Ultrastructure of the new neuromuscular junctions formed during reinnervation of rat soleus muscle by a “foreign” nerve

Summary In rats the fast fibular nerve was transposed to the slow soleus muscle outside the original innervation band. Formation of new neuromuscular junctions was induced by cutting the soleus nerve after different periods of time. The morphological maturation of these junctions was studied by electron microscopy.New neuromuscular junctions do not form when the original innervation is left intact.Three to five days after denervation, vesicle-laden terminal boutons contact muscle fibers with only the basal lamina of the latter intervening. Three weeks after denervation, most boutons are larger and postsynaptic folds are present, although younger stages are also seen. Sixteen weeks after denervation, the neuromuscular junctions appear mature. This corresponds well with electrophysiological findings in the same material.The fully developed neuromuscular junctions sixteen weeks after denervation possess postsynaptic folds similar to those of normal fast muscle fibers. This suggests that the fast fibular nerve rather than the slow soleus muscle fibers determines the morphology of the postsynaptic folds.Possible trophic neuromuscular interactions are discussed.The authors are indebted to Mrs. Jorunn Line Vaaland, Miss Bjørg Riber, and Miss Berit Branil for technical assistance. Dr. T. Lømo and Dr. C. Slater have contributed constructive criticism and advice     

Anomeric preference of glucose utilization in rat erythrocytes

The -anomer of glucose relative to the -anomer was more rapidly metabolized into lactate by rat erythrocytes at 37° C (/ ratio = ca. 1.3): the amounts of - and -D-glucose metabolized into lactate during 3 min were 0.21 and 0.27 mol/gHb, respectively. Also, the transport of -D-glucose into erythrocytes was more rapid than that of -D-glucose: the amounts of - and -D-glucose transported into erythrocytes during 3 min were approximately 3.5 and 5.0 mol/gHb, respectively. Glucose phosphorylation by rat erythrocyte hexokinase (i.e., a possible rate-limiting step in glycolysis) occurred at higher velocities with the -anomer than with the a-anomer (/ ratio = 1.28). The Km value of hexokinase for either anomer of glucose was 53 M. The glucose concentrations in erythrocytes incubated with - and -D-glucose reached about 1 mM in 1 min, indicating that hexokinase is almost completely saturated with glucose within less than 1 min. The results suggest that glucose phosphorylation and glucose transport are major and minor determinants, respectively, for the anomeric preference of glucose utilization in rat erythrocytes.     

Photoreactive fatty acid analogues that bind to the rat liver fatty-acid binding protein: 11-(5′-azido-salicylamido)-undecanoic acid derivatives

Summary Photoreactive probes for the hydrophobic pocket of the liver fatty acid-binding protein, 11-(5-azido-salicylamido)-undecanoic acid (5 ASU) and its acetyl ester (Ac5 ASU), were synthesized and their interaction with the protein was assessed. Fatty acid-binding proteins are closely related proteins which are abundantly expressed in tissues with active lipid metabolism. A simple model that assumes that the protein possesses a single kind of sites fitted the binding of radioiodinated 5 ASU to L-FABP satisfactorily. The apparent dissociation constant, 1.34×10^–7 M, evidenced a slightly higher affinity than that reported for C16–C20 fatty acids. Consistent with the binding curve, 5 ASU effectively competed with palmitic acid for the hydrophobic sites and the effect was nearly complete for concentrations of 1 gmM; oleic acid, in turn, displaced the radiolabelled probe. Irradiation at 366 nm of¹²⁵I-5 ASU bound to L-FABP caused the covalent cross-linking of the reagent. The amount of radioactivity covalently bound reached a maximum after 2 min thus agreeing with the photo-activation kinetics of the unlabelled compound that evidenced a t_1/2 of 31.1 sec. The yield with which probes bound to L-FABP became covalently linked to the protein, appraised after SDS-PAGE of irradiated samples, was estimated as 23 and 26 per cent for 5 ASU and Ac5 ASU respectively. In turn, irradiation of L-FABP incubated with 5ASU or Ac5 ASU resulted in the irreversible loss of about one fourth its ability to bind palmitic acid. Both results, taken together, suggested that the derivatives are linked to the protein through the sites for fatty acids. When cross-linking of¹²⁵I-5 ASU was performed after incubation with delipidated cytosol and products were analyzed by SDS-PAGE, a band was visualized in a position similar to that of purified L-FABP.Abbreviations FABP Fatty Acid-Binding Protein - L-FABP Hepatic FABP - I-FABP Intestinal FABP - C-FABP Cardiac FABP - 5 ASU-11 (5-azido-salicylamido)-undecanoic acid - Ac5 ASU-11 (O-acetyl-5-azido-salicylamido)-undecanoic acid     

