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PubMed1996

Ascaris lumbricoides aggregation in relation to child growth status, delayed cutaneous hypersensitivity, and plant anthelmintic use in Madagascar.

The relationships between Ascaris lumbricoides worm burden, growth status, general delayed cutaneous hypersensitivity (DCH) response, and plant anthelmintic use were investigated in a 12-mo prospective survey of 663 children, 4-10 yr old, living in the Ranomafana rainforest, Madagascar. Initial fecal examinations revealed prevalences of 93% for A. lumbricoides, 55% for Trichuris trichuria, and 27% for hookworm. After anthelmintic treatment and a 12-mo reinfection period, 428 of the children participated in worm expulsion studies using pyrantel pamoate, revealing an overdispersed A. lumbricoides worm population, mean = 19.2 (SD = 20.4). Malnutrition was common with 72% of the children growth stunted, 61% underweight, and 6% wasted. The children were also skin tested to recall antigens for DCH, with 94% reacting. The DCH immune response was significantly decreased in the malnourished children; however, DCH was not reduced in relation to increasing worm intensity. Growth status, growth velocity, and triceps skinfold did not vary significantly in relation to A. lumbricoides worm burden. Traditional plant anthelmintic treatment was effective in significantly reducing worm intensity. This study indicates that, in human communities where the children are predominately stunted, A. lumbricoides does not aggregate in the most malnourished or immunodepressed children.

Analysis of Variance↗
PubMed2005

Amyloid fibril formation of alpha-synuclein is accelerated by preformed amyloid seeds of other proteins: implications for the mechanism of transmissible conformational diseases.

Alpha-synuclein is one of the causative proteins of familial Parkinson disease, which is characterized by neuronal inclusions named Lewy bodies. Lewy bodies include not only alpha-synuclein but also aggregates of other proteins. This fact raises a question as to whether the formation of alpha-synuclein amyloid fibrils in Lewy bodies may occur via interaction with fibrils derived from different proteins. To probe this hypothesis, we investigated in vitro fibril formation of human alpha-synuclein in the presence of preformed fibril seeds of various different proteins. We used three proteins, Escherichia coli chaperonin GroES, hen lysozyme, and bovine insulin, all of which have been shown to form amyloid fibrils. Very surprisingly, the formation of alpha-synuclein amyloid fibril was accelerated markedly in the presence of preformed seeds of GroES, lysozyme, and insulin fibrils. The structural characteristics of the natively unfolded state of alpha-synuclein may allow binding to various protein particles, which in turn triggers the formation (extension) of alpha-synuclein amyloid fibrils. This finding is very important for understanding the molecular mechanism of Parkinson disease and also provides interesting implications into the mechanism of transmissible conformational diseases.

Amyloid↗
PubMed1994

Colonic mucosal replacement by syngeneic small intestinal stem cell transplantation.

A novel method of colonic mucosal replacement by transplantation of disaggregated small intestinal epithelium is described. Thirty-one inbred rats had the ascending colon isolated, and surgical mucosectomy was performed on the "free" loop. Epithelial cell aggregates were isolated from postnatal small intestine using collagenase and dispase digestion, then 20 microL of the cell suspension was "seeded" over the denuded colonic muscle of 25 recipient rats. Six control rats had surgical mucosectomy only. All loops were retrieved after 14 days for histologic examination. Stem cell lineage studies were used with selective staining protocols to identify enterocytes, goblet cells, entero-endocrine cells, and Paneth cells. A neomucosa with typical small bowel morphology including crypts and villi and all four stem cell lineages was regenerated by transplanted cells on the colonic muscle in 19 of 25 (76%) recipients. Control loops showed no epithelial regrowth confirming total mucosectomy. With appropriate stromal support, transplanted small intestinal stem cells have the capacity to re-epithelialize denuded colonic muscle with small bowel neomucosa.

Animals↗
PubMed1980

Isolation of a morphologically-transformed epithelial cell-line from rat kidney following an in vivo dose of dimethylnitrosamine.

