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PubMed1989

Inhibitory effect of stearidonic acid (18:4 n-3) on platelet aggregation and arachidonate oxygenation.

The effect of stearidonic acid (18:4 n-3) present in fish and some plant oils, such as black currant seed oil, was studied on human platelets. When added to platelets simultaneously with collagen, arachidonic acid or endoperoxide mimetic U46619, 18:4 n-3 appeared as a weak inhibitor of platelet aggregation. In addition, 18:4 n-3 did not alter the metabolism of exogenous arachidonic acid. In contrast, when preincubated with platelets after precoating onto albumin, 18:4 n-3 inhibited platelet aggregation induced by thrombin, collagen, arachidonic acid or U46619, and was as potent as eicosapentaenoic acid (20:5 n-3) tested under similar conditions. Stearidonic acid also altered the endogenous arachidonate oxygenation stimulated by low doses of thrombin, but to a significantly lesser extent than did 20:5 n-3. It seems therefore that, in addition to competing with endogenous arachidonate metabolism, 18:4n-3 may affect platelet aggregation by another mechanism.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed2005

Force microscopy studies of fibronectin adsorption and subsequent cellular adhesion to substrates with well-defined surface chemistries.

Molecular force spectroscopy was used to study the mechanical behavior of plasma fibronectin (FN) on mica, gold, poly(ethylene glycol), and -CH(3), -OH, and -COOH terminated alkanethiol self-assembled monolayers. Proteins were examined at two concentrations, one resulting in a saturated surface with multiple intermolecular interactions referred to as the aggregate state and another resulting in a semiaggregate state where the proteins were neither completely isolated nor completely aggregated. Modeling of the force-extension data using two different theories resulted in similar trends for the fitted thermodynamic parameters from which insight into the protein's binding state could be obtained. Aggregated proteins adsorbed on hydrophobic surfaces adopted more rigid conformations apparently as a result of increased surface denaturation and tighter binding while looser conformations were observed on more hydrophilic surfaces. Studies of FN in a semiaggregate state showed heterogeneity in the model's thermodynamic parameters suggesting that, in the early stages of nonspecific adsorption, multiple protein conformations exist, each having bound irreversibly to the substrate. Proteins in this state all demonstrated a more rigid conformation than in the corresponding aggregate studies due to the greater number of substrate contacts available to the protein. Finally, the force spectroscopy experiments were examined for any biocompatibility correlation by seeding substrates with human umbilical vascular endothelial cells. As predicted from the models used in this work, surfaces with aggregated FN promoted cellular deposition while surfaces with FN in a semiaggregate state appeared to hinder cellular deposition and growth. The atomic force microscope's use as a means for projecting surface biocompatibility, although requiring additional testing, does look promising.

Adsorption↗
PubMed1998

Identification of three novel unique proteins in seed oil bodies of sesame.

Plant seeds store triacylglycerols in discrete organelles called oil bodies. An oil body preserves a matrix of triacylglycerols surrounded by a monolayer of phospholipids embedded with abundant structural proteins termed oleosins and probably some uninvestigated minor proteins of higher molecular mass. Three polypeptides of 27, 37, and 39 kDa (temporarily denominated as Sop1, Sop2, and Sop3) were regularly co-purified with seed oil bodies of sesame. Comparison of amino acid composition indicated that they were substantially less hydrophobic than the known oleosins, and thus should not be aggregated multimers of oleosins. The results of immuno-recognition to sesame proteins extracted from subcellular fractions of mature seeds, various tissues, and oil bodies purified from different stages of seed formation revealed that these three polypeptides were unique proteins gathered in oil bodies, accompanying oleosins and triacylglycerols, during the active assembly of the organelles in maturing seeds. Both in vivo and in intro, immunofluorescence labeling using secondary antibodies conjugated with FITC (fluorescein isothiocyanate) confirmed the localization of these three polypeptides in oil bodies.

Amino Acids↗
PubMed1975

Inability to detect Chlamyodomonas microtubule assembly in vitro: possible implications to the in vivo regulation of microtubule assembly.