Endogeneous interferon α/β produced by murine Kupffer cells augments liver-associated natural killing activity

Summary Nonparenchymal liver cells from untreated C3HeB/FeJ mice, when incubated in medium containing-10% fetal bovine serum or portal serum, produced significant amounts of interferon alpha/beta (IFN/). In contrast, other cell populations (spleen, mononuclear blood cells and peritoneal cells) from C3HeB/FeJ mice or nonparenchymal liver cells from other strains of mice (C3H/HeJ, germ-free C3H/HeN and C57Bl/6J) produced little or no detectable IFN in fetal bovine serum under the same culture conditions. The cells in the nonparenchymal liver cell population responsible for IFN/ production were adherent, phagocytic, silica-sensitive, carbonyl-iron-sensitive, and Thy1.2^–, presumably Kupffer cells or resident liver macrophages. IFN/ production by cultured Kupffer cells was not observed if medium containing fetal bovine serum or portal serum was treated with polymyxin B or if Kupffer cells were cultured in serum-free medium. This suggested that small amounts of endotoxin in fetal bovine or portal serum stimulated Kupffer cells to produce IFN/. Possibly, Kupffer cells are in a different state of activation/maturation than peritoneal and splenic macrophages since the sensitivity of resident Kupffer cells from C3HeB/FeJ mice to the stimulatory effects of endotoxin. The endogenous production of IFN/ by Kupffer cells from C3HeB/FeJ mice can augment liver-associated natural killer (NK) activity against YAC-1 cells (4 h) and induce liver-associated cytotoxic activity, not restricted by the major histocompatibility complex, against NK resistant P815 mastocytoma cells (18 h).This work was supported by National Institutes of Health grant CA28835, VA Merit Grant and by the Margaret Duffy and Robert Cameron Troup Fund Abbreviations used: NPC, nonparenchymal liver cells; FBS, fetal bovine serum; IFN, interferon; -AsGm-1, anti-asialo-GM1; -Thy1.2, anti-Thy1.2; Hepes, 4-(2-hydroxyethyl)1-piperazineethanesulfonic acid; HBSS, Hanks' balanced salt solution; GBSS, Gey's balanced salt solution; SRBC, sheep red blood cells; Ab, mouse anti-SRBC; NK, natural killer; MHC, major histocompatibility complex; polyI·polyC, polyinosinec·polycytidylic acid     

Metabolism, not autoxidation, plays a role in α-oxoaldehyde- and reducing sugar-induced erythrocyte GSH depletion: Relevance for diabetes mellitus

Erythrocyte and lens reduced glutathione (GSH) levels are often lower in patients with diabetes whereas erythrocyte dicarbonyl levels are often higher. We hypothesise that high plasma carbohydrates may be metabolised by glycolytic and pentose phosphate pathways to form -oxoaldehydes, which deplete cellular GSH. Our aims were: (1) to compare the effectiveness of various carbohydrates or metabolites at depleting erythrocyte GSH, (2) to determine if GSH loss is related to the autoxidation or metabolism of carbohydrates. It was found that erythrocyte GSH was depleted by 50% (ED-50) at t = 2.5 h when erythrocytes were incubated with the following: methylglyoxal (MG) 23 M, glyoxal 75 M, DL-glyceraldehyde 299 M, deoxyribose 606 M, xylitol 626 M, and ribose 2 mM. The glycolytic inhibitors, sodium arsenate and KF prevented ribose, deoxyribose, xylitol and MG-induced GSH depletion in erythrocytes over 2 h. However, the antioxidant trolox and the ferric chelator detapac did not affect MG-induced GSH depletion. These data suggest that the carbohydrates or glyceraldehyde were metabolised to form carbonyls such as MG which depleted erythrocyte GSH as a result of catalysis by glyoxalase I. None of the carbohydrates were autoxidised to carbonyls over this time period. We speculate that as a result of GSH depletion, subsequent glycoxidative stress affects erythrocyte function and contributes to diabetic complications.     

