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At least 793 records · Page 44Linked to original sources
PubMed2002

Functional properties of oat globulin modified by a calcium-independent microbial transglutaminase.

Oat globulin was modified by a calcium-independent microbial transglutaminase (TG). The TG-polymerized protein had higher solubility than the control at acidic pH and had improved water- and fat-binding properties. Incubation of 10% (w/v) oat globulin dispersions in the presence of TG at 37 degrees C led to the formation of a well-developed viscoelastic gel network with a microstructure characterized by thick strands and large clusters. The TG-induced gels had higher modulus values, lower loss tangent values, and lower frequency dependency than the heat-induced gels. The TG-induced gel system has the characteristics of classical polymer gel with permanent "chemical" cross-links, whereas the heat-denatured system has the characteristics of a temporary "physical" gel with breakable cross-links. Fourier transform infrared spectroscopy showed marked shift and intensity changes in several major bands, suggesting pronounced changes in protein conformation during TG-induced gelation. Aggregation of protein molecules was also indicated by the progressive increases in two infrared bands (1679-1682 and 1622-1625 cm(-)(1)) associated with the formation of intermolecular beta-sheets and strands. Results suggest that new food polymers with unique functionality can be produced from oat globulin treated with TG and that elastic gels can be formed near neutral pH, instead of the alkaline pH required for thermally induced oat globulin gels.

Avena↗
PubMed2006

Start-up of the Anammox process in a membrane bioreactor.

The start-up of an Anammox process was studied in a membrane sequencing batch reactor (MSBR) in which a submerged hollow fibre membrane module was used to retain the biomass. The reactor was seed with Anammox biomass and fed using the Van de Graaf medium. During a first operating stage, salt precipitation was observed and interfered with microbial activity and caused a decrease of the nitrogen removal rate of the reactor from 100 to only 10 mgl(-1) per day. Salt precipitation was avoided by diminishing adequately the Ca and P concentrations of the Van de Graaf medium during the last operating stage. This action increased quickly the activity of the system, and nitrogen removal rate reached up to 710 mgl(-1) per day with almost full nitrite removal. Sporadic flotation of the sludge was observed in the MSBR. The use of the membrane avoided biomass wash-out from the system. Moreover, a surprising fact was that Anammox biomass did not grow in flocs in the MSBR, but in granules. This fact showed that this kind of microorganisms have a trend to grow in aggregates. Results indicated that the use of the MSBR could be a suitable system for nitrogen removal by using the Anammox reaction.

Ammonia↗
PubMedDate not supplied

Plant NAD-dependent glutamate dehydrogenase. Purification, molecular properties and metal ion activation of the enzymes from Lemna minor and Pisum sativum.

Glutamate dehydrogenase (L-glutamate: NAD+ oxidoreductase (deaminating) EC 1.4.1.2) has been purified to homogeneity from Lemna minor and seeds of Pisum sativum. As established by polyacrylamide gel electrophoresis the Pisum-enzyme constitutes a multiple pattern of seven charge isoenzymes whereas the Lemna enzyme shows one single protein band. Molecular weights of 230 000 were calculated for both enzymes by sedimentation equilibrium measurements. (Pisum-enzyme) and comparative gel filtration (Lemna-enzyme). Sodium dodecyl sulfate gel electrophoresis and electron microscopic observations revealed that both enzymes are composed of four identical subunits (molecular weight 58 500) arranged in a tetraedric structure. The amino acid compositions of both enzymes are similar to those of various hexameric glutamate dehydrogenases. The N-terminal amino acid of the Pisum-enzyme is alanine. Both enzymes require Ca2+ for maximal catalytic activity. For the Lemna-enzyme the K0.5 values for Ca2+ are 22 microM (NAD+-dependent reaction), respectively. Ca2+ which to some extent can be replaced by Zn2+ does not affect the enzyme aggregation but seems to govern a reversible equilibrium between catalytically active and inactive enzyme forms.

Amino Acids↗
PubMed1998

Purification and characterization of a lectin from seeds of Vatairea macrocarpa Duke.