In an in vivo-in vitro system, a rat kidney epithelial cell-line was isolated into continuous culture 48 h after a single administration intraperitoneally of dimethylnitrosamine (DMN). The dose used, 60 mg/kg, was known to induce approximately a 40% incidence of renal cortical epithelial umors, in addition to a much higher incidence of renal mesenchymal tumors. The cell-line was established from several persisting islands of epithelial cells which were cultured in Leibowitz medium supplemented with various-growth enhancing agents and periodically, the antifibroblastic agent cishydroxyprolin. Through succeeding subcultures, the cells retained an epithelial morphology and at confluence piled-up into 3-dimensional ridges and domes above the monolayer. The cells were characterized by continuous growth in culture, the ability to form colonies at very low seeding rates, loss of anchorage-dependence as determined by growth in semi-solid media and formation of multicellular aggregates in suspension culture, and agglutination in the presence of conconavalin A. These altered properties of growth and behavior indicate that the renal epithelial cells constitute a morphologically transformed line. As such, they may represent the same target cell population from which DMN-induced epithelial tumors arise in the rat kidney.

Animals↗
PubMed1995

The core Alzheimer's peptide NAC forms amyloid fibrils which seed and are seeded by beta-amyloid: is NAC a common trigger or target in neurodegenerative disease?

BACKGROUND: NAC is a 35-amino-acid peptide which has been isolated from the insoluble core of Alzheimer's disease (AD) amyloid plaque. It is a fragment of alpha-synuclein (or NACP), a neuronal protein of unknown function. We noted a striking sequence similarity between NAC, the carboxyl terminus of the beta-amyloid protein, and a region of the scrapie prion protein (PrP) which has been implicated in amyloid formation. RESULTS: NAC was prepared by chemical synthesis and was found to form amyloid fibrils via a nucleation-dependent polymerization mechanism. NAC amyloid fibrils effectively seed beta 1-40 amyloid formation. Amyloid fibrils comprising peptide models of the homologous beta and PrP sequences were also found to seed amyloid formation by NAC. CONCLUSIONS: The in vitro model studies presented here suggest that seeding of NAC amyloid formation by the beta-amyloid protein, or seeding of amyloid fibrils of the beta-amyloid protein by NAC, may occur in vivo. Accumulation of ordered NAC aggregates in the synapse may be responsible for the neurodegeneration observed in AD and the prion disorders. Alternatively, neurodegeneration may be caused by the loss of alpha-synuclein (NACP) function.

Amino Acid Sequence↗
PubMed2002

Adaptive immune responses of legumin nanoparticles.

Legumin is one of the main storage proteins in the pea seeds (Pisum sativum L.) and the molecules of this protein have the capacity of binding together to form nanoparticles after aggregation and chemical cross-linkage with glutaraldehyde. The aim of this work was to study the adaptive immune response of legumin nanoparticles in rats. Following intradermal immunisation with the native protein legumin and legumin nanoparticles of about 250 nm, the humoral and cell-mediated immune responses were analysed in rats. The humoral responses against legumin and legumin nanoparticles were examined by western blot and ELISA analysis. Both techniques clearly showed that sera from rats immunised with legumin strongly expressed antibodies against this protein. On the contrary, serum samples from rats inoculated with legumin nanoparticles did not contain detectable amounts of antibodies. These results may be explained by a reduction on the antigenic epitopes of the protein induced by the glutaraldehyde used during the cross-linking step. Concerning the cell-mediated response, neither legumin nor legumin nanoparticles stimulated an immunogenic response. This absence of response of spleen lymphocytes for legumin and legumin nanoparticles may be explained by a cytostatic effect of legumin which was corroborated by the evaluation of the middle phase of cell apoptose. In fact, both legumin and legumin nanoparticles are potent inductors of a cytostatic phenomenon and showed a significant increase of the chromatin condensation (p < 0.05) as compared with control.