It has been demonstrated that the in vitro assembly of microtubules from Chlamydomonas preparations does not occur under a wide range of conditions, including those efficacious for mammalian brain tubulin. This incompetence of Chlamydomonas extracts to form microtubules is independent of the tubulin concentration, the presence of added nucleotides or an added seed, temperature, or the concentration of divalent cation. However, an amorphous aggregate was observed under certain conditions, who composition was mainly tubulin. The in vitro reassembly of microtubules in gerbil brain extracts is inhibited by Chlamydomonas preparations. Fractionation of the Chlamydomonas extracts by column chromatography suggests that the inhibitory component is Chlamydomonas tubulin itself. The mechanism of this inhibition is unknown, but reassembly experiments indicate that the 2 types of tubulins cannot copolymerize. We suggest that the Chlamydomonas tubulin, derived from a cytoplasmic pool, requires to be activated prior to its in vivo polymerization into microtubules.

Animals↗
PubMed1997

Contact of sarcoma cells with aligned fibroblasts accelerates their displacement: computer-assisted analysis of tumour cell locomotion in co-culture.

The shape and locomotion of rat sarcoma XC cells on glass, polystyrene, and confluent monolayer cultures of aligned human skin fibroblasts were studied with quantitative, computer-assisted methods. The cell shape depended upon the substratum; the sarcoma cells seeded on fibroblasts assumed polarized shapes. The tumour cells emigrating from aggregates and in sparse cultures showed random locomotion when plated on glass or on the polystyrene surface of tissue culture dishes in isotropic conditions. However, when sarcoma cell aggregates were plated onto underlying aligned fibroblasts, the sarcoma cells showed contact guidance, migrating along the long axes of fibroblasts. Simultaneously, suppression of migration normal to the axis of fibroblasts orientation was observed. The sarcoma cells displaced a few times faster on aligned fibroblasts than under isotropic conditions in control cultures. This fast displacement was found to result from the less frequent cell turnings and straightening of cell trajectories (i.e., from klinokinesis), and not from an acceleration of cell movement and the longer cell tracks (i.e., not from orthokinesis). The presented results support the suggestion of Abercrombie (M. Abercrombie. 1979. Nature (London). 281: 259-262.) that tumour cells may be guided by the underlying normal cells when invading surrounding tissues and forming metastases.

Animals↗
PubMed1999

[Structural characteristics of osteopontin for calcium oxalate crystal].

BACKGROUND: I investigated which structural segment of osteopontin (OPN), a matrix component of urinary stones, is significantly related to the formation of urinary stones. METHODS: I prepared several kinds of OPNs under various conditions and compared the effects of these OPNs on calcium oxalate (CaOx) crystal using RI counts obtained by the seed crystal method and diluted urine method. Furthermore, I performed scanning electron microscopic (SEM) observation of CaOx crystals used in these experiments and evaluated the effects of OPN based on morphological changes in CaOx crystals. The following OPNs were used in this study: human recombinant OPN (rOPN), human native OPN (nOPN) purified from human milk, denatured OPN (dOPN) obtained by adding organic solvent during the course of nOPN purification, and asiaro OPN (aOPN) obtained by removing sialic acid after enzymatic digestion of nOPN. RESULTS: When the effects of OPNs (15 micrograms/ml) were evaluated by the seed crystal method, the following inhibitory activities were observed: nOPN (82%), aOPN (56%), dOPN (49%) and rOPN (15%). When the effects of OPNs (150 micrograms/ml) were evaluated by the undiluted urine method, the following inhibitory activities were observed: nOPN (38%), aOPN (27%), dOPN (21%) and rOPN (0%). Furthermore, using nOPN, I performed SEM observation of CaOx crystals and found that nOPN mainly inhibited CaOx crystal aggregation. CONCLUSION: Since the inhibitory activity of nOPN was observed not only in the seed crystal method, but also in the undiluted urine method, it was suggested that nOPN may play an important role in the living body during the course of urinary stone formation. Moreover, the inhibitory activity of OPN was not due to its primary structure, but it was closely related to its higher-order structure and side chains including sialic acid. Furthermore, it was clarified that the inhibitory activity of OPN mainly resulted from inhibition of CaOx crystal aggregation rather than growth inhibition in these crystals.