A lectin from Vatairea macrocarpa Duke seeds (VML) was isolated using affinity chromatography on a guar gum column. The lectin, a glycoprotein without erythrocyte specificity, displays specificity to galactose and some derivatives. On SDS-polyacrylamide gels, V. macrocarpa seed lectin is composed of two major high-Mr bands of 34 and 32 kDa and two minor low-Mr bands of 22 and 13 kDa. N-Terminal sequencing showed that the 34, 32, and 13 kDa products possess identical N-terminal sequence, which display best similarity with the N-terminal portion of Robinia pseudoacacia lectins (RPL). On the other hand, the N-terminal sequence of the 22 kDa band can be aligned with an internal sequence of RPL starting at residue 149 of the cDNA-derived sequence. These data indicate that, like other leguminous lectins, VML is made up of a mixture of one-chain 30-35 kDa glycoforms and of 22 and 13 kDa endogenous C- and N-terminal fragments. Size-exclusion chromatography indicated that, at neutral pH, VML is predominantly a dimeric (70 kDa) protein, although tetramers (115 kDa) and larger aggregates (300 kDa) were also present.

Amino Acids↗
PubMed2003

Simulations of human lysozyme: probing the conformations triggering amyloidosis.

A natural mutant of human lysozyme, D67H, causes hereditary systemic nonneuropathic amyloidosis, which can be fatal. In this disease, insoluble beta-stranded fibrils (amyloids) are found in tissues stemming from the aggregation of partially folded intermediates of the mutant. In this study, we specifically compare the conformation and properties of the structures adopted from the induced unfolding, at elevated temperature, using molecular dynamics. To increase the sampling of the unfolding conformational landscape, three 5 ns trajectories are performed for each of the wild-type and mutant D67H proteins resulting in a total of 30 ns simulation. Our results show that the mutant unfolds slightly faster than the wild-type with both wild-type and mutant proteins losing most of their native secondary structure within the first 2 ns. They both develop random transient beta-strands across the whole polypeptide chain. Clustering analysis of all the conformations shows that a high population of the mutant protein conformations have a distorted beta-domain. This is consistent with experimental results suggesting that this region is pivotal in the formation of conformations prone to act as "seeds" for amyloid fiber formation.

Amyloidosis, Familial↗
PubMed2001

Immobilization of heparin to EDC/NHS-crosslinked collagen. Characterization and in vitro evaluation.

In the present study, heparin immobilization to a non-cytotoxic crosslinked collagen substrate for endothelial cell seeding was investigated. Crosslinking of collagen using N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) resulted in a material containing 14 free primary amino groups per 1000 amino acid residues (E/N14C). At a fixed molar ratio NHS:EDC of 0.6, the amount of heparin covalently immobilized to E/N14C increased with increasing molar ratios of EDC to heparin carboxylic acid groups (Hep-COOH), to a maximum of approximately 5-5.5 wt% at a ratio of 2. Upon incubation in cell culture medium of endothelial cells, 4 to 7% of the immobilized heparin was released during 11 days. Immobilization of increasing amounts of heparin to E/N14C progressively reduced activation of contact activation proteases. Optimal anticoagulant activity, as measured by thrombin inhibition, was obtained after heparin immobilization using a ratio of EDC to Hep-COOH of 0.2-0.4 (14-20 mg heparin immobilized per gram of collagen). Platelets deposited to (heparinized) E/N14C showed only minor spreading and aggregation, although heparin immobilization slightly increased the number of adherent platelets. The results of this study suggest that heparin immobilization to EDC/NHS-crosslinked collagen may improve the in vivo blood compatibility of this material.

Biocompatible Materials↗
PubMed2004

Surfactant phospholipid changes after antigen challenge: a role for phosphatidylglycerol in dysfunction.

