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At least 469 records · Page 26Linked to original sources
PubMed2002

DNA from bird-dispersed seed and wind-disseminated pollen provides insights into postglacial colonization and population genetic structure of whitebark pine (Pinus albicaulis).

Uniparentally inherited mitochondrial (mt)DNA and chloroplast (cp)DNA microsatellites (cpSSRs) were used to examine population genetic structure and biogeographic patterns of bird-dispersed seed and wind-disseminated pollen of whitebark pine (Pinus albicaulis Engelm.). Sampling was conducted from 41 populations throughout the range of the species. Analyses provide evidence for an ancestral haplotype and two derived mtDNA haplotypes with distinct regional distributions. An abrupt contact zone between mtDNA haplotypes in the Cascade Range suggests postglacial biogeographic movements. Among three cpSSR loci, 42 haplotypes were detected within 28 cpSSR sample populations that were aggregated into six regions. Analysis of molecular variance (amova) was used to determine the hierarchical genetic structure of cpSSRs. amova and population pairwise comparisons (FST ) of cpSSR, and geographical distribution of mtDNA haplotypes provide insights into historical changes in biogeography. The genetic data suggest that whitebark pine has been intimately tied to climatic change and associated glaciation, which has led to range movements facilitated by seed dispersal by Clark's nutcracker (Nucifraga columbiana Wilson). The two hypotheses proposed to explain the genetic structure are: (i) a northward expansion into Canada and the northern Cascades in the early Holocene; and (ii) historical gene flow between Idaho and the Oregon Cascades when more continuous habitat existed in Central Oregon during the late Pleistocene. Genetic structure and insights gained from historical seed movements provide a basis on which to develop recovery plans for a species that is at risk from multiple threats.

Animals↗
PubMed2000

Unfolding of multimeric proteins in presence of denaturants. A case study of helianthinin from Helianthus annuus L.

Helianthinin (11S), a multisubunit protein from Sunflower Seeds (Helianthus annuus L.) dissociates to its monomer (2S) through a trimeric (7S) intermediate as a function of guanidine hydrochloride (GuHCl) and guanidine thiocyanate (GuHSCN) concentration. Measurements of viscosity, velocity sedimentation patterns and spectroscopic parameters of the protein in presence of these denaturants both at equilibrium and as a function of time clearly suggest that the dissociation, unfolding and aggregation of this multimeric protein occur sequentially. The unfolding of the protein in guanidinium salts has two transitions with the first transition occurring between native to intermediate and the second transition occurring between intermediate state and unfolded state. The midpoint concentration for the major transition is 1.75 M for GuHCl and 0.8 M for GuHSCN. Evaluation of this data suggests that during the process of denaturation the simultaneous unfolding of acidic and basic subunits of the protein takes place. At intermediate concentrations of denaturant namely 1.6 M of GuHCl or 1.1 M of GuHSCN the aggregation of the protein was found to be maximum. The results suggests the possibility of a mechanism for the dissociation, denaturation and unfolding of multimeric proteins in presence of the chemical denaturants.

2S Albumins, Plant↗
PubMed2000

Review: modulating factors in amyloid-beta fibril formation.

Amyloid formation is a key pathological feature of Alzheimer's disease and is considered to be a major contributing factor to neurodegeneration and clinical dementia. Amyloid is found as both diffuse and senile plaques in the parenchyma of the brain and is composed primarily of the 40- to 42-residue amyloid-beta (Abeta) peptides. The characteristic amyloid fiber exhibits a high beta-sheet content and may be generated in vitro by the nucleation-dependent self-association of the Abeta peptide and an associated conformational transition from random to beta-conformation. Growth of the fibrils occurs by assembly of the Abeta seeds into intermediate protofibrils, which in turn self-associate to form mature fibers. This multistep process may be influenced at various stages by factors that either promote or inhibit Abeta fiber formation and aggregation. Identification of these factors and understanding the driving forces behind these interactions as well as the structural motifs necessary for these interactions will help to elucidate potential sites that may be targeted to prevent amyloid formation and its associated toxicity. This review will discuss some of the modulating factors that have been identified to date and their role in fibrillogenesis.

