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R Bassi

Publications and source records attributed to R Bassi.

At least 19 recordsLinked to original sources
PubMed1992

Characterization of chlorophyll a/b proteins of photosystem I from Chlamydomonas reinhardtii.

In this study we have isolated the chlorophyll a/b-binding proteins from a photosystem I preparation of the green alga Chlamydomonas reinhardtii and characterized them by N-terminal sequencing, fluorescence, and absorption spectroscopy and by immunochemical means. The results indicate that in this organism, the light-harvesting complex of photosystem I (LHCI) is composed of at least seven distinct polypeptides of which a minimum number of three are shown to bind chlorophyll a and b. Both sequence homology and immunological cross-reactivity with other chlorophyll-binding proteins suggest that all of the LHCI polypeptides bind pigments. Fractionation of LHCI by mildly denaturing methods showed that, in contrast to higher plants, the long wavelength fluorescence emission typical of LHCI (705 nm in C. reinhardtii) cannot be correlated with the presence of specific polypeptides, but rather with changes in the aggregation state of the LHCI components. Reconstitution of both high aggregation state and long wavelength fluorescence emission from components that do not show these characteristics confirm this hypothesis.

Amino Acid Sequence↗
PubMed1992

Formation of free sphingosine and ceramide from exogenous ganglioside GM1 by cerebellar granule cells in culture.

Cerebellar granule cells differentiated in culture were incubated with ganglioside [3H-Sph]GM1 in order to have it inserted into the plasma membrane and metabolized. Among the formed metabolites radioactive sphingosine and ceramide were identified. [3H]Ceramide started to be measurable after 10 min of incubation (pulse), and [3H]sphingosine after 15 min. Their concentrations increased with pulse time, and, after a 1-hour pulse, with chase time. After a 1-hour pulse with 2 x 10(-6) M [3H-Sph]GM1 followed by a 4-hour chase, the amount of [3H]sphingosine and [3H]ceramide formed were 0.04 and 0.4 pmol/10(6) cells, respectively. Particularly the ability to produce sphingosine was higher in differentiated than in undifferentiated cells. It is concluded that ganglioside turnover contributes to the maintenance of the intracellular levels of free sphingosine and ceramide.

Cells, Cultured↗
PubMed1992

A supramolecular light-harvesting complex from chloroplast photosystem-II membranes.

In this study, we report on the composition of a photosystem-II antenna preparation which contains three chlorophyll-a/b proteins (CP), CP29, CP24 and light-harvesting complex (LHC) II obtained from Zea mays grana membranes as previously described [Dainese, P. & Bassi, R. (1991) J. Biol. Chem. 266, 8136-8142]. We demonstrate that the three chlorophyll proteins are present in the preparation with a 3:3:9 molar ratio and that they form a supramolecular antenna complex which represents one third of the photosystem-II antenna system. Phosphorylation experiments show that this complex is involved in the mechanism of regulation of excitation-energy distribution between photosystems: phosphorylation of the membranes induces dissociation of the LHCII moiety from the CP29-CP24 moiety and changes in the aggregation state of LHCII components of the CP29-CP24-LHCII complex. The LHCII subpopulations of the complex are shown to be distinct from the total LHCII population by isoelectrofocusing analysis. On the basis of these data and in the light of the stoichiometry of photosystem-II chlorophyll-binding proteins, we propose a model for the organization of photosystem-II antenna system.

Chloroplasts↗
PubMed1992

Ionic permeability of the mitochondrial outer membrane.

The ionic permeability of the outer mitochondrial membrane (OMM) was studied with the patch clamp technique. Electrical recording of intact mitochondria (hence of the outer membrane (OM], derived from mouse liver, showed the presence of currents corresponding to low conductances (less than 50 pS), as well as of four distinct conductances of 99 pS, 152 pS, 220 pS and 307 pS (in 150 mM KCl). The latter were voltage gated, being open preferentially at positive (pipette) potentials. Very similar currents were found by patch clamping liposomes containing the isolated OM derived from rat brain mitochondria. Here a conductance of approximately 530 pS, resembling in its electrical characteristics a conductance already attributed to mitochondrial contact sites (Moran et al. 1990), was also detected. Immunoblot assays of mitochondria and of the isolated OM with antibodies against the outer membrane voltage-dependent anion channel (VDAC) (Colombini 1979), showed the presence of the anion channel in each case. However, the typical electrical behaviour displayed by such a channel in planar bilayers could not be detected under our experimental conditions. From this study, the permeability of the OMM appears different from what has been reported hitherto, yet is more in line with that multifarious and dynamic structure which apparently should belong to it, at least within the framework of mitochondrial biogenesis (Pfanner and Neupert 1990).