Lipid profile of rat myelin subfractions

Myelin from adult rat brains was separated on a discontinuous sucrose gradient into three subfractions. Analysis of light, heavy and membrane fraction lipid classes was performed by HPTLC and densitometry while fatty acid composition was determinated by GLC. The more interesting results observed are: i) the membrane fraction resembles in its lipid and fatty acid composition other cell membranes (particulary oligodentrocytes); ii) light and heavy myelin are quite similar between them but the former has a higher content of sphingomyelin, a lower hydroxy/nohhydroxy cerebrosides ratio and a lower content of monoenoic fatty acids than the heavy subfraction. The results obtained could explain the different structures observed in each myelin subfraction since fatty acid composition, hydroxy fatty acids, sphingomyelin and cholesterol play a key role in the stability and structure of membranes.     

The protective role of DL-α-lipoic acid in the oxidative vulnerability triggered by Aβ-amyloid vaccination in mice

Recent reports indicate that -amyloid peptide (A) vaccine based therapy for Alzheimers disease (AD) may be on the horizon. There are however, concerns about the safety of this approach. Immunization with A has several disadvantages, because it crosses the blood brain barrier and cause inflammation and neurotoxicity. The present work is aimed to study the protective effective of -lipoic acid (LA) in the oxidative vulnerability of -amyloid in plasma, liver, spleen and brain, when A fibrils are given intraperitoneally in inflammation induced mice. Result shows that reactive oxygen species (ROS) in the astrocytes of inflammation induced mice along with A (IA) has shown 2.5-fold increase when compared with LA treated mice. The increased level of lipid peroxidase (LPO) (p < 0.05) and decreased antioxidant status (p < 0.05) were observed in the plasma, liver, spleen and brain of IA induced mice when compared with LA treated mice.Data shows that there were no significant changes observed between the control and LA treated mice. Our biochemical and histological results highlight that significant oxidative vulnerability was observed in IA treated mice, which was prevented by LA therapy. Our findings suggest that the antioxidant effect of LA when induced with A may serve as a potent therapeutic tool for inflammatory AD models. (Mol Cell Biochem 270: 29–37, 2005)     

Interaction of alpha-lactalbumin with mini-alphaA-crystallin

A-Crystallin can function like a molecular chaperone. We have recently shown that residues 71-88 in A-crystallin represent the chaperone active site of the protein. A peptide containing the sequence of A-crystallin sequence DFVIFLDVKHFSPEDLTVK (mini A-crystallin) by itself displays the antiaggregation property of A-crystallin. We have prepared a complex of reduced -lactalbumin and mini-A-crystallin and investigated the nature, conformation, and properties of the complex by dynamic light scattering, HPLC analysis, CD spectroscopy, and fluorescence studies. Although mini-A was able to prevent the precipitation of reduced -lactalbumin, large aggregates (50-500 nm) of the complex were formed during the assay. Amino acid composition estimation revealed that -lactalbumin and mini-A-crystallin were present in 1:2 ratio in the aggregates. During our study significant red shift in the Trp fluorescence emission maximum and an increase in Bis-ANS binding to the mini A-crystallin-bound -lacatalbumin were observed. The CD spectra of the complex showed a significant loss of -helical content but the -sheet content appeared to be less affected, indicating the molten-globule state of the reduced lactalbumin in the complex. These data show that the active site of A-crystallin by itself can maintain a significantly denatured and unfolded protein in soluble form.     