Calcium Oxalate↗
PubMed1992

5c,11c,14c-eicosatrienoic acid and 5c,11c,14c,17c-eicosatetraenoic acid of Biota orientalis seed oil affect lipid metabolism in the rat.

The effects of 5c,11c,14c-eicosatrienoic acid (20:3BSO) and 5c,11c,14c,17c-eicosatetraenoic acid (20:4BSO), polyunsaturated fatty acids (PUFA) contained in Biota orientalis seed oil (BSO), on lipid metabolism in rats were compared to the effects of fats rich in linoleic acid (LA) or alpha-linolenic acid (ALA) under similar conditions. The potential effect of ethyl 20:4BSO as an essential fatty acid also was examined in comparison with the ethyl esters of LA, ALA and gamma-linolenic acid (GLA). BSO- and ALA-rich fat decreased the concentration of plasma total cholesterol, high density lipoprotein cholesterol, triglyceride and phospholipid as compared to LA-rich fat. BSO was more effective in reducing plasma cholesterol concentrations than was the ALA-rich fat. Dietary BSO markedly decreased the hepatic triglyceride concentration as compared to the LA-rich or ALA-rich fats. Aortic production of prostaglandin I2 tended to decrease in rats fed BSO or ALA-rich fat compared to those fed the LA-rich fat. Adenosine diphosphate-induced platelet aggregation was similar in the three groups. The proportion of arachidonic acid (AA) in liver phosphatidylcholine (PC) of rats fed BSO was lowest compared to that of rats fed ALA-rich or LA-rich fats. Administration of 20:4BSO, ALA or GLA to essential fatty acid-deficient rats decreased the ratio of 20:3n-9 to AA in liver PC to the same extent; administration of LA was more effective. The results indicate that the effects of specific PUFA contained in BSO on lipid metabolism are different from those of LA and ALA. It is also suggested that 20:4BSO may exhibit some essential fatty acid effects.

Adipose Tissue↗
PubMed2000

Misfolding and aggregation of vacuolar glycoproteins in plant cells.

Phaseolin and lectin-related polypeptides, the abundant oligomeric glycoproteins of bean seeds, are synthesized on the endoplasmic reticulum (ER) and then transported to the storage vacuole via the Golgi apparatus. Glycosylation and folding are among the major modifications these proteins undergo in the ER. Although a recurrent role of N-glycosylation is on protein folding, in previous studies on common bean (Phaseolus vulgaris) seeds we demonstrated that the oligosaccharide side-chains are not required for folding, intracellular transport and activity of storage glycoproteins. We show here that in lima bean (Phaseolus lunatus), incubation of the developing cotyledon with tunicamycin to prevent glycosylation has a dramatic effect on the intracellular transport of the storage glycoproteins. When lacking their glycans, phaseolin and lectin-related polypeptides misfold and are retained in the ER as mixed aggregates to which the chaperone BiP irreversibly associates. The lumen of the ER becomes enlarged to accommodate the aggregated polypeptides. Intracellular transport of legumin, a naturally unglycosylated storage protein, is mostly unaffected by the inhibitor, indicating that the observed phenomenon specifically occurs on glycoproteins. Furthermore, recombinant lima bean phaseolin synthesized in tobacco protoplasts is also correctly folded and matured in the presence of tunicamycin. To our knowledge, this is the first report that describes in detail the block of intracellular transport of vacuolar glycoproteins in plant cells due to aggregation following glycosylation inhibition.

Biological Transport↗
PubMed1971

Norepinephrine stinulated increase of cyclic AMP levels in developing mouse brain cell cultures.

Norepinephrine causes a four- to sixfold increase in the intracellular level of cyclic AMP (adenosine 3', 5'-monophosphate) reaggregated brain cell cultures derived from embryonic mouse brain. The cyclic AMP level of adult brain is increased by norepinephrine; however, embryonic mouse brain does not show a cyclic AMP response. The aggregate cultures thus demonstrate an event of differentiation very similar to that seen in vivo.