In asthma, inflammation-mediated surfactant dysfunction contributes to increased airway resistance, but the mechanisms for dysfunction are not understood. To test mechanisms that alter surfactant function, atopic asthmatics underwent endobronchial antigen challenge and bronchoalveolar lavage (BAL). BAL fluids were sequentially separated into cells, surfactant, and supernatant, and multiple end points were analyzed. Each end point's unique relationship to surfactant dysfunction was determined. Our results demonstrate that minimum surface tension (gamma(min)) of surfactant after antigen challenge was significantly increased with a spectrum of responses that included dysfunction in 6 of 13 asthmatics. Antigen challenge significantly altered the partitioning of surfactant phospholipid measured as a decreased ratio of large surfactant aggregates (LA) to small surfactant aggregates (SA), LA/SA ratio. Phosphatidylglycerol (PG) was significantly reduced in the LA of the dysfunctional asthmatic BALs. There was a corresponding significant increase in the ratio of phosphatidylcholine to PG, which strongly correlated with both increased gamma(min) and decreased LA/SA. Altered surfactant phospholipid properties correlated with surfactant dysfunction as well or better than either increased eosinophils or protein. Secretory phospholipase activity, measured in vitro, increased after antigen challenge and may explain the decrease in surfactant PG. In summary, alteration of phospholipids, particularly depletion of PG, in the LA of surfactant may be an important mechanism in asthma-associated surfactant dysfunction.

Allergens↗
PubMed1995

Early pulmonary recurrence of non-Hodgkin's lymphoma after autologous marrow transplantation: evidence for reinfusion of lymphoma cells?

Disease recurrence is a major cause of failure after autologous bone marrow transplantation for Hodgkin's disease or non-Hodgkin's lymphoma. Relapse usually occurs at sites of previous involvement. The patient described here died of massive pulmonary involvement with Ki-1 antigen (CD30)-positive immunoblastic lymphoma 2 months after transplantation with unpurged autologous marrow. This relapse in a previously uninvolved organ prompted resectioning of the pre-storage marrow biopsy and resulted in identification of one small aggregation of malignant cells. A review of open lung biopsies and necropsies of autologous marrow recipients treated in Seattle identified no other patients with pulmonary malignancy who lacking previous lung tumor or evidence of contiguous pulmonary and mediastinal involvement. These observations raise questions about the assessment of pre-harvest marrow involvement and the need for marrow purging. This case also suggests that organ and tissue localization of malignant cells may be determined by abnormally expressed 'homing' ligands.

Adolescent↗
PubMed2004

Metabolites of lesser grain borer in grains.

Many volatile alcohol and ester metabolites of the lesser grain borer (LGB, Rhyzopertha dominica) cultured on wheat grain were identified. Volatiles from infested samples at 80 degrees C were collected on Tenax absorbent, thermally desorbed, and analyzed by gas chromatography (GC) using infrared (IR) and mass (MS) detectors for component identification. A solid-phase microextraction (SPME) technique was used to analyze selected samples with a GC-MS system set up for obtaining chemical ionization mass spectra. SPME was also used in a synthesis process required to identify ester metabolites. Predominant compounds in LGB-infested grains were 2-pentanol and its esters of 2-methyl-2-pentenoic (A) and 2,4-dimethyl-2-pentenoic (B) acids, which are known aggregation pheromones, dominicalures 1 and 2. 2-Pentanol esters of saturated A, beta-keto- and beta-hydroxy derivatives of A and B, homologues of A and B, and acid moieties lacking the 2-methyl substitution were found. Other straight- and branched-chain secondary alcohols and their esters were also observed. Reexamination of GC-MS-IR data acquired in previous investigations of LGB cultured on sorghum grain and commercial samples in a grain odor study showed the presence of many LGB metabolites in addition to the known dominicalures.

Animals↗
PubMed1999

Only multimeric hensin located in the extracellular matrix can induce apical endocytosis and reverse the polarity of intercalated cells.

