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PubMed2001

Effects of tomato extract on human platelet aggregation in vitro.

Among all fruits tested in vitro for their anti-platelet property, tomato had the highest activity followed by grapefruit, melon, and strawberry, whereas pear and apple had little or no activity. Tomato extract (20-50 microl of 100% juice) inhibited both ADP- and collagen-induced aggregation by up to 70% but could not inhibit arachidonic acid-induced platelet aggregation and concomitant thromboxane synthesis under similar experimental conditions. The anti-platelet components (MW <1000 Da) in tomatoes are water soluble, heat stable and are concentrated in the yellow fluid around the seeds. The active fractions were separated using gel filtration and HPLC. The aqueous fraction (110 000 xg supernatant) of tomatoes containing anti-platelet activity was subjected to gel filtration column chromatography (Biogel P2 column). The activity was fractionated into two peaks, peak-3 and peak-4 (major peak). Subsequently, peak-4 was further purified by HPLC using a reversed-phase column. NMR and mass spectroscopy studies indicated that peak F2 (obtained from peak 4) contained adenosine and cytidine. Deamination of peak F2 with adenosine deaminase almost completely abolished its anti-platelet activity, confirming the presence of adenosine in this fraction. In comparison, deamination of peak-4 resulted in only partial loss of inhibitory activity while the activity of peak-3 remained unaffected. These results indicate that tomatoes contain anti-platelet compounds in addition to adenosine. Unlike aspirin, the tomato-derived compounds inhibit thrombin-induced platelet aggregation. All these data indicate that tomato contains very potent anti-platelet components, and consuming tomatoes might be beneficial both as a preventive and therapeutic regime for cardiovascular disease.

Cells, Cultured↗
PubMed1990

Salt-soluble lectins of corn grain.

Lectins extracted from corn (Zea mays L.) kernel with Tris-HCl buffer pH 7.5 were isolated from the crude extract by affinity chromatography on Sepharose 6B-N-acetyl-d-galactosamine and Sepharose 6B-methylalpha-d-mannoside, and also by lectin affinity chromatography using concanavalin A and Lens culinaris lectin as ligands. According to preferential monosaccharide specificity, salt-soluble lectins of corn seed comprise at least two distinct types: N-acetyl-d-galactosamine-interactive and mannose-interactive lectins. The extracted lectins are unstable, with a tendency to form aggregates during storage.

Journal Article↗
PubMed2002

Proteomic analysis of the endoplasmic reticulum from developing and germinating seed of castor (Ricinus communis).

Endoplasmic reticulum (ER) has been prepared and analysed from germinating and developing castor bean endosperm. A combination of one- and two-dimensional (1-D and 2-D) gel electrophoresis was used to study the complexity of sample and protein differences between the two stages. The ER of the developing oilseed is central to the synthesis, sorting and storage of protein and lipid reserves while the germinating seed is concerned with their degradation. Sample complexity has been reduced by separation of ER proteins into lumenal, peripheral membrane and integral membrane subfractions. Membrane proteins pose specific problems in aggregation and binding to passive surfaces. We have overcome this by collection of membranes at density gradient interfaces and by silanization of plastic ware. Several major components have been identified from 1-D gels by N-terminal sequencing and matrix-assisted laser desorption/ionization (MALDI) peptide mass fingerprints. These include protein disulphide isomerase (PDI), calreticulin and developing-ER-specific oleate-12-hydroxylase involved in the biosynthesis of ricinoleic acid. In excess of 300 spots are detectable in each developmental fraction by high sensitivity 2-D gels. This is the first 2-D electrophoretic analysis of plant ER. These gels reveal significant differences between germinating and developing ER. Preparative loading 2-D gels of germinating ER have been run and 14 selected spots characterized by quadrupole time of flight tandem mass spectrometry (Q-TOF MS/MS). Ten of these proteins were assigned function on the basis of identity with existing castor database entries, or by homology with other species. Two proteins, aspartate proteinase precursor and N-carbamyl-L-aminohydrolase-like protein, appear to be absent from developing profiles. Most of the proteins identified are concerned with roles in protein processing and storage, and lipid metabolism which occur in the ER. Data from three of the assigned spots included unidentified peptides indicating the presence of more than one protein in these spots following 2-D electrophoresis. More extensive analysis will have to await developments in genomics but the basic separation technologies to simplify sample identity for a plant ER preparation have been established.

Amino Acid Sequence↗
PubMed1986

Secretion of a lipolytic protein aggregate by barley aleurone and its dissociation by starchy endosperm.