Age Factors↗
PubMed1989

The extracellular matrix modulates the response of PC12 cells to nerve growth factor: cell aggregation versus neurite outgrowth on 3-dimensional laminin substrata.

PC12 cells attach well to plastic culture dishes coated with laminin, collagen, polylysine or a basement membrane extract (2-dimensional substrata) and, in the presence of NGF, extend short neurites within 1-2 days. However, on gels (3-dimensional substrata) reconstituted from a basement membrane extract (RBM), PC12 cells attach extending short processes transiently and within one day, form networks of small aggregates interconnected by process-bearing cells. By 3 days the network collapses into large aggregates that, in media supplemented with NGF, extended a halo of neurites resembling dorsal root ganglia in culture. Time-lapse video recordings indicate that cell motility on RBM gel is accompanied by extensive blebbing as well as extension of processes that attach to and pull together neighbouring cells. These cellular events may contribute to the disruption of the gel underneath aggregates that is apparent when cultures are stained with Coomasie Blue. Ultrastructural studies indicated that aggregates often have zonula adherens-type junctions where cell bodies and processes come in contact. PC12 cells seeded onto gels of laminin alone behave essentially the same as on RBM gels, whereas on collagen gels they behave as on 2-dimensional substrata and extended neurites rather than aggregate. The extent of aggregation increases with greater cell density and is enhanced significantly by NGF. Antisera to NGF reduce the NGF-enhancement of aggregation but do not block aggregation in the absence of NGF. Dibutyryl cAMP or epidermal growth factor, which stimulate process extension and cell division respectively, do not enhance aggregation. However, 3A3, a monoclonal antibody to a laminin/collagen receptor on PC12 cells and antibodies (Fab fragments) to the neural cell adhesion molecule both inhibit cell aggregation.

Antibodies↗
PubMed1989

Relationship between Cottonseed Malate Synthase Aggregation Behavior and Suborganellar Location in Glyoxysomes and Endoplasmic Reticulum.

Malate synthase (EC 4.1.3.2) (MS), an enzyme unique to the glyoxylate cycle, was studied in cotyledons of dark-grown cotton (Gossypium hirsutum, L.) seedlings. MS has generally been regarded as a peripheral membrane protein in glyoxysomes and believed by some to be synthesized on rough ER. Immunocyto-chemical localization of MS in both in situ and isolated cottonseed glyoxysomes, however, showed that MS was located throughout the matrix of glyoxysomes, not specifically associated with their membranes. Biochemical data also supported matrix localization. Isolated glyoxysomes were diluted in variously-buffered salt solutions (200 millimolar KCl or 100 millimolar K-phosphate) or detergents (0.1% Triton X-100, 10 millimolar deoxycholate, or 1.0% Triton X-114) and centrifuged to pellet membranes. Greater than 70% of the MS was recovered in supernatants after treatment with salt solutions, whereas generally less than 30% was released following detergent treatments. MS in pellets derived from glyoxysomes burst in low ionic strength buffer solutions was aggregated (observed on rate-zonal gradients). MS released following salt treatments was the 20S nonaggregated form indicating that salt solutions either disaggregated (or prevented aggregation of) glyoxysomal MS rather than releasing it from membranes. We confirmed reports by others that MS comigrated with ER (NADH: cytochrome c reductase) in sucrose (20-40% w/w) gradients buffered with 100 millimolar Tricine (pH 7.5) after 3 hours centrifugation. However, cottonseed MS did not comigrate with ER in gradients buffered with 10 millimolar Hepes (pH 7.0) or 20 millimolar K-phosphate (pH 7.2) after 3 hours centrifugation, or after 22 hours centrifugation in Tricine or Hepes. Collectively, our data with cotton seeds indicate that MS is not a peripheral membrane protein, and that the aggregation behavior of MS (in various buffers) very likely has led to misinterpretations of its putative associations with ER and glyoxysomal membranes.

Journal Article↗
PubMed2005

Anti-thrombotic effect of proanthocyanidin, a purified ingredient of grape seed.