Animals↗
PubMed1992

The role of the ganglioside lipid moiety in the process of ganglioside-cell interactions.

The role of the ceramide moiety of gangliosides, together with the deriving aggregative properties of ganglioside in solution, in the process of ganglioside-cell interactions was studied. The natural GM1(stearoyl) and the synthetic GM1(acetyl), containing the stearoyl and acetyl groups as the acyl moiety, respectively, were used in binding experiments to rat cerebellar granule cells. Regardless of the cell culture conditions, such as the presence of absence of fetal calf serum, the association of GM1(acetyl) to the cells was much greater than that of GM1(stearoyl). GM1(acetyl) was present in the incubation medium as monomers. After incubation, a large part of the total GM1(acetyl) associated to cells, 76-93% depending on the experimental conditions, was removed by washing with protein solutions. The remaining associated ganglioside was not removed by repeating washing with protein solutions or trypsin treatments and was considered as a component of the membrane. The cell association of GM1(stearoyl), present in solution as monomers as well as micelles, could be classified as serum-labile, trypsin-labile and trypsin-stable. The trypsin-stable form of association, corresponding to the molecules stably inserted into the membrane, was proportionally higher, the proportions varying with increasing incubation time and decreasing ganglioside concentration. This form of association was particularly high when incubation was performed in the presence of fetal calf serum. Incubation experiments performed with a mixture of GM1(stearoyl) and GM1(acetyl) in a molar ratio which allowed their presence in the medium as monomers as well as mixed micelles, led to a ganglioside association suggesting that besides the aggregative properties of the molecule other ganglioside properties are involved in the ganglioside-cell interaction process.

Animals↗
PubMed1991

Lateral redistribution of cytochrome b6/f complexes along thylakoid membranes upon state transitions.

The cytochrome b6/f complex operates in photosynthetic electron transfer either in linear electron flow from photosystem II to photosystem I or in cyclic flow around photosystem I. Using membrane fractionation and immunocytochemistry, we show a change in lateral distribution of cytochrome b6/f complexes along the thylakoid membranes during state transitions. This change is seen in maize as well as in the green algae Chlamydomonas reinhardtii. When either of the two organisms were adapted to state II in vivo, the proportion of cytochrome b6/f complexes found in the photosystem I-enriched stroma lamellae regions was significantly larger than after adaptation to state I. A similar observation was made upon state I to state II transitions done in vitro by illuminating, in the presence of ATP, broken maize chloroplasts prepared from dark-adapted leaves. This reorganization of the electron-transfer chain is concurrent with the change in light-energy distribution between the two photosystems, which requires lateral displacement of light-harvesting complex II. That the changes in lateral distribution of both cytochrome b6/f and light-harvesting II complexes seen upon state transition in vitro similarly required addition of exogenous ATP, suggests that the change in cytochrome b6/f organization also depends on kinase activity. The increased concentration of cytochrome b6/f complexes in the vicinity of photosystem I in state II is discussed in terms of an increase in cyclic electron flow, thus favoring ATP production. Because transition to state II can be triggered in vivo by ATP depletion, we conclude that state transitions should be regarded not only as a light-adaptation mechanism but also as a rerouting of photosynthetic electron flow, enabling photosynthetic organisms to adapt to changes in the cell demand for ATP.

Chlamydomonas↗
PubMed1991

Cerebellar granule cells in culture exhibit a ganglioside-sialidase presumably linked to the plasma membrane.

Cerebellar granule cells differentiated in culture were incubated with ganglioside [3H-Sph]GD1a in order to have it inserted into the plasma membrane, internalized by endocytosis, and metabolized. The metabolites formed included GM1, product of GD1a desialosylation. No GM1 or other metabolites were present in the incubation medium, whereas with the lysosomal apparatus blocked by chloroquine, or GD1a endocytosis prevented at 4 degrees C, the only metabolite formed was GM1. These results suggest that GD1a desialosylation did not occur either extracellularly or intracellularly but likely, at the membrane level. Similar results were obtained with [3H-Gal]GD1b, whereas no degradation of [3H-NeuAc]GM1 took place in the presence of chloroquine or at 4 degrees C. In conclusion, cerebellar granule cells express in vivo a sialidase, presumably located on the cell surface, that affects GD1a and GD1b but not GM1.