When an intercalated epithelial cell line was seeded at low density and allowed to reach confluence, it located the anion exchanger band 3 in the apical membrane and an H+-ATPase in the basolateral membrane. The same clonal cells seeded at high density targeted these proteins to the reverse location. Furthermore, high density cells had vigorous apical endocytosis, and low density cells had none. The extracellular matrix of high density cells was capable of inducing apical endocytosis and relocation of band 3 to the basolateral membrane in low density cells. A 230-kDa extracellular matrix (ECM) protein termed hensin, when purified to near-homogeneity, was able to reverse the phenotype of the low density cells. Antibodies to hensin prevented this effect, indicating that hensin is necessary for conversion of polarity. We show here that hensin was synthesized by both low density and high density cells. Whereas both phenotypes secreted soluble hensin into their media, only high density cells localized it in their ECM. Analysis of soluble hensin by sucrose density gradients showed that low density cells secreted monomeric hensin, and high density cells secreted higher order multimers. When 35S-labeled monomeric hensin was added to high density cells, they induced its aggregation suggesting that the multimerization was catalyzed by surface events in the high density cells. Soluble monomeric or multimeric hensin did not induce apical endocytosis in low density cells, whereas the more polymerized hensin isolated from insoluble ECM readily induced it. These multimers could be disaggregated by sulfhydryl reagents and by dimethylmaleic anhydride, and treatment of high density ECM by these reagents prevented the induction of endocytosis. These results demonstrate that hensin, like several ECM proteins, needs to be precipitated in the ECM to be functional.

Anion Exchange Protein 1, Erythrocyte↗
PubMed2006

Multiphase modelling of cell behaviour on artificial scaffolds: effects of nutrient depletion and spatially nonuniform porosity.

This paper contains analysis of a recently formulated multiphase model for the growth of biological tissue that comprises motile cells and water inside a rigid scaffold material. The model is extended here to include a term describing cell proliferation which is mediated by the supply of a diffusible nutrient and to include the case where the scaffold porosity varies in space. Numerical solutions of the model equations are presented for different values of the parameters. Comparison is drawn between the different types of growth that arise when using static or dynamic methods for seeding the scaffold with cells. Analytical solutions are presented for the limiting cases in which the coefficient of drag between the cells and the scaffold is very large or zero. In the limit of large time, solutions reveal preferential tissue growth in the vicinity of the scaffold edge due to depletion of nutrient by the cells, consistent with experimental results. However, it is shown that reducing the coefficient of drag between the scaffold and the cells overcomes the effects of nutrient depletion by increasing cell mobility, thereby leading to improved uniformity of the cell distribution within the scaffold.

Algorithms↗
PubMed1990

Effect of different calcium channel blockers on angiotensin II- and vasopressin-induced prostacyclin biosynthesis in vascular smooth muscle cells.

To gain insight with regard to the mode of action of calcium antagonists on the vasculature, we examined the effects of nifedipine, isradipine, felodipine, verapamil, gallopamil, and amlodipine on vasoconstrictor-induced prostacyclin synthesis in vitro. Cultured rat aortic smooth muscle cells were seeded after two to four passages in multiwell plates. After washing of the culture medium and a preincubation period, the cells were exposed for 1 h to either angiotensin II (Ang II) or arginine-vasopressin (AVP) at increasing concentrations between 10(-10)-10(-6) M with or without each calcium antagonist tested at 10(-6) M. At the end of the incubation period, the medium was aspirated, centrifuged, and assayed for its content of protein and of 6-keto-PGF1 alpha by radioimmunoassay. Ang II induced a 15-fold increase and AVP induced a fivefold increase of 6-keto-PGF1 alpha at 10(-6) M. None of the various calcium channel blockers tested showed a significant effect on this agonist-stimulated production of 6-keto-PGF1 alpha. Consequently, calcium-channel blockers with different chemical structure, although known to inhibit agonist-induced vasoconstriction, appear to preserve vasoconstrictor-induced production of prostacyclin, a potent vasodilator and an inhibitor of platelet aggregation.

6-Ketoprostaglandin F1 alpha↗
PubMed1976

Lectin-receptor interactions in liposomes. II. Interaction of wheat germ agglutinin with phosphatidylcholine liposomes containing incorporated monosialoganglioside.

