1,721,017 research outputs found
CALORIMETRIA DIFFERENZIALE A SCANSIONE DI MISCELE FOSFOLIPIDICHE DA MEMBRANE DI GLOBULI ROSSI
No full textNegli ultimi anni particolare attenzione è stata posta alle relazioni tra funzione delle membrane, le loro proprietà chimico-fisiche e la composizione in acidi grassi, prevalentemente della frazione fosfolipidica considerato il fatto che fosfolipidi, trovati nelle membrane biologiche, derivano le loro proprietà idrofobiche dagli acidi grassi costituenti.
Le correlazioni più frequentemente analizzate sono quelle tra componenti di membrana e i loro acidi grassi modificati, in vivo, da alterazioni della composizione lipidica della dieta o, in vitro, mediante opportune modifiche del mezzo di coltura.
Somministrando diete diverse a bovine da latte abbiamo creato una “banca” di campioni di membrane di globuli rossi a diversa composizione lipidica (1, 2).
Scopi di questo studio sono stati: 1) l’analisi della composizione delle miscele di fosfolipidi estratti dalle membrane, 2) la valutazione delle correlazioni tra composizione e proprietà termotropiche delle miscele.
I lipidi totali sono stati estratti dagli eritrociti bovini con miscele di cloroformio/metanolo in diversi rapporti e le miscele lipidiche sono state frazionate mediante cromatografia su colonna.
Le singole specie fosfolipidiche sono state separate in HPTLC e quantificate mediante densitometria a scansione.
La composizione in acidi grassi dei fosfolipidi di membrana è stata valutata mediante gas-liquido cromatografia e dispersioni acquose tamponate sono state esaminate mediante calorimetria differenziale a scansione.
Per ogni campione è stato calcolato il contenuto di acidi grassi saturi (S), insaturi (U) e l’indice di insaturazione (UI).
I principali acidi grassi presenti (mediamente ≥ 10% in peso) sono risultati: C16:0, C18:0, C18:1, C18:2 e C24:0.
Le miscele di fosfolipi mostrano una o due transizioni di fase a temperature comprese tra 17.7 e 40.0 °C. La prima temperatura di transizione è compresa tra 17.7 e 26.0 °C.
UI e U/S non risultano essere correlati al contenuto percentuale delle diverse specie molecolari di fosfolipidi (PC, PE, SM, PS+PI) e di PC+SM, costituenti principali del foglietto esterno del doppio strato lipidico.
Esiste, invece, una correlazione lineare tra la prima temperatura di transizione ed UI (P < 0.05) e U/S (P < 0.01). La prima temperatura di transizione diminuisce all’aumentare di UI o del rapporto U/S.
References
1. G. Monticelli, S. Rapelli, G. Montorfano, P. Magistretti and B. Berra- “Red blood cell membrane composition following diet manipulation in the cow” Riv. Ital. Sostanze Grasse 67: 507-515 (1990)
2. G. Monticelli, M. Masserini,G. Lercker, T. Beringhelli, P. Marciani, E. Calappi and B. Berra – “Red blood cell membrane composition following diet manipulation in the cow. II: phospholipid fatty acid distribution and physico-chemical characteristics of membrane and its constituents” Riv. Ital. Sostanze Grasse 69: 189-199 (1992
The thermotropic behaviour of red blood cell membrane phospholipids
No full textPhospholipids are the major constituents of biological membranes and the lipid structural organization is important to membrane function. In this study we have examined, by differential scanning calorimetry (DSC), the thermal behaviour of phospholipids extracted from bovine red blood cell membrane and related the observed characteristics to the phospholipid classes in the mixture. Phospholípíd classes were separated by thin layer chromatography. DSC was performed on aqueous buffer dispersions (pH 7.4) approx. 3 mM. For all phospholipid samples at least one, or sometimes two transitions were observed. The calorimetric scans show the presence of a broad transition between 0 and 30 °C (midpoint of the transition at 24.1 - 24.5 °C) whereas the second phase transition occurs at a transition temperature of 32.4 - 38.1 °C. The analyzed membranes contained the same amounts of phosphatidylcholine (PC; 6 - 9 % w/w of total phospholipids) and phosphatidylethanolamine (PE; 24 – 32 %) but different percentual contents of sphingomyelin (SM; 16 – 31 %), which contributes to rigidification, and phosphatidylserine + phosphatidylinositol (PS + PI; 30 – 44 %) which may be considered as fluidizers, similar to lecithin. In all the analyzed samples practically identical temperatures at the first phase transition were obtained. The second phase transition, when occurring, was at very different temperatures. No direct correlation has been found between this second phase transition and the SM/PC ratio also if variations of this ratio in artificial bilayers and biological membranes have a pronounced effect on the system properties. The second phase transition appeared in samples characterized by an high value of the (PS + PI)/PC ratio (> 5.4) and the transition temperature increased with the value of this ratio. Further investigations seem to be necessary to consider the importance of the fatty acid composition of the studied mixtures on the thermotropic behaviour
Differential scanning calorimetry of erythrocyte reconstituted membrane