Amino acid profiles in some scented rice varieties

Summary Twelve scented (basmati) and one non-scented variety were analysed for their amino acid composition. The essential amino acid profiles of scented varieties when compared with non-scented, revealed that these varieties exhibited higher values, which ranged from 2.82 to 4.86 gm/100 gm protein for lysine, 1.92 to 3.13 for methionine, 1.67 to 4.23 for tyrosine, 3.65 to 4.91 for phenylalanine, 5.50 to 8.95 for leucine, 2.25 to 3.40 for isoleucine, 2.84 to 3.46 for threonine, 3.36 to 5.33 for valine. When these values were compared to FAO recommended standards, it was observed that most of the scented varieties had comparable or superior values, while varieties such as, Type 3, Basmati sufaid 100, Likitimachi, Randhunipagalu and Basmati 370 showed superior lysine, phenylalanine, leucine, and methionine content. These observations suggest that the scented varieties posses better amino acid profiles and exhibit superior nutritional qualities, which could be utilised in breeding varieties with improved amino acid composition.     

Mode of uptake of sterol by Arthrobacter simplex

The mechanism of uptake of water-insoluble -sitosterol by a newly isolated strain of Arthrobacter simplex SS-7 was studied. The production of an extracellular sterol-pseudosolubilizing protein during growth of A. simplex on -sitosterol was demonstrated by isolating the factor from the cell-free supernatant and its subsequent purification by Sephadex G-150 column chromatography. The M _r of the purified sterol-pseudosolubilizing protein determined by SDS–PAGE was 19kDa. The rate of sterol pseudosolubilization (5.2×10^–3g l^–1h^–1) could not adequately account for the rate of sterol uptake (72×10^–3g l^–1h^–1) and the specific growth rate (56×10^–3 h^–1). However in the unfavourable growth condition, when the cells were treated with sodium azide at the level of 30–60% of MIC, the sterol pseudosolubilization accounted for nearly 74% of the total growth containing 96% free cells. Cellular adherence to substrate particles was found to play an active role in the normal growth of the strain on -sitosterol. Unlike sodium acetate-grown cells, whose surface activity was negligible (60mNm^–1), the sterol-grown cells had strong surface activity (40mNm^–1). The high lipid content and long chain fatty acids in the cell-wall of -sitosterol-grown cells probably contribute to the high sterol adherence activity of the cells.     

A simple method for maintaining high multiplication of Narcissus shoot cultures in vitro

A simple method for stimulating and maintaining high in vitro multiplication of Narcissus shoot clump cultures was developed. Shoot clumps were subjected either to normal cutting where leaves were trimmed to 20 mm in length at the beginning of each culture passage or to severe cutting where shoot clumps were cut down to the basal plate region removing all green tissue. Severe cutting at the beginning of each culture passage initially doubled the leaf multiplication, compared to normal cutting, but the difference between cutting treatments declined in successive passages. The improvement in leaf multiplication was maintained when shoot clumps were subjected to severe cutting only at every other culture passage, with no cutting in the alternate recovery passages. In vitro multiplication was increased by severe cutting in all seven Narcissus cultivars which were tested.Abbreviations NAA-1 naphthylacetic acid - BAP benzylaminopurine     

Cellular fatty acid composition of six fastidious,gramnegative, xylem-limited bacteria from plants

Composition of cellular fatty acids was determined for strains of fastidious, Gramnegative, xylem-limited bacteria causing or associated with Pierce's disease of grapevine, phony disease of peach, plum leaf scorch, stunt of ragweed, elm leaf scorch, and periwinkle wilt. The most abundant fatty acids were straight-chain 150, 160, 170, and 180, unsaturated 161, 181, and unsaturated 17-and 19=carbon homologs. Minor fatty acids included straightchain 120, 140, 190, and 200; an unsaturated 15-carbon homolog; hydroxy-substituted 2-OH 120, 3-OH 120, and 3-OH 140; and branched chain iso-140 and iso-200. Cyclopropane acids were not detected. Physiological age had no effect on fatty acid composition. Class analysis of data indicated relative uniformity within the group. Saturated even-carbon chains comprised 31%–42%, unsaturated acids 41%–52%, saturated odd-carbon chains 10%–18%, hydroxysubstituted chains 2%–7%, and branched-chains 1%–4% of total fatty acids. The ratio of saturated-unsaturated acids ranged from 0.8 to 1.2.     

