Partial purification of tightly bound mitochondrial hexokinase from maize (Zea mays L.) root membranes

In mammals, hexokinase (HK) is strategically located at the outer membrane of mitochondria bound to the porin protein. The mitochondrial HK is a crucial modulator of apoptosis and reactive oxygen species generation. In plants, these properties related to HK are unknown. In order to better understand the physiological role of non-cytosolic hexokinase (NC-HK) in plants, we developed a purification strategy here described. Crude extract of 400 g of maize roots (230 mg protein) contained a specific activity of 0.042 µmol G6P min-1 mg PTN-1. After solubilization with detergent two fractions were obtained by DEAE column chromatography, NC-HK 1 (specific activity = 3.6 µmol G6P min-1 mg PTN-1 and protein recovered = 0.7 mg) and NC-HK 2. A major purification (yield = 500-fold) was obtained after passage of NC-HK 1 through the hydrophobic phenyl-Sepharose column. The total amount of protein and activity recovered were 0.04 and 18%, respectively. The NC-HK 1 binds to the hydrophobic phenyl-Sepharose matrix, as observed for rat brain HK. Mild chymotrypsin digestion did not affect adsorption of NC-HK 1 to the hydrophobic column as it does for rat HK I. In contrast to mammal mitochondrial HK, glucose-6-phosphate, clotrimazole or thiopental did not dissociate NC-HK from maize (Zea mays) or rice (Oryza sativa) mitochondrial membranes. These data show that the interaction between maize or rice NC-HK to mitochondria differs from that reported in mammals, where the mitochondrial enzyme can be displaced by modulators or pharmacological agents known to interfere with the enzyme binding properties with the mitochondrial porin protein.

Solubilization of human erythrocyte membranes by ASB detergents

Understanding the membrane solubilization process and finding effective solubilizing agents are crucial challenges in biochemical research. Here we report results on the interaction of the novel linear alkylamido propyl dimethyl amino propanosulfonate detergents, ASB-14 and ASB-16, with human erythrocyte membranes. An estimation of the critical micelle concentration of these zwitterionic detergents (ASB-14 = 100 µM and ASB-16 = 10 µM) was obtained using electron paramagnetic resonance. The amount of proteins and cholesterol solubilized from erythrocytes by these detergents was then determined. The hemolytic activities of the ASB detergents were assayed and the detergent/lipid molar ratios for the onset of hemolysis (Re sat) and total lysis (Re sol) were calculated, allowing the determination of the membrane binding constants (Kb). ASB-14 presented lower membrane affinity (Kb = 7050 M-1) than ASB-16 (Kb = 15610 M-1). The amount of proteins and cholesterol solubilized by both ASB detergents was higher while Re sat values (0.22 and 0.08 detergent/lipid for ASB-14 and ASB-16, respectively) were smaller than those observed with the classic detergents CHAPS and Triton X-100. These results reveal that, besides their well-known use as membrane protein solubilizers to enhance the resolution of two dimensional electrophoresis/mass spectrometry, ASB-14 and ASB-16 are strong hemolytic agents. We propose that the physicochemical properties of ASB detergents determine their membrane disruption efficiency and can help to explain the improvement in the solubilization of membrane proteins, as reported in the literature.

Structural properties of lipid reconstructs and lipid composition of normotensive and hypertensive rat vascular smooth muscle cell membranes

Multiple cell membrane alterations have been reported to be the cause of various forms of hypertension. The present study focuses on the lipid portion of the membranes, characterizing the microviscosity of membranes reconstituted with lipids extracted from the aorta and mesenteric arteries of spontaneously hypertensive (SHR) and normotensive control rat strains (WKY and NWR). Membrane-incorporated phospholipid spin labels were used to monitor the bilayer structure at different depths. The packing of lipids extracted from both aorta and mesenteric arteries of normotensive and hypertensive rats was similar. Lipid extract analysis showed similar phospholipid composition for all membranes. However, cholesterol content was lower in SHR arteries than in normotensive animal arteries. These findings contrast with the fact that the SHR aorta is hyporeactive while the SHR mesenteric artery is hyperreactive to vasopressor agents when compared to the vessels of normotensive animal strains. Hence, factors other than microviscosity of bulk lipids contribute to the vascular smooth muscle reactivity and hypertension of SHR. The excess cholesterol in the arteries of normotensive animal strains apparently is not dissolved in bulk lipids and is not directly related to vascular reactivity since it is present in both the aorta and mesenteric arteries. The lower cholesterol concentrations in SHR arteries may in fact result from metabolic differences due to the hypertensive state or to genes that co-segregate with those that determine hypertension during the process of strain selection.