Amyloid beta-Peptides↗
PubMed2004

Odontoblasts induced from mesenchymal cells of murine dental papillae in three-dimensional cell culture.

In an organ culture system under a three-dimensional microenvironment that provides the conditions needed for odontoblast differentiation, a row of odontoblasts can be induced (Kikuchi et al. 1996, 2001). Therefore, in a newly designed three-dimensional cell culture system that fulfils the conditions necessary for odontoblast differentiation (Kikuchi et al. 2002), we examined whether dental papilla cells in rat mandibular incisors could differentiate into tubular dentine-forming cells. In our previously established organ culture system, CM-Dil-labeled cells that were microinjected into isolated dental papillae were replaced by a row of odontoblasts. In a three-dimensional cell culture system, which consists of two kinds of type I collagen in the upper layer over multi-layered cells seeded onto collagen containing Matrigel in the lower layer and which acts as a structural meshwork, dental papilla cells were incubated as multi-layered cells in an artificial extracellular matrix (ECM). The cells aggregated to form a cell mass and invaginated as a cell mass into the ECM. The cells also extended fine fibrillar processes into the ECM. With regard to invagination, the proteolytic activities of matrix metalloproteinase-2 (MMP-2)/membrane type 1-matrix metalloproteinase (MT 1-MMP) were observed on the outer multi-layers of cells within a cell mass adjacent to the ECM. The cell mass progressively shrank to about one-half to one-third of its original diameter and was organized as a tissue surrounded by a newly secreted ECM, like dental pulp-dentine. The cells adjacent to the secreted ECM were constructed as a row of polarized columnar cells. They extended slender processes into the new ECM, which is characteristic of tubular matrix. Dentine sialophosphoprotein (DSPP) and dentine matrix protein 1 (DMP 1) genes, which are specific for odontoblast differentiation, were expressed in an aggregated cell mass where tubular matrix-forming cells were induced. Furthermore, the tubular matrix became mineralized under prolonged culture. These results imply that the putative progenitor cells/stem cells residing in dental papillae can differentiate into odontoblasts under appropriate conditions in vitro.

Animals↗
PubMed2005

Efficient system of artificial oil bodies for functional expression and purification of recombinant nattokinase in Escherichia coli.

Nattokinase, a serine protease, and pronattokinase, when expressed in Escherichia coli, formed insoluble aggregates without enzymatic activity. For functional expression and purification, nattokinase or pronattokinase was first overexpressed in E. coli as an insoluble recombinant protein linked to the C terminus of oleosin, a structural protein of seed oil bodies, by an intein fragment. Artificial oil bodies were reconstituted with triacylglycerol, phospholipid, and the insoluble recombinant protein thus formed. Soluble nattokinase was subsequently released through self-splicing of intein induced by temperature alteration, with the remaining oleosin-intein residing in oil bodies and the leading propeptide of pronattokinase, when present, spontaneously cleaved in the process. Active nattokinase with fibrinolytic activity was harvested by concentrating the supernatant. Nattokinase released from oleosin-intein-pronattokinase exhibited 5 times higher activity than that released from oleosin-intein-nattokinase, although the production yields were similar in both cases. Furthermore, active nattokinase could be harvested in the same system by fusing pronattokinase to the N terminus of oleosin via a different intein linker, with self-splicing induced by 1,4-dithiothreitol. These results have shown a great potential of this system for bacterial expression and purification of functional recombinant proteins.

Bacillus subtilis↗
PubMed1979

[Kinetics of the growth of Ca oxalate crystals from supersaturated solutions].