Inhibition of protein synthesis enhances the lytic effects of tumor necrosis factor α and interferon γ in cell lines derived from gynecological malignancies

Summary Few clinical responses have occurred in preliminary studies using the cytokines tumor necrosis factor (TNF) or interferon (IFN) in cancer patients. This may be related to the observation that many malignant cell lines are resistant to lysis by these cytokinesin vitro. Resistance to lysis by TNF or IFN in many cells is controlled by a protein-synthesis-dependent mechanism, such that when protein synthesis is inhibited cells become sensitive to lysis by these cytokines. Because there is some evidence that TNF and IFN act through different lytic mechanisms and are opposed by different resistance mechanisms, we treated a panel of eight cell lines, five derived from human cervical carcinomas (ME-180, MS751, SiHa, HT-3, and C-33A) and three derived from ovarian carcinomas (Caov-3, SK-OV-3, and NIH: OVCAR-3) with both TNF and IFN to determine whether such combination treatment might maximizein vitro cell lysis. Our results showed that pretreatment with IFN followed by exposure to TNF in the presence of protein synthesis inhibitors increased lysis of seven of the eight cell lines above that seen with either TNF or IFN and inhibitors of protein synthesis. Only the cell line C-33A was resistant to lysis by TNF and IFN, when exposed to these agents both alone and in combination with protein synthesis inhibitors. Clinically, combining the cytokines TNF and IFN with protein synthesis inhibitors may maximize thein vivo lytic effects of these cytokines.Supported by American Cancer Society Career Development Award 90-221     

The composition of Erythrosins, Fluorescein, Phloxine and Rose Bengal: a study using thin-layer chromatography and solvent extraction

Synopsis Commercial samples of Erythrosin B (CI 45430), Erythrosin Y (CI 45425), Fluorescein (CI 45350), Phloxine (CI 45410) and Rose Bengal (CI 45440) have been analysed by thin-layer chromatography. The Erythrosins were found to be mixtures consisting in the main of 4-iodofluorescein, 4,5-di-iodofluorescein, 2,4,5-triiodofluorescein and 2,4,5,7-tetraiodofluorescein, in some instances together with 2,4,5-tri-iodo-4,5,6,7-tetrachlorofluorescein and 2,4,5,7-tetraiodo-4,5,6,7-tetrachlorofluorescein. Samples of Fluorescein were mixtures of the nominal dye usually with traces of several unidentified, fluorescent components. Those of Phloxine consisted mainly of mixtures of 4-bromo-4,5,6,7-tetrachlorofluorescein, 4,5-dibromo-4,5,6,7-tetrachlorofluorescein, 2,4,5-tribromo-4,5,6,7-tetrachlorofluorescein and 2,4,5,7-tetrabromo-4,5,6,7-tetrachlorofluorescein, often with 4,5,6,7-tetrachlorofluorescein Samples of Rose Bengal were mixtures of 4-iodo-4,5,6,7-tetrachlorofluorescein, 4,5-di-iodo-4,5,6,7-tetrachlorofluorescein, 2,4,5-tri-iodo-4,5,6,7-tetrachlorofluorescein and 2,4,5,7-tetraiodo-4,5,6,7-tetrachlorofluorescein together with some unidentified components.Most of the commercial dye samples gave an insoluble residue when extracted with methanol. This residue was usually inorganic carbonate or halide. Some possible practical consequences of the various impurities are discussed.     