Animals↗
PubMed1991

Subunit stoichiometry of the chloroplast photosystem II antenna system and aggregation state of the component chlorophyll a/b binding proteins.

Photosystem (PS) II membranes, obtained by the method of Berthold et al. (Berthold, D. A., Babcock, G. T., and Yocum, C. F. (1981) FEBS Lett. 134, 231-234), have been fractionated by a sucrose gradient ultracentrifugation method which allows the quantitative separation of the three major chlorophyll binding complexes in these membranes: the chlorophyll (chl) a binding PSII reaction center core, the major light-harvesting complex II, and the minor chl a/b proteins called CP26, CP29, and CP24. Each fraction has been analyzed for its subunit stoichiometry by quantitative sodium dodecyl sulfate-polyacrylamide gel electrophoresis methods. The results show that 12 mol of light-harvesting complex II and 1.5 mol of each of the minor chl a/b proteins are present per mol of the PSII reaction center complex in PSII membranes. These data suggest a dimeric organization of PSII, in agreement with a recent crystallographic study (Bassi, R., Ghiretti Magaldi, A., Tognon, G., Giacometti, G. M., and Miller, K. (1989) Eur. J. Cell Biol. 50, 84-93) and imply that such a dimeric complex is served by antenna chl a/b proteins whose minimal aggregation state includes three polypeptides. This was confirmed by covalent cross-linking of purified antenna complexes.

Centrifugation, Density Gradient↗
PubMed1991

Identification and characterization of the major components of the Oncorhynchus mykiss egg chorion.

The extracellular coat surrounding the fish egg, commonly called the chorion, is a primary envelope that confers biochemical and morphological identity typical of the species. Purified chorions can be easily isolated from either oocytes or ovulated eggs. The aim of this work was to analyze the macromolecular composition of the various chorion components in Oncorhynchus mykiss (Salmonids). SDS-PAGE analysis of purified chorion showed a reproducible pattern of four major components (129, 62, 54, and 47 kD), representing about 80% of total chorion proteins. The 129 and 47 kD polypeptides were periodic-acid Schiff (PAS) and concanavalin A positive. After chemical and enzymatic deglycosylation treatments only the 129 and 47 kD components proved to be glycosylated and to belong to the "asparagine-linked" glycoprotein family. Furthermore, peptide mapping performed on isolated polypeptides showed comigrating fragments on SDS-PAGE. These results suggest that the four main chorion polypeptides might share common structural features.

Animals↗
PubMed1991

Exogenous gangliosides GD1b and GD1b-lactone, stably associated to rat brain P2 subcellular fraction, modulate differently the process of protein phosphorylation.

GD1b and GD1b-lactone (GD1b-L) gangliosides bind to the same extent to a P2 crude membrane preparation from rat brain. After 30 min of incubation with 10(-4), 10(-5) and 10(-6) M solutions of ganglioside, 1,800, 450, and 100 pmol of ganglioside/mg of protein, respectively, were found to be stably associated to the P2 fraction. This association modifies the phosphorylation process of the P2 membrane proteins in a dose-dependent manner, the maximal effect being reached at a ganglioside association of 1.85 nmol/mg of protein and in large part at 450 pmol/mg of protein. The effects of GD1b and GD1b-L on the phosphorylation of five proteins, showing apparent molecular masses of 17, 20, 36, 41, and 44 kDa, were different after 0.5 min of phosphorylation reaction as well as after 15 min. After 0.5 min of reaction, in the presence of stably associated GD1b, the phosphorylation of the 36-, 41-, and 44-kDa proteins was increased with reference to the control, whereas the phosphorylation of the 17- and 20-kDa proteins was decreased. GD1b-L exerted qualitatively similar effects only on the 44-, 41-, and 36-kDa proteins and to a strongly reduced degree. After 15 min of reaction, only the phosphorylation of the 36-kDa protein was stimulated by GD1b; GD1b-L exerted a similar effect, but to a low degree.

Animals↗
PubMed1990

Further studies on the changes of chicken brain gangliosides during prenatal and postnatal life.