In vitro cultivation of canine multipotent mesenchymal stromal cells on collagen membranes treated with hyaluronic acid for cell therapy and tissue regeneration

Support structures for dermal regeneration are composed of biodegradable and bioresorbable polymers, animal skin or tendons, or are bacteria products. The use of such materials is controversial due to their low efficiency. An important area within tissue engineering is the application of multipotent mesenchymal stromal cells (MSCs) to reparative surgery. The combined use of biodegradable membranes with stem cell therapy may lead to promising results for patients undergoing unsuccessful conventional treatments. Thus, the aim of this study was to test the efficacy of using membranes composed of anionic collagen with or without the addition of hyaluronic acid (HA) as a substrate for adhesion and in vitro differentiation of bone marrow-derived canine MSCs. The benefit of basic fibroblast growth factor (bFGF) on the differentiation of cells in culture was also tested. MSCs were collected from dog bone marrow, isolated and grown on collagen scaffolds with or without HA. Cell viability, proliferation rate, and cellular toxicity were analyzed after 7 days. The cultured cells showed uniform growth and morphological characteristics of undifferentiated MSCs, which demonstrated that MSCs successfully adapted to the culture conditions established by collagen scaffolds with or without HA. This demonstrates that such scaffolds are promising for applications to tissue regeneration. bFGF significantly increased the proliferative rate of MSCs by 63% when compared to groups without the addition of the growth factor. However, the addition of bFGF becomes limiting, since it has an inhibitory effect at high concentrations in culture medium.

Antioxidant effect of 4-nerolidylcatechol and α-tocopherol in erythrocyte ghost membranes and phospholipid bilayers

4-Nerolidylcatechol (4-NC) is found in Pothomorphe umbellataroot extracts and is reported to have a topical protective effect against UVB radiation-induced skin damage, toxicity in melanoma cell lines, and antimalarial activity. We report a comparative study of the antioxidant activity of 4-NC and α-tocopherol against lipid peroxidation initiated by two free radical-generating systems: 2,2′-azobis(2-aminopropane) hydrochloride (AAPH) and FeSO4/H2O2, in red blood cell ghost membranes and in egg phosphatidylcholine (PC) vesicles. Lipid peroxidation was monitored by membrane fluidity changes assessed by electron paramagnetic resonance spectroscopy of a spin-labeled lipid and by the formation of thiobarbituric acid-reactive substances. When lipoperoxidation was initiated by the hydroxyl radical in erythrocyte ghost membranes, both 4-NC and α-tocopherol acted in a very efficient manner. However, lower activities were observed when lipoperoxidation was initiated by the peroxyl radical; and, in this case, the protective effect of α-tocopherol was lower than that of 4-NC. In egg PC vesicles, malondialdehyde formation indicated that 4-NC was effective against lipoperoxidation initiated by both AAPH and FeSO4/H2O2, whereas α-tocopherol was less efficient in protecting against lipoperoxidation by AAPH, and behaved as a pro-oxidant for FeSO4/H2O2. The DPPH (2,2-diphenyl-1-picrylhydrazyl) free-radical assay indicated that two free radicals were scavenged per 4-NC molecule, and one free radical was scavenged per α-tocopherol molecule. These data provide new insights into the antioxidant capacity of 4-NC, which may have therapeutic applications for formulations designed to protect the skin from sunlight irradiation.

Ultrafiltration performance of PVDF, PES, and cellulose membranes for the treatment of coconut water (Cocos nucifera L.)

Ultrafiltration (UF) inhibits the enzymatic activity which is responsible for color changes of coconut water without the need for heat treatment. In the present study, UF performance in terms of the permeate flux and enzymatic retention of the coconut water was evaluated at laboratory unit (LU) and pilot unit (PU). The membranes studied were polyethersulfone 150 kDa (UP150), polyvinylidene fluoride 150 kDa (UV150) and cellulose 30 kDa (UC030). The UP150 membrane showed the best permeate flux. The UC030 membrane showed the lowest flux, but it resulted in 100% enzymatic retention, while the other membranes showed enzymatic retentions between 71 and 85%. The application of the UC030 in the pilot unit (PU) resulted in a flux value higher than that obtained in the LU due to the tangential velocity effect. The UC030 membrane has proved adequate for industrial applications.