Stimulation by hexose esters of lactate production by rat erythrocytes,insensitivity to 3-O-methyl-D-glucose and inhibition by 2-deoxy-D-glucose and its tetraacetic ester

Selected esters of D-glucose were recently proposed as tools to provide the sugar to cells, whilst bypassing the carrier system for hexose transport across the plasma membrane. In the present study, -D-glucose pentaacetate, -D-glucose pentaacetate, -D-mannose pentaacetate and, to a lesser extent, 6-O-acetyl-D-glucose, all tested at a 1.7 mM concentration, were found to increase lactate production above basal value in rat erythrocytes. Over 90 min incubation, the increment in lactate production ranged from about 1.2 (-D-glucose pentaacetate) to 0.6 (6-O-acetyl-D-glucose) mol/l of erythrocytes. Little or no change in lactate production was observed in cells exposed to -L-glucose pentaacetate, -D-glucose pentaethylsuccinate, -D-galactose pentaacetate or -D-galactose pentaacetate. The metabolic response to -D-glucose pentaacetate was resistant to 3-O-methyl-D-glucose (10-80 mM) which suppressed, however, that evoked by D-glucose. D-mannoheptulose (10 mM) virtually failed to affect the response to D-glucose and its pentaacetate ester. On the contrary, 2-deoxy-D-glucose (10.6 mM) inhibited to the same relative extent (55% decrease) lactate production in erythrocytes exposed to either unesterified D-glucose or -D-glucose pentaacetate. The tetraacetic ester of 2-deoxy-D-glucose was more efficient than unesterified 2-deoxy-D-glucose in inhibiting lactate production from -D-glucose pentaacetate. It is proposed that selected esters of saccharides represent useful tools to bypass defects in hexose transport, and to increase their nutritional or therapeutic efficiency.     

Identification of subunits required for the catalytic activity of the F1-ATPase

F₁() complexes containing equimolar ratios of the and subunits have been shown to function as active ATPases, whereas individually isolated and subunits show no real ATPase activity. These results indicate that the single-copy subunits are not required for F₁-ATPase activity. The minimal F₁()-core complexes exhibit, however, lower rates and some different properties from those of their parent whole F₁ or ₃₃ complexes. It is therefore concluded that for obtaining a full spectrum of the characteristic functional properties of an F₁-ATPase the presence of the F₁- subunit is also required. The implications of these findings on the subunit location of both catalytic and noncatalytic nucleotide binding sites is discussed.     

The β-1,3-glucan-binding protein from the crayfish Pacifastacus leniusculus,when reacted with a β-1,3-glucan,induces spreading and degranulation of crayfish granular cells

Summary A -1,3-glucan-binding protein (GBP) was purified from crayfish plasma, and incubated with laminarin (L), a -1,3-glucan. The GBP reacted with laminarin (GBP-L) induced strong spreading and partial degranulation of isolated and separated crayfish granular haemocytes. However, neither the GBP nor laminarin alone induced any changes in the crayfish granular cells. When monolayers of granular haemocytes were incubated with 20 g of GBP-L, more than 82% of the haemocytes were affected. The activity of GBP-L on granular cells was dose-dependent and a plateau was reached at 10 g of GBP-L. The degranulation of crayfish haemocytes induced by GBP-L seemed to occur by a regulated exocytosis, since it was strongly inhibited by specific blockers of this process such as SITS or calmidazolium. Monospecific anti-GBP antibodies also totally blocked the effect of GBP-L on crayfish granular cells. Indirect immunofluoresence staining demonstrated that the GBP-L could bind to the surface of granular cells, whereas GBP did not bind or bound very weakly to the haemocyte surface.     