Several factors influencing the nucleation and growth of Ca-oxalate crystals from metastable and instable solutions were studied in some detail. Factors of interest were the absolute concentration of calcium respectively oxalate, the quotient oxalate/calcium, repeated additions of calcium and/or oxalate, the presence or absence of crystal seeds, the agitation respectively stagnation of the metastable Ca-oxalate solution, the duration of crystallization, etc. The striking findings are the eminent role of oxalate in the formation of big crystals and crystal aggregates, the distinct inhibition of crystal growth at higher and very high calcium concentrations, as well as the substantial crystal enlargement at the presistent oxalate load.

Calcium Oxalate↗
PubMed1999

Small angle X-ray scattering of wheat seed-storage proteins: alpha-, gamma- and omega-gliadins and the high molecular weight (HMW) subunits of glutenin.

Small angle X-ray scattering in solution was performed on seed-storage proteins from wheat. Three different groups of gliadins (alpha-, gamma- and omega-) and a high molecular weight (HMW) subunit of glutenin (1Bx20) were studied to determine molecular size parameters. All the gliadins could be modelled as prolate ellipsoids with extended conformations. The HMW subunit existed as a highly extended rod-like particle in solution with a length of about 69 nm and a diameter of about 6.4 nm. Specific aggregation effects were observed which may reflect mechanisms of self-assembly that contribute to the unique viscoelastic properties of wheat dough.

Gliadin↗
PubMed1976

Cleavage initiation activities of microtubules and in vitro reassembled tubulins of sperm flagella.

Cleavage of fish (Oryzias latipes) eggs was induced by injection of heterologous (sea urchin Hemicentrotus pulcherrimus and oyster Crassostrea gigas) sperm microtubules. Cleavage initiating CI activity of microtubules was higher in 3% PVP suspension than in 6% BSA, and not affected significantly by the concentration of microtubules themselves. The CI activity of microtubules suspended in 3% PVP was comparatively stable in the frozen state. Heat-treatment at more than 55 degrees C resulted in the loss of most or all of their CI activity. Such activity was observed in side-by-side aggregates of tubulin linear polymers of sea urchin (Hemicentrotus pulcherrimus) spermatozoa but not in dispersed linear polymers or tubulin dimers. Microtubules with CI activity seem to participate in initiating cleavage as astral centers, or a "seed" for polymerization of ooplasmic tubulins in activated eggs.

Animals↗
PubMed2004

Biocompatibility of two commercial forms of mineral trioxide aggregate.

AIM: To examine the biocompatibility of two commercial forms of mineral trioxide aggregate (MTA), by evaluating the morphology of an established cell line. METHODOLOGY: The two cements were cast on glass cover slips and cured for 1 or 28 days. Saos-2 osteosarcoma cells were trypsinized and seeded at a density of 1 x 10(5) cells and were then placed in medium over the material-coated coverslips for 1, 5 and 7 days. After these time intervals the media were discarded and the cells fixed. Cell morphological investigation was performed by scanning electron microscopy at various magnifications ranging from x 250 to x 500. The biocompatibility of cement constituents, alusilicate flux and bismuth oxide was also investigated. RESULTS: All cement samples cured for 1 day showed a confluent cell monolayer after 5 and 7 days. The response to both materials was similar. Materials cured for 28 days showed incomplete cell confluence after 1 and 5 days. Alusilicate flux and bismuth oxide did not demonstrate biocompatibility. CONCLUSIONS: The 1-day cured samples of two commercial forms of MTA showed good biocompatibility. However, the 28-day cured samples were less biocompatible after 1 and 5 days.

Aluminum Compounds↗
PubMed1993

Investigations of the architecture of tamarind seed polysaccharide in aqueous solution by different scattering techniques.

The architecture of tamarind seed polysaccharide (TSP) has been investigated by light scattering (LS), small angle X-ray scattering (SAXS) and synchrotron radiation scattering (SRSAXS). The experimental data show that TSP in aqueous solution consists of multi stranded aggregates, with a high degree of particle stiffness. The angular dependence of the scattered intensity is typical for wormlike chains. Data evaluation on the basis of this model yields a statistical Kuhn segment length lK = 150 nm. The cross sectional radius of gyration is estimated as Rgcs = 6.0 +/- 0.5 A, which is more than twice the value, published for single stranded polysaccharides. Correspondingly, the experimental value of the linear mass density, measured by LS, is about five times higher than the theoretical value calculated from the primary structure.

