible MAPK, but some oocytes were mature, and the majority of these oocytes contained MAPK. Incubation of oocytes resulted in a significant increase in the proportion of matured oocytes and in the percentage of oocytes containing MAPK in both the matured and not matured groups. Addition of IGF-I to the culture medium increased the proportion of matured oocytes, addition of leptin decreased it, and ghrelin and obestatin did not oocyte maturation. Addition of hormones did not affect the expression of MAPK in either immature or mature oocytes. PD98059, when given alone, suppressed the maturation and accumulation of MAPK in both mature and immature oocytes. When given together with hormones, PD98059 was able to reduce the stimulatory effect of IGF-I, to invert the inhibitory action of leptin to stimulatory and to induce the stimulatory action of ghrelin and obestatin on meiosis. IGF-I, ghrelin and obestatin, but not leptin, when given together with PD98059, increased the accumulation of MAPK in both immature and mature oocytes. Association of nuclear maturation and expression of MAPK in oocytes before, but not after culture, as well as the prevention of oocyte maturation by MAPK blocker suggests the involvement of MAPK-dependent intracellular mechanisms in the promotion of reinitiation, but not completion of meiosis. The effect of hormonal additions on meiosis of oocytes suggests that IGF-I is a stimulator, leptin can be an inhibitor, while ghrelin and obestatin probably do not control oocyte maturation. The ability of PD98059 to modify the effect of hormones on oocyte maturation and on MAPK expression suggests possible interference of hormones and MAPK-dependent intracellular mechanisms in oocytes. However, no influence of hormones on MAPK and lack of association between action of hormones and PD98059 on MAPK and meiosis suggest that MAPK is probably not a mediator of effect of IGF-I, leptin, ghrelin and obestatin on porcine oocyte nuclear maturation.     

L’apport de l’imagerie hybride TEMP/TDM dans la prise en charge du cancer différencié de la thyroïde

IntroductionSingle photon emission computed tomography combined with a low dose computed tomography (SPECT/CT), is a hybrid imaging integrating functional and anatomical data. The purpose of our study was to evaluate the contribution of the SPECT/CT over traditional planar imaging of patients with differentiated thyroid carcinoma (DTC).MethodsPost-therapy iodine 131 (¹³¹I) whole-body scan followed by cervico-thoracic SPECT/CT, were performed in 100 patients with DTC.ResultsAmong these 100 patients followed for a predominantly papillary DTC, planar imaging and SPECT/CT, were perfectly concordant in 70% of patients and discordant in the remaining 30%. The use of fusion imaging SPECT/CT compared to conventional planar imaging allowed us to correct our therapeutic approach in 27% (27/100 patients), according to the protocols of therapeutic management of our institute.ConclusionSPECT/CT is a hybrid imaging modality which provides better identification and more correct anatomic localization of the foci of radioiodine uptake with impact on therapeutic management.     

Catalase activity in developing seedling of opium poppyPapaver somniferum L

Catalase is an enzyme unique to glyoxysomes in developing poppy seedlings. Catalase activity is very low in endosperm and in embryo of germinating poppy seeds. During postgerminative growth and development the enzyme activity increases rapidly with maximum in endosperm on day 2 and in developing seedling on day 3. A rapid decline of enzyme activity parallells the extension growth of poppy seedlings. Three electrophoretic forms of catalase have been detected in isolated glyoxysomes and partially purified catalase preparation. Electron microscopic observation indicates the presence of catalase in glyoxysomes of parenchyma cells of poppy seedling cotyledons. Numerous lipid bodies and electron-dense deposits in vacuoles are the most characteristic feature of these cells.     

Novel coumarin derivatives as potent acetylcholinesterase inhibitors: insight into efficacy,mode and site of inhibition

The inhibitory efficacy of two substituted coumarin derivatives on the activity of neurodegenerative enzyme acetylcholinesterase (AChE) was assessed in aqueous buffer as well as in the presence of human serum albumin (HSA) and compared against standard cholinergic AD drug, Donepezil (DON). The experimental data revealed the inhibition to be of non-competitive type with both the systems showing substantial inhibitory activity on AChE. In fact, one of the tested compounds Chromenyl Coumarate (CC) was found to be better inhibitor (IC₅₀ = 48.49 ± 5.6 nM) than the reference drug DON (IC₅₀ = 74.13 ± 8.3 nM), unequivocally amplifying its importance. The structure of the compound was found to play a vital role in the inhibitory efficiency, validating previous Structure Activity Relationship (SAR) reviews for coumarin. The mechanism of inhibition remained impervious when the experimental medium was switched from aqueous buffer to HSA, albeit noticeable change in the inhibition potency of the compound 3, 3′- Methylene-bis (4-hydroxy coumarin) (MHC) (38%) and CC (35%). Both the coumarin derivatives were observed to bind to the peripheral anionic site (PAS) of AChE and also found to displace the fluorescence marker thioflavinT (ThT) from AChE binding pocket. All experimental observations were seconded by molecular docking and MD simulation results. The inferences drawn in this study form a foundation for further investigation on these compounds; magnifying the probability of their usage as AD drugs and re-emphasizes the significance of drug delivery media while considering the inhibition potencies of targeted drugs.     

Évaluation de l’irradiation additionnelle des patients de médecine nucléaire en imagerie hybride TEMP/TDM

One hundred and ten consecutive patients and 130 SPECT/CT examinations were involved in this retrospective study that focused on the evaluation of the excess of dose contributed by the CT to the patient during the SPECT/CT explorations, for routine examinations in nuclear medicine. The average age of patients was 53 years. In this study, it appeared that irradiation induced by a low dose CT combined with a SPECT is low compared to that of a diagnostic CT. The main risk on patients is the occurrence of radiation-induced cancer. In our study, this increased risk induced by the additional CT with low dose settings in line with SPECT examination, is not significant and does not exceed 0.026%. By weighing the diagnostic value of SPECT/CT examination with that of a stand-alone SPECT examination dosimetric “incremental cost” is justified because of its direct clinical benefit conveyed to the patient.