13Cα and 13Cβ chemical shifts as a tool to delineate β-hairpin structures in peptides

Unravelling the factors that contribute to the formation and the stability of -sheet structure in peptides is a subject of great current interest. A -hairpin, the smallest -sheet motif, consists of two antiparallel hydrogen-bonded -strands linked by a loop region. We have performed a statistical analysis on protein -hairpins showing that the most abundant types of -hairpins, 2:2, 3:5 and 4:4, have characteristic patterns of ¹³C and ¹³C conformational shifts, as expected on the basis of their and angles. This fact strongly supports the potential value of ¹³C and ¹³C conformational shifts as a means to identify -hairpin motifs in peptides. Their usefulness was confirmed by analysing the patterns of ¹³C and ¹³C conformational shifts in 13 short peptides, 10–15 residues long, that adopt -hairpin structures in aqueous solution. Furthermore, we have investigated their potential as a method to quantify -hairpin populations in peptides.     

The irreversibility of inner mitochondrial membrane permeabilization by Ca2+ plus prooxidants is determined by the extent of membrane protein thiol cross-linking

We have previously shown that mitochondrial membrane potential () drop promoted by prooxidants and Ca²⁺ can be reversed but not sustained by ethylene glycol-bis(-aminoethylether)-N,N,N,N-tetraacetic acid (EGTA) unless dithiothreitol (DTT), a disulfide reductant, is also added [Valle, V. G. R., Fagian, M. M., Parentoni, L. S., Meinicke, A. R., and Vercesi, A. E. (1993).Arch. Biochem. Biophys. 307, 1–7]. In this study we show that catalase or ADP are also able to potentiate this EGTA effect. When EGTA is added long after (12 min) the completion of swelling or elimination, no membrane resealing occurs unless the EGTA addition was preceded by the inclusion of DTT, ADP, or catalase soon after was collapsed. Total recovery by EGTA is obtained only in the presence of ADP. The sensitivity of the ADP effect to carboxyatractyloside strongly supports the involvement of the ADP/ATP carrier in this mechanism. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized membrane proteins shows that protein aggregation due to thiol cross-linkage formed during drop continues even after is already eliminated. Titration with 5,5-dithio-bis(2-nitrobenzoic acid) supports the data indicating that the formation of protein aggregates is paralleled by a decrease in the content of membrane protein thiols. Since the presence of ADP and EGTA prevents the progress of protein aggregation, we conclude that this process is responsible for both increased permeability to larger molecules and the irreversibility of drop. The protective effect of catalase suggests that the continuous production of protein thiol cross-linking is mediated by mitochondrial generated reactive oxygen species.     

Two novel isoneolacto-undecaglycosylceramides carrying Galα1→3Lewisx on the 6-linked antenna and N-acetylneuraminic acidα2→3 or Galactose α1→3 on the 3-linked antenna, expressed in porcine kidney

Three sialosylated and three neutral glycosphingolipids sharing a common iso-neolacto core were isolated from porcine kidney cortex. They were purified by preparative HPTLC, and were characterized by partial exoglycosidase hydrolysis followed by thin layer chromatography and immunostaining with anti-Gal13Gal, anti-type 2 lactosamine and anti-Lewisx antibodies, methylation analysis, MALDI-TOF mass spectrometry and 1H-NMR spectroscopy. Among neutral glycolipids, one was a known structure, VI3VI3(Gal)2-iso-nLc8Cer, and two were novel structures differing by the number of Gal3Lewisx determinants: VI3VI3(Gal)2V3Fuc-iso-nLc8, and VI3VI3(Gal)2 V3V3(Fuc)2-iso-nLc8. The single Gal3Lewis x determinant was found on the 6-linked antenna. Among sialosylated glycolipids, two had been previously found in other species and tissues, VI3VI3(NeuAc)2-iso-nLc8, and VI3NeuAcVI3Gal-iso-nLc8. A novel structure was discovered presenting a Gal3Lewisx determinant on the 6-linked antenna and a N-acetylneuraminic acid on the 3-linked antenna, VI3NeuAcVI3GalV3Fuc-iso-nLc8. These results indicate that, in vivo, the porcine kidney 3fucosyltransferase synthesizes the Gal3Lewisx determinant, acting on the 6-linked before the 3-linked Gal3neolactosamine, and appears unable to synthesize the sialosylated Lewisx determinant on neolactoseries glycolipids.