The developmental profiles of the gangliosides and those of the fatty acids and long-chain bases of the total ganglioside mixture of the brain of chicken were followed from the 10th day of incubation to the 63rd posthatching day. One O-acetylated polysialoganglioside that seems specific of the earlier embryonic stage and up to 21 alkali-stable components could be recognized by high-resolution two-dimensional TLC procedures and quantified by computer-assisted two-dimensional TLC densitometry. Besides a number of gangliosides identified by co-chromatography with reference standards, 10 were of unidentified structure, and within these 4 did not belong to the gangliotetrahexosyl series. Throughout embryonic life, the ceramide portion of gangliosides was found to contain the long-chain base species with 18 carbons. Those with 20 carbons, approximately 10% of the total, were found to be present only after hatching.

Alkalies↗
PubMed1990

Relationship between the regulation of membrane enzyme activities by gangliosides and a possible ganglioside segregation in membrane microdomains.

Laser and neutron scattering experiments showed that in mixed micelles of ganglioside GM2 and GT1b, a membrane mimicking system, the segregation of gangliosides may occur spontaneously. Photolabeling experiments using nitrophenylazide containing ganglioside GM1 proved that gangliosides added to cells in culture enter the cell and bind to its membrane as components of microdomains, which specifically interact with a protein of about 30 kDa. This suggests that ganglioside segregation may be a natural phenomenon. Gangliosides when added to granule cells in culture led to increase in protein phosphorylation, the effect exerted being related to the amount of ganglioside molecules inserted stably into the cell lipid layer and an increase of 0.7% of the cell original ganglioside content promoted an increase of 57% in the incorporation of 32P into cell membrane proteins. From the above results a possible relationship between ganglioside segregation and involvement of ganglioside in enzyme activity control is suggested.

Animals↗
PubMed1989

Lactonization of GD1b ganglioside under acidic conditions.

Gangliosides that contain the disialosyl residue alpha-Neu5Ac-(2--8)-alpha-Neu5Ac-(2--3)- can lactonize in the presence of traces of acid and this reaction has been studied in detail on GD1b [beta-Gal-(1--3)-beta-GalNAc-(1 --4)-[alpha-Neu5Ac-(2--8)-alpha-Neu5Ac-(2 --3)]-beta-Gal-(1--4)-beta-Glc-1--1)-Cer]. Lactonization occurs rapidly at a proton-ganglioside molar ratio of less than 1. At equilibrium, the ratio of GD1b to its lactone is 3:7. The data suggest the possibility that a proton-driven lactonization of gangliosides may occur in vivo.

Animals↗
PubMed1989

[Changes in the levels of ACTH and cortisol after passive exposure to cigarette smoke in smokers and non-smokers].

Plasma ACTH and cortisol levels were studied in smokers and non smokers, (exposed or not to smoke of the environment), after passive exposure to cigarette smoking. Non smokers, usually not exposed to smoke, show a rise in both hormones, whereas smokers and non smokers commonly exposed to smoke don't show any change in ACTH and cortisol levels. These data suggest that nicotine acts as an acute stimulus on the hypophysis-adrenal axis even passively inhaled.

Adrenocorticotropic Hormone↗
PubMed1989

[Oral administration of nicotine and plasma levels of ACTH and cortisol in smokers and non-smokers].

Plasma ACTH and cortisol levels after oral administration of nicotine (chewing gum containing nicotine 2 mg) in short and long time (10 and 45 min) were studied in smokers and non smokers. Non smokers after short time administration showed significant rise in ACTH and cortisol. No modifications were seen in the other groups of subjects. These data confirm that nicotine stimulates hypophysis-adrenal axis in non smokers and that this effect is connected with nicotine contents of cigarettes rather than other volatile substances.

Administration, Oral↗
PubMed1989

[ACTH and cortisol after cigarette smoke exposure during the dexamethasone suppression test in smokers and non-smokers].

In this research the effect of nicotine, (smoke of cigarette), was studied in smokers and non smokers during dexamethasone inhibitory test (1 mg h 23.00). ACTH and cortisol plasma levels, physiologically suppressed at 08.00, increased, after 30 min from smoking, only in group of non smokers. These data suggest that nicotine, in non smokers, could induce a maximum stimulus on diencephalic structures, so to overcome the inhibition of dexamethasone.

Adrenocorticotropic Hormone↗