Animals↗
PubMed1991

[Studies of sodium pentosan polysulphate (SPP) therapy on the urinary stone disease. Experimental studies on the calcium oxalate crystallization and the effects in healthy subjects].

Concerning with the inhibitory activity of sodium pentosan polysulphate (SPP) on calcium oxalate stone formation, the following three experiments were carried out. 1) The inhibitory activities for calcium oxalate crystal formation, aggregation and growth in vitro, 2) the effects on the deposition of calcium oxalate crystals in rats treated with ethylene glycol, and 3) the effects of oral administration to human subjects on the metastable limit of urinary calcium oxalate and on the inhibitory activity of the urine for calcium oxalate crystal growth. The results were as follows: 1. The degree of the calcium oxalate crystal formation measured by an aggregometer was decreased by addition of more than 1 mM of SPP. 2. The inhibitory activity of SPP on calcium oxalate crystal aggregation was measured using a Coulter Counter. The aggregation was inhibited at a low concentration of SPP (0.02 microM). 3. Calcium oxalate crystal growth was measured using a seeded crystal method with [14C] oxalic acid. SPP inhibited the crystal growth proportionally to the concentration of SPP (0.1 microM-100 microM). 4. Renal tubular deposition of calcium oxalate crystals was marked in rats that orally received ethylene glycol (0.4%), which was expected to be suppressed by intramuscular administration of SPP (30 mg/kg/day or 60 mg/kg/day). 5. Oral administration of SPP (500 mg/day) to five healthy male subjects for five days resulted a tendency to increase of the metastable limit of the urinary calcium oxalate as measurement by an aggregometer, but the inhibitory activity of the urine for calcium oxalate crystal growth, which was measured by seeded crystal method with [14C] oxalic acid, showed no variable changes.

Adult↗
PubMedDate not supplied

A quantitative assay for intercellular aggregation.

In an earlier communication (Munn et al., J Immunol. Methods 166: 11-25, 1993), we presented the initial development of a quantitative assay for monitoring the rates of cellular aggregation based on digital image processing and video microscopy. This study describes some important enhancements and modifications to the procedure. A new index is introduced to characterize the three-dimensional morphology of the aggregates. This index is based on temporal changes in the projected area of the cells and cell aggregates during the course of the experiment. By drawing an analogy with the kinetic theory of gases, we have also introduced a procedure to normalize for variations in cell seeding density among different experiments. In addition, the image analysis technique has been improved by introducing a background subtraction algorithm to remove illumination defects and an adaptive segmentation procedure. These improvements allowed us to completely automate the image analysis procedure, thus minimizing user intervention and improving the reproducibility of the measurements. The enhanced visual assay is evaluated using some recent results from our studies on homotypic lymphocyte aggregation.

Cell Aggregation↗
PubMed2003

The role of cell seeding density and nutrient supply for articular cartilage tissue engineering with deformational loading.

OBJECTIVE: Functional tissue engineering (FTE) of articular cartilage involves the use of physiologically relevant mechanical signals to encourage the growth of engineered constructs. The goal of this study was to determine the utility of deformational loading in enhancing the mechanical properties of chondrocyte-seeded agarose hydrogels, and to investigate the role of initial cell seeding density and nutrient supply in this process. DESIGN: Chondrocyte-seeded agarose hydrogels were cultured in free-swelling conditions or with intermittent deformational loading (10% deformation, 1 Hz, 1 h on/ 1 h off, 3 h per day, five days per week) over a two-month culture period. Disks were seeded at lower (10 million cells/ml) and higher (60 million cells/ml) seeding densities in the context of a greater medium supply than previous studies (decreasing the number of cells/ml feed medium/day) and with an increasing concentration of fetal bovine serum (10 or 20% FBS). RESULTS: Under these more optimal nutrient conditions, at higher seeding densities and high serum concentration (20% FBS), dynamically loaded constructs show >2-fold increases in material properties relative to free-swelling controls. After two months of culture, dynamically loaded constructs achieved a Young's modulus of approximately 185 kPa and a dynamic modulus (at 1 Hz) of approximately 1.6 MPa, with a frequency dependent response similar to that of the native tissue. These values represent approximately 3/4 and approximately 1/4 the values measured for the native tissue, respectively. While significant differences were found in mechanical properties, staining and bulk measurements of both proteoglycan and collagen content of higher seeding density constructs revealed no significant differences between free-swelling and loading groups. This finding indicates that deformational loading may act to increase material properties via differences in the structural organization, the production of small linker ECM molecules, or by modulating the size of macromolecular proteoglycan aggregates. CONCLUSIONS: Taken together, these results point to the utility of dynamic deformational loading in the mechanical preconditioning of engineered articular cartilage constructs and the necessity for increasing feed media volume and serum supplementation with increasing cell seeding densities.