Acidic and basic lysophospholipase activities (LPL) have been separated by ion-exchange chromatography of barley extracts. The basic activity predominates in the starchy endosperm of germinating barley and in the medium of hormone-stimulated half-seeds; the acidic activity is the predominant form in the medium of hormone-stimulated aleurone layers. Addition of either starchy endosperm or EDTA to the acidic activity produces the basic activity. The two activities display the same pH optimum and have similar Km values. Inactivation profiles of LPLs with immunoglobulin G (IgG) prepared against the purified basic LPL are the same. The acidic LPL obtained from the incubation medium from stimulated aleurone layers appears in the void volume on gel filtration with Bio-Gel P100. Acid phosphatase and alpha-amylase in the same incubation medium appear at their expected elution volumes on this column. Gel filtration in the presence of EDTA results in the acidic activity eluting in a volume characteristic of the basic LPL (Mr, 40,000). On Bio-Gel P300 the acidic activity peak is centered at Mr, 160,000. SDS-gel electrophoresis of fractions across this peak shows a simple distribution of proteins eluting with Mr greater than or equal to 160,000. The potential role of an aggregate in the secretion of lipolytic proteins is discussed.

Chromatography, Gel↗
PubMed1997

Adenosine 5'-triphosphate is required for the assembly of 11S seed proglobulins in vitro.

Seed protein proglobulins were synthesized from cDNAs in reticulocyte lysates. Most proglobulins were recovered as trimers when translation rates were low, but mostly monomers were recovered at high translation rates. The prevalence of monomers was accompanied by elevated amounts of insoluble protein recovered at the bottom of sucrose density gradients. Apyrase treatment of translation mixtures after synthesis, but before significant assembly occurred, drastically reduced trimer assembly and increased the proportion of insoluble aggregate. These observations indicated that ATP is required for protein folding and/or trimer assembly. The appearance of insoluble aggregated protein when rates of synthesis were elevated or when ATP was absent suggested that protein misfolding had occurred. Trimer assembly was stimulated when wheat germ translation mixtures defective in supporting efficient trimer assembly were supplemented with fractions isolated from endoplasmic reticula of developing pea (Pisum sativum) seeds. Molecular chaperones are likely involved in folding and/or assembly of proglobulin trimers both in reticulocyte lysates and in seeds. Consistent with this hypothesis, trimer formation was reduced when carboxymethylated bovine albumin and alpha-casein, considered to mimic proteins with extended chain and molten globular conformations and thereby compete for Hsp70- and Hsp60-type molecular chaperones, respectively, were introduced into translation mixtures.

Adenosine Triphosphate↗
PubMed1978

Dissociation, aggregation of sesame alpha-globulin in nonionic detergent solution.

Nonionic detergents Triton X-100 and Brij 36T induce dissociation and aggregation of the protein sesame alpha-globulin above the critical micelle concentrations (cmc) of the detergents. Spectrophotometric titration in Triton shows no change in the pKInt value of the tyrosyl groups at 1x10-3 M detergent where both dissociation and aggregation of the protein are observed. Fluorescence measurement does not indicate any change in the environment of the tryptophan groups of the protein in Brij. Viscosity measurements show no major conformational change of the protein in the detergent solution. Binding measurements suggest that perhaps micelles of the detergent predominantly bind to the protein. The detergent micelles preferentially bind to the exposed hydrophobic surfaces of the protein subunits. The association of the protein detergent complex through electrostatic interaction is probably responsible for the formation of the aggregates.

Alpha-Globulins↗
PubMed1990

Cabbage seed protease inhibitor: a slow, tight-binding inhibitor of trypsin with activity toward thrombin, activated Stuart factor (factor Xa), activated Hageman factor (factor XIIa), and plasmin.

An inhibitor of procoagulant and fibrinolytic enzymes was derived from cabbage seeds by a procedure using acetone precipitation, ion-exchange chromatography, and gel filtration. The cabbage seed inhibitor was a 10-Kd monomeric protein with intrachain disulfide bonds. This preparation prevented clot formation in whole blood and blocked the ability of thrombin to induce clot formation in plasma and to induce platelet aggregation. A number of proteases were inhibited, as demonstrated by using purified enzymes in amidolytic assays. Tight-binding inhibition was observed for activated Stuart factor (factor Xa) and plasmin. Inhibition of thrombin and activated Hageman factor (factor XIIa) was observed with a molar excess of inhibitor. No inhibition was detected for activated plasma thromboplastin antecedent (factor XIa), plasma kallikrein, or C1 esterase. Reaction progress curves for trypsin indicated slow, tight-binding inhibition, with an apparent inhibition constant in the nanomolar range or less. The electrophoretic mobility of trypsin was altered by the inhibitor in nondenaturing polyacrylamide gel electrophoresis (PAGE) but not in sodium dodecyl sulfate (SDS)-PAGE, indicating noncovalent bonding. Only partial reversal of trypsin inhibition could be demonstrated by washing the inhibitor from enzyme immobilized on solid beads. A dot-blot technique with cabbage seed inhibitor was capable of detecting 10 ng nitrocellulose-bound trypsin. The dot-blot technique also appeared capable of detecting plasmin. These findings demonstrated the potential utility of this inhibitor as a probe for detection of tightly bound proteases. In summary, cabbage seed extracts contain an inhibitor with activity toward a broad range of proteases important to hemostasis. To our knowledge, this agent represents the first inhibitor isolated from a plant source that inhibits thrombin.