Light↗
PubMed1985

Defective adhesion to extracellular matrix leads to altered social behaviour in cultured fibroblasts.

We describe the properties of variant mouse fibroblasts selected for poor adhesion to growth substratum containing subcellular matrix accumulated by adherent cells at confluence. The variant cells adhere to virgin plastic and grow normally to confluence in the presence of serum. After subculture and reseeding onto the same surface the cells initially adhere, but after a further 2 days of growth they retract into aggregates and detach. If the aggregates are dispersed and cells reseeded onto the same surface they remain rounded. However, if the same cells are added back to virgin plastic they adhere and grow normally. The retraction can be abolished by treating the subcellular matrix-coated plastic with papain. This behaviour therefore reflects the ability of the cells to modify the composition of the underlying substratum during growth. The variant cells also exhibit retraction 2 days after seeding on a surface previously containing wild-type cells at confluence, while wild-type cells do not retract on subcellular matrix deposited by variants. This shows that the variant behaviour arises not from a deficiency in the subcellular matrix, but from an alteration in the adhesive capacity of the cells. The results are interpreted in terms of three putative adhesion mechanisms: cell-cell adhesion in confluent monolayers and aggregates; 'early' type cell-substratum adhesion, which occurs during culture on virgin plastic; and 'late' type cell-substratum adhesion occurring on surfaces containing accumulated subcellular matrix. The variant phenotype is characterized by a deficiency in the last of these. It is also associated with an increased ability to grow in suspension culture at high dilution.

Animals↗
PubMed1997

The toxicity of the Alzheimer's beta-amyloid peptide correlates with a distinct fiber morphology.

In an attempt to elucidate the relationship among aggregation properties, fiber morphology, and cellular toxicity several beta-amyloid peptides (A beta) were prepared according to a standardized procedure. Peptides either carried mutations inside the membrane anchor segment around amino acid position 35 or their carboxy terminus was shortened from 42 to 41, 40, or 39 amino acids. The time-dependent self-assembly of monomeric A beta into fibers was simultaneously monitored by electron microscopy, circular dichroism spectroscopy, analytical ultracentrifugation, and A beta-mediated cellular toxicity using the reduction of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to measure cell viability. The transition of A beta monomers into fibers was analyzed by more than 600 electron micrographs. Distinct morphological changes from seed-like structures to immature and mature fibers were observed. Seeds were of spherical appearance. Immature fibers were typically elongated structures with a rough surface and with varying thickness depending on the A beta sequence. Mature fibers were characterized by a periodic variation of their thickness along the fiber axis. The proportion of these different structures and the total amount of aggregated A beta was amino acid sequence-dependent. Wild-type A beta 1-42 and its oxidized derivative carrying a methionine sulfoxide residue at position 35 showed the highest rate of fiber formation and exerted toxic activity in the MTT assay at very low nanomolar concentrations. The fibers formed by these two peptides were predominantly of the mature type. In contrast, carboxyl-terminus truncated peptides A beta 1-41, A beta 1-40, and A beta 1-39 or most A beta 1-42 derivatives mutated around amino acid position 35 showed a reduced aggregation rate, the immature fibers predominated, and the toxicity was orders of magnitude lower. Thus, a correlation can be drawn among the chemical structure, aggregation properties, fiber morphology, and cellular toxicity.

Amino Acid Sequence↗
PubMed1994

Purification and physicochemical characterization of soyatoxin, a novel toxic protein isolated from soybeans (Glycine max).