INTRODUCTION: Moderate and regular consumption of wine reduces the risk of acute coronary thrombotic events. The mechanism of the anti-thrombotic effect of wine is not clear. Extract or purified ingredients of grapes have not yet been studied for anti-thrombotic effect. MATERIALS AND METHODS: Anti-thrombotic effect of proanthocyanidin, a highly purified ingredient of grape seed, was assessed by a shear-induced thrombosis test in vitro and by a laser-induced thrombosis test in the mouse carotid artery, in vivo. RESULTS AND CONCLUSIONS: Intravenously (20 mg/kg body weight, BW) or orally (2 x 200 mg/kg BW) administered proanthocyanidin significantly inhibited the laser-irradiation induced thrombus formation in the carotid artery (both P=0.01). Subsequent to oral administration of proanthocyanidin, in vitro platelet reactivity to shear stress has been inhibited. The latter suggests that the in vivo anti-thrombotic effect of proanthocyanidin may be due to a direct inhibitory effect on platelets.

Animals↗
PubMed1999

A molecular investigation of true dominance in Huntington's disease.

Huntington's disease (HD) is thought to show true dominance, since subjects with two mutant alleles have been reported to have similar ages at onset of disease compared to heterozygous sibs. We have investigated this phenomenon using a cell culture model. Protein aggregate formation was used as an indicator for pathology, as intraneuronal huntingtin inclusions are associated with pathology in vitro and in vivo. We showed that cytoplasmic and nuclear aggregates are formed by constructs comprising part of exon 1 of huntingtin with 41, 51, 66, or 72 CAG repeats, in a rate that correlates with repeat number. No inclusions were seen with 21 CAG repeat constructs. Mutant and wild type huntingtin fragments can be sequestered into inclusions seeded by a mutant huntingtin. Wild type huntingtin did not enhance or interfere with protein aggregation. The rate of protein aggregation was dose dependent for all mutant constructs tested. These experiments suggested a model for the dominance observed in HD; the decrease in the age at onset of a mutant homozygote may be small compared to the variance in the age at onset for that specific repeat number in heterozygotes. Our experiments also provide a model, which may explain the different repeat size ranges seen in patients and healthy controls for the different polyglutamine diseases.

Alleles↗
PubMed1993

T cell activation by clustered tyrosine kinases.

Many cellular recognition events in the immune system are initiated by aggregation of cell surface receptors that lack intrinsic protein-tyrosine kinase activity. Receptor-associated kinases related to the src protooncogene product have been found to be essential for cellular activation and may interact with the cytoplasmic domains of the antigen receptor chains. We show here that anti-CD16 antibody-mediated clustering of chimeric transmembrane proteins bearing a CD16 extracellular domain and a Src family kinase intracellular domain is not sufficient to initiate a cellular activation signal in T cells, whereas clustering of similar chimeras bearing Syk or ZAP-70 kinase sequences triggers calcium mobilization. Aggregation of the Syk chimera alone, or coaggregation of chimeras bearing Fyn and ZAP-70 kinases, suffices to initiate cytolytic effector function. The pattern of tyrosine phosphorylation induced by clustering of the Syk chimera is similar to the pattern induced by aggregation of T cell receptor.

Amino Acid Sequence↗
PubMed1996

Seeding of A beta fibril formation is inhibited by all three isotypes of apolipoprotein E.

Apolipoprotein E is immunochemically localized to amyloid plaque in Alzheimer's brains, and the allelic distribution of ApoE in individuals is associated with a disposition toward Alzheimer's disease. We show here that all three ApoE isotypes exhibit a strong and specific ability to inhibit both nucleation and seeding of fibril formation by the A beta peptide in vitro. A beta (1-40) depleted of aggregates requires long incubation times before the onset of fibril formation, but addition of very low levels of A beta fibrils to such reactions is sufficient to reduce or eliminate this lag time. ApoE added to such seeded reactions extends the lag time in a dose-dependent manner, so that higher levels of seeding require higher levels of ApoE to achieve a given delay time to reaction onset. This effect is observed with all three isotypes produced in Escherichia coli, as well as with plasma-derived ApoE and the N-terminal domain of ApoE3 produced in E. coli. In contrast, bovine serum albumin and the four-helix bundle protein interleukin-4 are poor inhibitors of seeding. ApoE3 can also inhibit fibril formation by A beta (1-42). The three full-length isotypes of ApoE produced in E. coli are equipotent at inhibition. It is therefore possible that the genetics of ApoE and AD may fundamentally depend on the ability of ApoE to inhibit seeding but that the trends in the genetics must be related to something other than the specific activities of the native ApoE isoforms used in these studies. The data show ApoE to be the first member of a new class of fibril formation inhibitor that acts by blocking the seeding of fibril growth.