Brassica↗
PubMed1971

Polysaccharides in germination. Xyloglucans ( amyloids') from the cotyledons of white mustard.

Two xyloglucan fractions have been isolated from the cotyledons of resting white-mustard seeds, the first by extraction with hot EDTA, and the second by subsequent extraction with alkali or lithium thiocyanate. Although both appear to have the ;amyloid' type of structure in which chains of (1-->4)-linked beta-d-glucopyranose residues carry d-xylose-rich side chains through position 6, these side chains are rather different in structure in the two polysaccharide fractions, and the second or ;insoluble' xyloglucan has fewer of them. The side chains in both polysaccharides are also different from those in other seed amyloids, especially in having xylose linked through positions 3 and 4 (instead of through position 2 as usual) and in containing fucose residues. Both polysaccharides show the characteristic blue ;amyloid' colour with iodine in the presence of sodium sulphate, and it is suggested that this arises by the interaction of iodine molecules and possibly iodide ions within the interstices between aggregated xyloglucan chains. ;Soluble' xyloglucan is metabolized during germination and is presumed to have a reserve function. ;Insoluble' xyloglucan is metabolized less completely over the period studied but its lack of turnover during cell-wall differentiation indicates that it also is a reserve. These and other beta-(1-->4)-linked reserve polysaccharides of seeds might also have a structural function which is of particular value for the survival of the dormant seed.

Chromatography, Gas↗
PubMed1981

A new mitogenic D-galactosephilic lectin isolated from seeds of the coral-tree Erythrina corallodendron. Comparison with Glycine max (soybean) and Pseudomonas aeruginosa lectins.

The lectin of Erythrina corallodendron (Caesalpiniaceae) seeds was purified by heating, ammonium sulfate fractionation, and affinity chromatography on acid-treated Sepharose. The purified lectin is similar to the soybean lectin in being a glycoprotein of molecular weight around 110 000 - 120 000 and having D-galactosephilic activity. This lectin, like the soybean and Pseudomonas aeruginosa lectins, binds to D-galactosamine, N-acetyl-D-galactosamine, alpha- and beta-galactosides as well as to D-galactose. Like these lectins it absorbs onto either untreated or enzyme (papain or neuraminidase) treated human red blood cells, but exhibits a considerable mitogenic activity towards human lymphocytes (predominantly T cells) only after their treatment with neuraminidase. This mitogenic stimulation of lymphocytes is inhibited by D-galactose and its derivatives. Despite the great similarity between them, the E. corallodendron, soybean, and Pseudomonas lectins differ in regard to the intensity of their agglutinating activity towards erythrocytes obtained from different animals and human donors of diverse ABO blood groups. This phenomenon may be attributed to the difference in the affinities of the three lectins to the various D-galactose derivatives and to their molecular properties.

Acetylgalactosamine↗
PubMed2003

Prion domain interaction responsible for species discrimination in yeast [PSI+] transmission.

BACKGROUND: The yeast [PSI+] factor is transmitted by a prion mechanism involving self-propagating Sup35 aggregates. As with mammalian prions, a species barrier prevents prion transmission between yeast species. The N-terminal of Sup35 of Saccharomyces cerevisiae, necessary for [PSI+], contains two species-signature elements-a Gln/Asn-rich region (residues 1-41; designated NQ) that is followed by oligopeptide repeats (designated NR). RESULTS: In this study, we show that S. cerevisiae[PSI+] is transmissible through plasmid shuffling and cytoplasmic transfer to heterotypic Sup35s whose NQ is replaced with the S. cerevisiae NQ. In addition to homology, the N-terminal location is essential for NQ mediated susceptibility to [PSI+] transmission amongst heterotypic Sup35s. In vitro, a swap of NQ of S. cerevisiae Sup35 led to cross seeding of amyloid formation. CONCLUSIONS: These findings suggest that NQ discriminates self from non-self, and is sufficient to initiate [PSI+] transmission irrespective of whether NR is heterotypic. NR as well as NQ alone coalesces into existing [PSI+] aggregates, showing their independent potentials to interact with the identical sequence in the [PSI+] conformer. The role of NQ and NR in [PSI+] prion formation is discussed.