Physicochemical characterization and biological properties of a new toxic protein isolated from soybeans (Glycine max) is reported. The purification procedure consisted basically of ammonium sulfate fractionation, ion exchange, and affinity chromatographies, the latter being used for the removal of the seed's lectin and of its trypsin inhibitor. The highly purified protein, designated soyatoxin, is a single chain acidic protein (pI 4.4-4.6) of 21 kDa, dependent on reduced thiol groups to maintain its solubility and biological activities. The toxin is a metalloprotein containing iron, calcium, zinc, and magnesium. Soyatoxin is highly toxic to mice (LD50 7-8 mg/kg mouse body wt upon intraperitoneal injection). It produces dyspnoea, tonic-clonic convulsions, and flaccid paralysis prior to death of intraperitoneally injected mice. Furthermore, soyatoxin is immunologically related to another toxic protein (canatoxin), isolated from Canavalia ensiformis seeds, which is distinct from soyatoxin in containing 18 x 10 kDa noncovalently bound subunits. Some biological properties including acute intraperitoneal toxicity, canatoxin-like immunoreactivity, hemagglutination, trypsin inhibitory activity, induction of platelet release reaction, and aggregation displayed by soyatoxin were studied and used to differentiate soyatoxin from soybean lectin and trypsin inhibitors.

Animals↗
PubMed1980

The effect of seed crystals on calcium oxalate nucleation.

Equimolar concentrations of calcium chloride and potassium oxalate were precipitated by a rapid mixing technique. The precipitated calcium oxalate particles were repeatedly counted at 15 min with a Coulter counter until reproducible total particle count and particle distribution curves were obtained. These values were compared with those found after the addition of various seed crystals and 10 per cent urine to the system. Calcium oxalate seed crystals markedly enhanced the nucleation rate of calcium oxalate (1), although only an estimated 16 per cent acted as effective nucleators. The addition of sodium acid urate crystals resulted in a small decrease in the number of calcium oxalate particles precipitated and fewer than 0.1 per cent of the crystals acted as effective nuclei. Seeds of agglomerated hydroxyapatite crystals also produced a small decrease in the number of calcium oxalate particles and fewer than 1 per cent of the seeds functioned as nucleators. The addition of 10 per cent urine to the system greatly increased the number of calcium oxalate particles produced although we are uncertain whether the urine provided effective nuclei or prevented particle aggregation. Our data indicate that, in a rapidly mixed system, neither sodium acid urate nor hydroxyapatite crystals are effective nucleators for the precipitation of calcium oxalate. The occurrence of epitaxial growth of calcium oxalate on these substances in urinary stones is therefore called into question.

Calcium Chloride↗
PubMed1990

Association and folding in legumin oligomers of lupin seed.

We studied the association and conformational behavior under native or denaturing conditions in the 12S in equilibrium with 7S oligomers of lupin legumin and in the modified 7S (m7S) oligomer, which has lost the capacity to make a 12S molecule. Circular dichroism (CD), gel filtration FPLC, and PAGE were used. The native m7S oligomer has more alpha helix and nearly the same amount of beta structure as the 12S in equilibrium with 7S preparation. Conditions that shift the equilibrium in the 12S in equilibrium with 7S system toward the 7S oligomer also make the secondary structure more similar to that of m7S molecules: higher negative ellipticity appears to be a peculiarity of 7S assemblies, whether they contain modified or unmodified monomers. Part of the helical components show low stability and disappear in 1 M urea. The CD and the separation behavior on increasing the urea concentration, and in 6 M guanidine HCl, denote similar multistep unfolding in both preparations. The 12S oligomer disassembles progressively: however, also under highly denaturing conditions, modified and unmodified preparations are mainly present in an associated form. Small amounts of monomer and aggregates were detected at high denaturant concentrations.

Chromatography, High Pressure Liquid↗
PubMed1987

Soybean hydrophobic protein. Isolation, partial characterization and the complete primary structure.