Bovine brain gangliosides incorporated into phospholipid liposomes provide receptors for wheat germ agglutinin. Purified monosialogangliosides were mixed with egg phosphatidylcholine, and unilamellar liposomes were generated. Addition of wheat germ agglutinin induced the liposomes to fuse, and gel filtration analysis revealed that the lectin was incorporated into the fused liposomes. The fusion process was studied by following the changes in the 90 degrees light scattering. Increasing the proportion of the monosialoganglioside in the liposomes was found to increase both the extent of the lectin-induced liposome fusion and the rate of the reaction; below a threshold of approx. 5 mol%, the process was extremely slow. The increase in light scattering could be prevented by the addition of the hapten inhibitor, N-acetyl-D-glucosamine (1 mM). Addition of the inhibitor, subsequent to the lectin, caused a partial decrease in light scattering due to the dissociation of unfused vesicle aggregates. Electron microscopic examination revealed that the ganglioside-containing liposomes were vesicles, 244 +/- 25 A (S.D.) in diameter. Upon addition of wheat germ agglutinin, the vesicles appeared to fuse to form larger vesicles, corresponding to dimers and trimers of the initial vesicles. Inhibition studies with a variety of monosaccharides indicated that the sialic acid moieties present in the ganglioside acted as the lectin-receptor sites. This was confirmed by the observation that wheat germ agglutinin did not interact with phosphatidylcholine vesicles containing desialyated ganglioside.

Binding Sites↗
PubMed1995

Culture of bovine oviduct epithelial cells (BOEC).

BACKGROUND: Bovine oviduct epithelial cells are widely used in co-culture experiments to improve early embryonic development and in vitro fertilization in embryo transfer programmes for domestic animals. METHODS: The present study compares different methods for harvesting and culture of bovine oviduct epithelial cells in order to optimize handling. Bovine oviduct epithelial cells were mechanically or enzymatically isolated and cultured on glass, on permeable membranes, or in suspension. Growth of the cells and their state of differentiation was examined by means of classical staining methods, immunohistochemistry and electron microscopy. RESULTS: Initial cell suspensions contained sheets of ciliated and nonciliated (secretory) cells; 24 h after seeding, free floating epithelial cells formed vesicles with cilia on their external surface. First adhesion of cells was seen 72 h after seeding. Later on, cells grew continuously and confluent monolayers were formed after 7 days. Results were identical after mechanical or enzymatical cell harvesting and were identical on both substrata tested, i.e., on glass and on permeable membranes. Light and electron microscopy proved the monolayers to resemble a polarized, simple, cuboidal to columnar epithelial membrane with intact junctional complexes and numerous apical microvilli. Their epithelial nature was established by immunostaining for cytokeratins. Cilia were missing and secretory granules were scarce. A layer of acidic glycoprotein material was demonstrated on the apical surface. Monolayers of bovine oviduct epithelial cells stored lipid droplets and large quantities of glycogen. About 50% of the seeded cells did not adhere but survived in the culture medium as free floating cells. These suspended cells maintained morphological criteria of differentiation (cilia and secretory granules) until day 12 of culture. Proliferation rates of cultivated cells were determined by counting mitoses and by immunostaining with MIB1 antibody. Results showed coincidence of rapid proliferation and morphological dedifferentiation of monolayers. Suspended cells, by contrast, did not proliferate but retained cellular differentiation under identical culture conditions. CONCLUSIONS: The results strongly suggest that monolayers of bovine oviduct epithelial cells will not fully substitute for original oviduct epithelium when used in co-culture experiments after in vitro fertilization.

Animals↗
PubMed2001

Resveratrol reduction of infarct size in Long-Evans rats subjected to focal cerebral ischemia.

Resveratrol is found in a wide variety of plant species. It is present in the seeds and skin of grapes and constitutes one of the major components of red wine. This study was undertaken to evaluate whether resveratrol could effectively suppress infarct size from the damaging effects of focal cerebral ischemia. The middle cerebral artery was occluded for 1 hr and 24 hr reperfusion in anesthetized Long-Evans rats. In pretreatment or treatment groups, resveratrol, at dosages of 10(-6), 10(-7), 10(-8) and 10(-9) g/kg, was intravenous injected 15 minutes before middle cerebral artery (MCA) occlusion or when the common carotid arteries clips were removed respectively. Pretreatment or treatment of resveratrol (10(-6), 10(-7), 10(-8) and 10(-9) g/kg) did not produce any changes in pH, blood gases, heart rate or mean arterial blood pressure, but it significantly reduced the total volume of infarction at the doses 10(-6) and 10(-7) g/kg. Our study suggests resveratrol is a potent neuroprotective agent in focal cerebral ischemia. Its beneficial effects may be related to its anti-platelet aggregation activity, vasodilating effect, antioxidant property or by all mechanisms together.