No full textThe thermal properties of biomembranes examined by dífferential calorimetry mimic the properties of bílayers formed from their isolated lipids or from synthetic lipids. In this study we have analyzed, by differential scanning calorimetry (DSC), the thermal behaviour of lipids extracted from bovine erythrocyte and of their reconstítuted mixtures. Membrane lipids were extracted from blood samples of 11 cows and separated in the three components: cholesterol, phospholipids and glycolipids. Phospholipid and glycolipid classes were determined by thin layer chromatography. Lipids from erythrocyte membrane of different animals (at controlled diet), at a given time, were pooled together at a proportional amount like in the original membranes. DSC was performed on aqueous buffer dispersions (pH 7.4) of the average lipid components but, ín some cases, the sample contained lipids ísolated from one animal. Average phospholipid sample exhibited two phase transitions: the first at 24 - 26 °C and the second at about 38 °C. Some scans of phospholípids from one animal show only the first transítíon which occurs between 23.1 and 25.4 °C whereas the second phase transition, when occurring, is at very different temperatures (32.4 - 38.1 °C). For all average glycolipid samples one transition was observed at about 35 °C. Mixtures of cholesterol and phospholipids show only one broad transition. In these míxtures the transition temperature is close to that of the second transition of phospholipid samples.
Some correlations between the transition temperatures and the amounts of different phospholipids in the samples have been tested but it seems important to consider the fatty acid composition of the studied míxtures to account for the different levels of saturated and unsaturated fatty acids
Change of ganglioside accessibility at the plasma membrane surface of cultured neurons, following Protein Kinase C activation
No full textWhile the mechanism of signal transduction across the plasma membrane from the exo- to the endoplasmic side has been extensively investigated, the possible return of messages back to the outer layer is less known. We studied the effect of protein kinase C activation on the ganglioside accessibility at the exoplasmic face of intact rat cerebellar granule cells in culture, using the enzyme sialidase as the probing molecule. Under the experimental conditions (1 milliunit/mL enzyme, 2 min incubation at 37 degrees C), only GT1b and GD1a gangliosides were partially affected by the enzyme (28.6 and 25.7% hydrolysis, respectively). After cell treatment with phorbol 12-myristate 13-acetate, inducing protein kinase C activation, GT1b and GD1a ganglioside susceptibility to sialidase was strongly decreased (8.6 and 15.9% hydrolysis, respectively). A reduction of ganglioside hydrolysis was also observed when protein kinase C activation was induced by cell treatment for 15 min with 100 mu M glutamate. On the contrary, accessibility did not vary when protein kinase C translocation was not effective (either in the absence of Ca2+ in the medium or using 1 mu M glutamate) or when the kinase activity was inhibited by staurosporine. These data suggest that following PKC activation, a key step of inbound transmembrane signaling, cell may dispatch outbound messages to the plasma membrane outer layer, changing the selective recognition and crypticity of glycolipids at the cell surface, possibly through a modulation of their segregation state
GM1 ganglioside-Triton X-100 mixed micelles. Transitions among different micellar species monitored by physicochemical and enzymatic methods
No full textAqueous dispersions of two amphiphiles, GM1 ganglioside and Triton X-100, in different proportions, were analysed for some physicochemical properties (surface tension, viscosity, consolution temperature) and for susceptibility to the action of galactose oxidase. By varying the molar ratio between the two components, well defined transitions among different micellar species were recorded by physicochemical measurements. Galactose oxidase was able to recognize the different species of mixed micelles, its kinetics displayed break points which exactly superimposed on those recorded, under the same conditions, by physicochemical methods
Characterization of biotinylated liposomes sensitive to temperature and pH: New tools for anti-cancer drug delivery
No full textWe describe a liposome formulation characterized by sensitivity to concurrent and small temperature and pH changes. Liposome permeability was assessed by monitoring the release of entrapped carboxyfluorescein (CF), using a fluorescence dequenching technique. The thermotropic behavior of the liposomes was investigated by differential scanning calorimetry. After 2 h at 37 degrees C in fetal calf serum, liposomes composed of a mixture of dipalmitoyl phosphatidylcholine/cholesterol/GM1 ganglioside/biotinoyl-dipalmitoyl phosphatidylethanolamine (100:20:6:0.25 molar ratio) released 8% CF at pH 7.4 and 12% CF at pH 6.7. At 41 degrees C the leakage was 72% at pH 7.4 and almost complete (99%) at pH 6.7. The pH and temperature sensitivity, with maximal release when the two circumstances occurred simultaneously, was confirmed by entrapping calcein or [C-14]glucose. The reasons for the bilayer sensitivity and the conditions for in vivo drug delivery are discussed