A 9000-Mr protein isolated from a 60% ethanolic extract of soybean (Glycine max) seeds has been characterized and fully sequenced. The protein consists of 80 amino acid residues with four disulfide bonds. It contains a large number of hydrophobic residues and lacks methionine, phenylalanine, tryptophan, lysine and histidine residues. The protein readily crystallizes from water but is quite soluble in aqueous organic solvents like 95% 1-propanol. It aggregates to form large molecules (above 80 kDa) under ordinary denaturing conditions, such as 6 M guanidine X HCl and 8 M urea. Sequence analysis showed that the amino-terminal four-fifths is extremely hydrophobic and most of the acidic residues exist as their amide forms, and only the carboxyl-terminal short segment is rather hydrophilic. A computer search for homology detected an unexpected similarity of this protein to rat prolactin; however, its significance could not be assessed and this protein appears to represent a hitherto unknown protein family. Although no biochemical activity could be detected, the existence in relatively high abundance (approx. 200 mg from 1 kg seeds) of this novel protein may suggest its physiological significance in the plant.

Amino Acid Sequence↗
PubMed2002

Optimization of cardiac cell seeding and distribution in 3D porous alginate scaffolds.

Cardiac tissue engineering has evolved as a potential therapeutic approach to assist in cardiac regeneration. We have recently shown that tissue-engineered cardiac graft, constructed from cardiomyocytes seeded within an alginate scaffold, is capable of preventing the deterioration in cardiac function after myocardial infarction in rats. The present article addresses cell seeding within porous alginate scaffolds in an attempt to achieve 3D high-density cardiac constructs with a uniform cell distribution. Due to the hydrophilic nature of the alginate scaffold, its >90% porosity and interconnected pore structure, cell seeding onto the scaffold was efficient and short, up to 30 min. Application of a moderate centrifugal force during cell seeding resulted in a uniform cell distribution throughout the alginate scaffolds, consequently enabling the loading of a large number of cells onto the 3D scaffolds. The percent cell yield in the alginate scaffolds ranged between 60-90%, depending on cell density at seeding; it was 90% at seeding densities of up to 1 x 10(8) cells/cm(3) scaffold and decreased to 60% at higher densities. The highly dense cardiac constructs maintained high metabolic activity in culture. Scanning electron microscopy revealed that the cells aggregated within the scaffold pores. Some of the aggregates were contracting spontaneously within the matrix pores. Throughout the culture there was no indication of cardiomyocyte proliferation within the scaffolds, nor was it found in 3D cultures of cardiofibroblasts. This may enable the development of cardiac cocultures, without domination of cardiofibroblasts with time.

Alginates↗
PubMed1994

Stimulatory effect of opioid peptides and naloxone on rat spinal cord cells in primary dissociated culture.

Opioid peptides leu-enkephalin, a synthetic analog of enkephalin dalargin and an opiate receptor blocker naloxone were studied for their morphological effect on the cells of dissociated cultures of rat spinal cord. Low density seeding of cells (3.10(5);6.10(5) cells/ml) on collagen substrate was performed to document that opioid peptides increase the number of cultured cells and neurite outgrowth and lead to the activation of the initiated processes of aggregate formation. Upon higher density of plating (5.10(6) cells/ml) with poly-L-lysine as a substrate, activation of the aggregate formation process was demonstrated, both opioid peptides and naloxone leading to an increase in the size of aggregates. Statistical treatment of the results obtained in this set of experiments documented that leu-enkephalin, dalargin and naloxone increased 2.2-, 2.2-2.6-, 2.4-fold, respectively, the size of aggregates compared to the control, i.e. the reaction of spinal cord cells to opioid peptides and opiate receptor blocker naloxon was unidirected. The total effect of opioid peptides and naloxon resulted in a 3.6-fold increase in the size of the aggregates compared to the control. The data obtained in this study allow the assumption that opioid peptides and naloxone, while activating spinal cord cells via receptors of a different type, manifest the properties of factors thus increasing survival and adhesion of spinal cord cells in culture.

Amino Acid Sequence↗