Animals↗
PubMed2003

Gelatin-chondroitin-hyaluronan tri-copolymer scaffold for cartilage tissue engineering.

The mechanism by which the cell synthesizes and secretes extracellular matrix (ECM) and is, in turn, regulated by the ECM is termed dynamic reciprocity. The aim of the present work was to produce a gelatin/chondoitin-6-sulfate/hyaluronan tri-copolymer to mimic natural cartilage matrix for use as a scaffold for cartilage tissue engineering. The scaffold produced had a uniform pore size of about 180 microm and adequate porosity of 75%. Porcine chondrocytes were seeded onto the tri-copolymer scaffold and cultured in Petri dishes or spinner flasks for 2, 3, 4, or 5 weeks. Chondrocytes were uniformly distributed in the scaffold in the spinner flask cultures, but less so in the Petri dish cultures. Secretion of ECM was found under histology examination. In spinner flask cultures, chondrocytes retained their phenotype for at least 5 weeks, as shown immunohistochemically, and synthesized type II collagen. These results show that gelatin/chondroitin sulfate/hyaluronan tri-copolymer has potential for use as a cartilage tissue engineering scaffold.

Animals↗
PubMed2000

Lmo2 and GATA-3 associated expression in intraembryonic hemogenic sites.

It is now widely accepted that hemopoietic cells born intraembryonically are the best candidates for the seeding of definitive hemopoietic organs. To further understand the mechanisms involved in the generation of definitive hemopoietic stem cells, we analysed the expression of the hemopoietic-related transcription factors Lmo2 and GATA-3 during the early steps of mouse development (7-12 dpc), with a particular emphasis on intraembryonic hemogenic sites. We show here that both Lmo2 and GATA-3 are present in the intraembryonic regions known to give rise to hemopoietic precursors in vitro and in vivo, suggesting that they act together at key points of hemopoietic development. (1) Lmo2 and GATA-3 are expressed in the caudal mesoderm during the phase of intraembryonic precursors determination. (2) A highly transient concomitant expression is observed in the caudal intraembryonic definitive endoderm, suggesting that these factors are involved in the specification of intraembryonic hemopoietic precursors. (3) Lmo2 and GATA-3 are expressed within the hemopoietic clusters located in the aortic floor during fetal liver colonisation. Furthermore, a strong GATA-3 signal allowed us to uncover previously unreported mesodermal aggregates beneath the aorta. A combined in situ and immunocytological analysis strongly suggests that ventral mesodermal GATA-3 patches are involved in the process of intraembryonic stem cell generation.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Characterization of oligomeric species in the fibrillization pathway of the yeast prion Ure2p.

The [URE3] prion of Saccharomyces cerevisiae shares many features with mammalian prions and poly-glutamine related disorders and has become a model for studying amyloid diseases. The development of the [URE3] phenotype is thought to be caused by a structural switch in the Ure2p protein. In [URE3] cells, Ure2p is found predominantly in an aggregated state, while it is a soluble dimer in wild-type cells. In vitro, Ure2p forms fibrils with amyloid-like properties. Several studies suggest that the N-terminal domain of Ure2p is essential for prion formation. In this work, we investigated the fibril formation of Ure2p by isolating soluble oligomeric species, which are generated during fibrillization, and characterized them with respect to size and structure. Our data support the critical role of the N-terminal domain for fibril formation, as we observed fibrils in the presence of 5 M guanidinium chloride, conditions at which the C-terminal domain is completely unfolded. Based on fluorescence measurements, we conclude that the structure of the C-terminal domain is very similar in dimeric and fibrillar Ure2p. When studying the time course of fibrillization, we detected the formation of small, soluble oligomeric species during the early stages of the process. Their remarkable resistance against denaturants, their increased content of beta-structure, and their ability to 'seed' Ure2p fibrillization suggest that conversion to the amyloid-like conformation has already occurred. Thus, they likely represent critical intermediates in the fibrillization pathway of Ure2p.

Chromatography, Gel↗