Alzheimer Disease↗
PubMed2002

Functional behavior of primary rat liver cells in a three-dimensional perfused microarray bioreactor.

We have previously described the design and operation of a microfabricated bioreactor that supports perfused 3D culture of liver cells and facilitates evolution of tissue-like morphological structures. Here, we describe the functional viability of cells maintained in this microarray bioreactor and examine the influence of different seeding protocols on the evolution of structure and function in comparison with static culture. Primary rat hepatocytes were seeded into the perfusion reactors either as single-cell suspensions immediately after isolation or as spheroidal aggregates formed over a 2- to 3-day period. Initial studies in which cells were cultured for 7 days postisolation revealed significantly greater functional activity and morphological stability of cells that were preaggregated for up to 3 days before seeding in the reactor, compared with direct seeding of single cells. Total albumin secretion and urea genesis rates in single-cell reactor cultures declined significantly during this initial culture period while remaining constant in preaggregated reactor cultures. Longer term studies indicate that rates of albumin secretion and urea genesis are maintained at constant levels through 15 days postisolation. These metabolic rates are an order of magnitude higher than observed for the same preaggregated structures cultured statically with comparable medium ratio and exchange conditions. The metabolic function data are supported by light microscopy images showing viable tissue structures, and electron microscopy images that reveal tight junctions, glycogen storage, and bile canaliculi.

Albumins↗
PubMed2004

Accelerated Abeta aggregation in the presence of GM1-ganglioside-accumulated synaptosomes of aged apoE4-knock-in mouse brain.

Aging and apolipoprotein E4 (apoE4) expression are strong risk factors for the development of Alzheimer's disease (AD); however, their pathological roles remain to be clarified. In the process of AD development, the conversion of the nontoxic amyloid beta-protein (Abeta) monomer to its toxic aggregates is a fundamental process. We previously hypothesized that Abeta aggregation is accelerated through the generation of GM1 ganglioside (GM1)-bound Abeta which acts as a seed for Abeta fibril formation. Here we report that GM1 level in detergent-resistant membrane microdomains (DRMs) of synaptosomes increased with age and that this increase was significantly pronounced in the apoE4- than the apoE3-knock-in mouse brain. Furthermore, we show that Abeta aggregation is markedly accelerated in the presence of the synaptosomes of the aged apoE4-knock-in mouse brain. These observations suggest that aging and apoE4 expression cooperatively accelerate Abeta aggregation in the brain through an increase in the level of GM1 in neuronal membranes.

Aging↗
PubMed2005

Chaperoning prions: the cellular machinery for propagating an infectious protein?

Newly made polypeptide chains require the help of molecular chaperones not only to rapidly reach their final three-dimensional forms, but also to unfold and then correctly refold them back to their biologically active form should they misfold. Most prions are an unusual type of protein that can exist in one of two stable conformations, one of which leads to formation of an infectious alternatively folded form. Studies in Baker's yeast (Saccharomyces cerevisiae) have revealed that prions can exploit the molecular chaperone machinery in the cell in order to ensure stable propagation of the infectious, aggregation-prone form. The disaggregation of yeast prion aggregates by molecular chaperones generates forms of the prion protein that can seed the protein polymerisation that underlies the prion propagation cycle. In this article, we review what we have learnt about the role of molecular chaperones in yeast prion propagation, describe a model that can explain the role of various classes of molecular chaperones and their co-chaperones, and speculate on the possible involvement of chaperones in the propagation of mammalian prions.

Cytosol↗