evidence for nonrandom distribution of GD1a ganglioside in rabbit brain microsomal membranes
No full textGD1a is the major ganglioside of rabbit brain microsomal membranes and occurs mainly with two molecular species, containing the C18:1 (62.3%) and C20:1 (37.7%) long-chain bases. The membranes were exposed to Vibrio cholerae (VC) sialidase under conditions where the enzyme hydrolyzed only GD1a (~9%), producing GM1 ganglioside, whereas the other gangliosides remained virtually unaffected. The long-chain-base analysis showed that newly-formed GM1 contained ~68% of the C20:1 molecular species. This indicates that VC sialidase did not randomly affect the two molecular species of GD1a but hydrolyzed preferentially the C20:1 one. In similar experiments, GD1a was inserted into the external layer of phosphatidylcholine vesicles and incubated with VC sialidase under conditions producing ~10% hydrolysis. Long-chain-base analysis showed that the proportion of C20:1 species in GM1 was 25.1% using vesicles composed of dipalmitoylphosphatidylcholine and 42.3% with egg phosphatidylcholine, whereas it was 39.2% in the starting GD1a. Therefore, in artificial membranes, VC sialidase acted preferentially on the C18:1 or C20:1 molecular species, depending on the length and unsaturation of the phospholipid fatty acids. Because VC sialidase is known to affect molecular dispersions more easily than packed aggregations of the gangliosidic substrate, the data suggest that in rabbit brain microsomal membranes the GD1a ganglioside molecular species carrying C20:1 long-chain base are more molecularly dispersed than those containing C18:1 long-chain base
Ganglioside lateralization in the brain of female rats
No full textThe ganglioside composition of the cerebral hemispheres of young and adult rats of either sex has been herein assessed for the first time, In females, the total ganglioside content at any age, the content of GM1, GD1a, and GD1b at 8 days, and the content of GM1, GD1b, GT1b, and GQ1b at 60 days were higher in the right than in the left hemisphere, In males, no difference was observed, Concerning the ceramide moiety, a difference was displayed by C18:1 long-chain base in GD1a, whose proportion was-higher in the left than in the right hemisphere of females aged 8 days, The comparison between homolateral hemispheres of rats of different sex revealed several differences, On average, in 8-day-old animals, the content of gangliosides was higher in females than in males, At 60 days the amount of gangliosides was on average lower in females than in males, even if with some exception, The data obtained with the current investigation show the existence of a ganglioside lateralization in rat brain, exclusively in females, and almost entirely at charge of the oligosaccharide portion, Moreover, age dependent changes of ganglioside pattern and content show a dependence on brain lateralizatio
Specific tritium labeling of gangliosides at the 3-position of sphingosines
No full textGM1 and GD1a gangliosides, treated with 2,3-dichloro-5,6-dicyano benzoquinone (DDQ) in the presence of Triton X-100 and in a toluene medium were specifically oxidized at the 3-position of sphingosine. The maximum reaction yield (65%) was obtained after 40 hours at 37 degrees C with the following molar ratio of reactants: ganglioside-Triton X-100-DDQ 1:70:125. The formation of the 3-keto derivatives of GM1 and GD1a was demonstrated by: a) the appearance of a sharp peak at 1700 cm-1 and of a broad band at 1250 cm-1 (typical of allylic ketones and of carbonyl groups, respectively) in the infra-red spectrum; b) the appearance of an absorption maximum at 230 nm, identical to that featured by 3-keto-cerebrosides, in the ultraviolet spectrum; c) the degradation of long chain bases during the process of release from gangliosides and derivatization for analysis by gas-liquid chromatography (expected for long chain bases carrying a keto group in the 3-position); and d) the quantitative transformation of 3-keto-GM1 and 3-keto-GD1a to GM1 and GD1a, respectively, upon NaBH4 reduction. Reduction of 3-keto-GM1 and 3-keto-GD1a with [3H]-NaBH4 produced 3H-labeled GM1 and GD1a. [3H]GM1 and [3H]GD1a maintained the same carbohydrate and fatty acid composition of the original GM1 and GD1a, and did not contain any saturated long chain bases. Direct proof that the label was at C-3 of long chain bases was given by reoxidation with DDQ, which completely removed the label, and by ozonolysis, after which label was retained on the oligosaccharide-containing fragment. More than 99% of incorporated radioactivity was carried by the long chain bases. The radiochemical purity of labeled gangliosides was greater than 95% and the specific radioactivity was 1.25 and 1.28 Ci/m mol for [3H]GM1 and [3H]GD1a, respectively
- …