Armoriaux étrangers. II « Armoriaux différens, particulièrement d'Italie », mss. et impr.

Contient : Noblesse d'Aragon, Biscaye, Bohême, Catalogne, Crète et Dalmatie, Guipuscoa, Irlande, Livonie, Majorque, Ostfrise, Poméranie, Prusse, Raguse ; « Tutte le famiglie che nel 1637 godono la nobiltà di Catania, raccolte da don Alberto Palizzi. » Impr. Palerme, Alfonso dell'Isola, 1638, placard [blasons gravés] ; « Quinta parte de' Lustri Ravennati, a P. D. Seraphino Pasolino, abbate congregationis [canonicorum regularium Lateranensium]... » Impr. S. l. n. d., [1684], in-4°, p. 215-239 [blasons gravés]

Fragments de mss.

Poésies (1, 8). — Lettres de Gautier de Mortagne (2), Pierre [Abélard] (6v), frère Eudes (59), Yves de Chartres (98). — Concile de Pont-Audemer en 1279 (10). — Ars dictaminis (20). — Sermons (31). — Homélie de S. Césaire (82v). — Canons (87). — Lettres formées (95v). — Lettre de L., abbé de Pouthières, sur la grammaire (107). — Gerbert sur Porphyre (109). — Questions sur l'Écriture Sainte (117). — Préface de Juvencus (119 et 95). — Traité d'Arnoul de Lisieux contre Girard d'Angoulême (121 et 145). — Libellus Hug. de S. Victore de virtute amoris (137). — Miracle de S. Martin par un clerc d'Utrecht (155).

Recueil de pièces, mss. et imprimées, sur l'histoire d'Amérique, formé par le P. Léonard de Ste-Catherine de Sienne, Augustin déchaussé.

Contient : Mémoire adressé au Roy par le sr Champlain. Impr. in-4° ; Notes sur le Canada et l'évêché de Quebec ; « Information concernant l'affaire de Darien en 1699 ; » copie de l'imprimé ; « Les costes aux environs de la rivière de Misisipi, découvertes par Mr de la Salle en 1683,... par N. de Fer. 1701. » Carte et texte gravés ; Pièces diverses sur le fleuve Mississipi, parmi lesquelles une copie de la « lettre de M. d'Iberville, ou relation de son voyage... à l'embouchure du fleuve de Mississipi... 1699 ; » — fol. 49 : « Journal du voyage fait [à l'embouchure de la rivière de Mississipi] par... M. d'Iberville et le chevalier de Surgères... » (1698-1699) ; — fol. 107 : « Croquis du Mississipy..., par le sr Tonty... 1700 ; » — fol. 108 : « Campagne du vaisseau de la Renommée, en 1699 et 1700, sur la rivière de Mississipi » ; — fol. 119 : « Extrait de la relation des avantures et voyages de Mathieu Sàgean, 1701 ; » — fol. 127 : « Letre historique touchant le Mississipi, écrite à Paris, le 6 aoust 1702, par M. de Remonville » ; « Relation du voyage du sr de Beauchesne au Chilli, dans la mer du sud de l'Amérique, par le destroit de Magellan... » (1698-1701) ; Autre relation du même ; « Mémoire pour la découverte et la conqueste des pays de Quivira et de Thequaye dans l'Amérique septentrionale » ; Copie d'une lettré anonyme sur un voyage de Porto-Rico à Carthagène et sur les combats livrés à la flotte anglaise (1702)

Répertoire des pièces concernant la Normandie dans les « Monstres et reveues... des chevaliers », « à la solde du Roy » (1363-1593), recueillies par Gaignières et conservées aujourd'hui à la Bibliothèque nationale, mss franç. 21945-21338 ; par LÉCHAUDÉ D'ANISY.

Contient : Copies de diverses pièces concernant l'histoire de Normandie, montres, etc., la plupart tirées des mss de la Bibliothèque nationale