Amino Acid Sequence↗
PubMed1997

Chondrocyte transplantation to articular cartilage explants in vitro.

The transplantation of chondrocytes has shown promise for augmenting the repair of defects in articular cartilage. This in vitro study examined the efficiency of the transplantation of bovine chondrocytes onto articular cartilage disks and the ability of the transplanted chondrocytes to subsequently synthesize and deposit proteoglycan. The radiolabeling of chondrocyte cultures with [3H]thymidine, followed by 4 days of chase incubation, resulted in the incorporation of 98% of the radiolabel into DNA (as assessed by susceptibility to DNase). At the end of the culture period, the [3H]DNA was stable, with a half-life of radioactivity loss into the medium of 73 days. With use of radiolabeled chondrocytes for quantitation, the efficiency of transplantation onto a cartilage substrate was 93 +/- 4% for seeding densities of as much as 650,000 cells per cm2 and a seeding duration of 1 hour. These findings were confirmed both by tracking cells stained with 5-chlormethylfluorescein diacetate and by quantitating DNA. During the 16 hours after seeding onto a cartilage substrate (in which the endogenous cells had been lysed by lyophilization), the transplanted cells synthesized sulfated proteoglycan in direct proportion to the number of cells seeded. Most (83%) of the newly synthesized proteoglycan was released into the medium rather than retained within the layer of transplanted cells and the recipient cartilage substrate. Comparative studies with lyophilized-rehydrated or live cartilage as the recipient substrate indicated a similar efficiency of chondrocyte seeding and proteoglycan synthesis by the seeded chondrocytes. The transplanted cells retained the chondrocyte phenotype, as judged by a high proportion of the [35S]macromolecules being in the form of aggrecan that was capable of aggregating with hyaluronan and link protein, as well as by immunostaining within and around the transplanted cells for type-II, but not type-I, collagen. These results indicate that the number of chondrocytes transplanted onto a cut cartilage surface greatly affects the level of matrix synthesis; this in turn may affect repair.

Animals↗
PubMed1998

Deposition of storage proteins.

Plants store amino acids for longer periods in the form of specific storage proteins. These are deposited in seeds, in root and shoot tubers, in the wood and bark parenchyma of trees and in other vegetative organs. Storage proteins are protected against uncontrolled premature degradation by several mechanisms. The major one is to deposit the storage proteins into specialized membrane-bounded storage organelles, called protein bodies (PB). In the endosperm cells of maize and rice prolamins are sequestered into PBs which are derived from the endoplasmic reticulum (ER). Globulins, the typical storage proteins of dicotyledonous plants, and prolamins of some cereals are transported from the ER through the Golgi apparatus and then into protein storage vacuoles (PSV) which later become transformed into PBs. Sorting and targeting of storage proteins begins during their biosynthesis on membrane-bound polysomes where an N-terminal signal peptide mediates their segregation into the lumen of the ER. After cleavage of the signal peptide, the polypeptides are glycosylated and folded with the aid of chaperones. While still in the ER, disulfide bridges are formed which stabilize the structure and several polypeptides are joined to form an oligomer which has the proper conformation to be either deposited in ER-derived PB or to be further transferred to the PSV. At the trans-Golgi cisternae transport vesicles are sequestered which carry the storage proteins to the PSV. Several storage proteins are also processed after arriving in the PSVs in order to generate a conformation that is capable of final deposition. Some storage protein precursors have short N- or C-terminal targeting sequences which are detached after arrival in the PSV. Others have been shown to have internal sequence regions which could act as targeting information. In some cases positive targeting information is known to mediate sorting into the PSV whereas in other cases aggregation and membrane association seem to be major sorting mechanisms.

Amino Acid Sequence↗
PubMed2005

Alginate microcapsules prepared with xyloglucan as a synthetic extracellular matrix for hepatocyte attachment.

In this study, xyloglucan (XG) was used as a new synthetic extracellular matrix (ECM) for primary mouse hepatocyte attachment in Ca-alginate (AL) capsules. The rates of hepatocytes adhesion onto collagen type I-, XG-coated and uncoated polystyrene (PS) surface were 89.1%, 91.1% and 25.5%, respectively, at 4 h after incubation at 37 degrees C. From the inhibition study in a cell adhesion assay, the adhesion rates of freshly isolated hepatocytes and preincubated hepatocytes with 20 mm galactose onto the XG-coated surface were 55.7 and 17.3%, respectively, after 30 min incubation at 37 degrees C. Flow cytometric analysis showed that the internalization of XG by freshly isolated hepatocytes was stronger than preincubated hepatocytes with 20 mm galactose. The concentration of XG in AL/XG capsules to perform the best liver-specific functions was 0.5 mg/ml, where the highest albumin secretion rates were obtained. The albumin secretion, ammonia elimination rates and cell viability of hepatocytes were slowly decreased with culture time in AL/XG capsules, whereas those were rapidly decreased in AL capsules, indication of the more rapid formation of hepatocyte spheroids in AL/XG capsules than in AL capsules. More than 70% of the seeded hepatocytes in AL/XG capsules participated in spheroid formation after 2 days, whereas most hepatocytes in AL capsules remained as single cells and only a few cells began to form aggregates after 3 days. Intercellular molecule genes, such as connexin (Cx) 32 and E-cadherin, of hepatocyte spheroids in AL or AL/XG capsules were detected by reverse transcriptase-polymerase chain reaction. Cx32 and E-cadherin genes in AL/XG capsules were more rapidly reexpressed and expressed, respectively, than in AL ones. The results suggest that the multicellular spheroid formation of hepatocytes can enhance the liver-specific functions in the three-dimensional space in the presence of XG as a new synthetic ECM owing to the specific interaction between the galactose moieties of XG and asialoglycoprotein receptors of hepatocytes.

Alginates↗
PubMed1993

Procoagulant activity of the MC28 fibrosarcoma cell line in vitro and in vivo.

Experimental evidence suggests that many tumours can activate blood coagulation and that such interaction is part of the pathology of metastatic tumour growth. This study aimed to study the procoagulant activity of the methylcholanthrene-induced (MC28) fibrosarcoma to determine whether coagulation activation by these cells could explain the previously reported effects of oral anticoagulants on lung seeding in this model. MC28 cells shortened the recalcification times of normal and factor VII-deficient plasma and directly activated factor X in a chromogenic assay, but did not aggregate platelets in vitro in either whole blood or platelet-rich plasma. Cellular coagulant activity was calcium-dependent, blocked by DFP and concanavalin A but not inhibited by iodoacetamide, E-64 or antibodies to human tissue factor or factor VII. Injection of viable MC28 cells into hooded Lister rats induced a decrease in platelet count (P < 0.001), plasma factor X (P < 0.001) and fibrinogen (P < 0.05) and a marked increase in plasma haemoglobin (P < 0.001). These effects were either not observed or were considerably less marked in heparinized or warfarinized animals. Injection of MC28 cells treated with concanavalin A in vitro completely abolished the clotting changes observed with untreated cells. In conclusion, MC28 cells possessed a potent factor X-activating serine proteinase procoagulant in vitro, which had some of the characteristics of a tissue factor/factor VIIa complex. In vivo, MC28 cells caused clotting activation and intravascular fibrin generation. Since thrombocytopenia was abolished by heparin and the cells lacked platelet aggregating activity in vitro, thrombocytopenia was probably secondary to intravascular coagulation and thrombin generation. The trigger for intravascular clotting activation appeared to be the cellular procoagulant activity since it was abolished by prior in vitro blockade of the latter with concanavalin A.

Animals↗
PubMed2000

Thermal stabilities of lupin seed conglutin gamma protomers and tetramers.

Various experimental approaches have been used in this work to assess the thermal stabilities of lupin seed conglutin gamma at two pH values, 4.5 and 7.5, at which the protein exists as a protomer and a tetramer, respectively. The patterns of thermal unfolding at the two pH values differed significantly; the tetramer aggregated and became insoluble, whereas the protomer was still soluble after thermal treatment. Also, the midpoint transition temperatures were dramatically different, being 60.3 and 75.1 degrees C for the protomer and tetramer, respectively. The behavior of conglutin gamma at neutral pH was also affected by disulfide formation/interchange, in that some unfolded protein molecules became covalently stabilized. More detailed analyses by differential scanning calorimetry and indirect fluorescence measurements, using 8-anilino-1-naphthalenesulfonic acid as a probe, confirmed the remarkable differences observed in the thermal stabilities of the two protein forms and allowed models for their unfolding patterns to be drawn.

Calorimetry, Differential Scanning↗