Animals↗
PubMed2007

Modification of the crystal habit of celecoxib for improved processability.

Crystallization is often used in the pharmaceutical industry for purification and isolation of drugs, and also as a means of generating polymorphs or isomorphs. The aim of this study was to investigate the role of extrinsic crystallization parameters on the crystallized product, with special emphasis on improving the mechanical properties of acicular celecoxib. Celecoxib isomorphs were prepared using different techniques (solvent crystallization and vapour diffusion) and crystallization conditions (solvents, stirring, degree of supersaturation, crystallization temperature and seeding). Powder X-ray diffractometry, spectroscopic and thermal methods were used to investigate physical characteristics of crystals. Growth kinetics and aggregation dynamics of crystallization in polar and non-polar solvents were simulated using a dynamic light scattering method. The quick appearance of broad peaks over the range of 10-8000 nm in chloroform during crystallization simulation studies indicated faster aggregation in non-polar solvents. Aspect ratio, flow, compressibility and surface area of recrystallized products were also determined. Surface topography was determined by atomic force microscopy and the lath-shaped crystals (aspect ratio of 2-4) exhibited a roughness index of 1.79 in comparison with 2.92 for needles. Overall, the lath-shaped isomorphs exhibited improved flow and better compressibility.

Calorimetry, Differential Scanning↗
PubMed1988

Glycosaminoglycans partially substitute for proteoglycans in spheroid formation of adult rat hepatocytes in primary culture.

Adult rat hepatocytes seeded in a noncoated plastic dish containing serum-free medium formed a monolayer within 24 h of culture. Those seeded in a dish coated with a proteoglycan fraction isolated from rat liver reticulin fibers attached to the dish but did not spread within 4 h, and then gradually assembled to form floating spherical aggregates (spheroids) with a diameter of 120 +/- 40 micron, within 72 h. The proteoglycan fraction appeared to contain dermatan sulfate, heparan sulfate and an unidentified glycosaminoglycan in its glycan moieties by glycosaminoglycan analysis after pronase digestion and high molecular weight proteoglycan molecules (mw: over 300,000 and about 200,000) by SDS-PAGE analysis. Cells seeded in dishes coated with these defined glycosaminoglycans and heparin assembled to form hemispheroids and multilayer islands, but not floating spheroids, within 72 h of culture. Dermatan sulfate had a stronger ability to induce hemispheroids than heparan sulfate or heparin. As the hemispheroid and multilayer islands were the intermediate form between monolayer and floating spheroids, the glycosaminoglycan moieties of the proteoglycan fraction were thought to participate in the formation of spheroid.

Animals↗
PubMed2001

[The adhesion and seeding of NIH3T3 fibroblast on PHB surfaces].

Biodegradable polymers have been widely used in the field of tissue engineering. The adhesive characteristic of polymer surfaces is critical for cell seeding and growth. The adhesion and growth of NIH3T3 fibroblast on poly-beta-hydroxybutrpic acid (PHB) was studied here, and the cell-surface adhesion strength was measured in a shear flow detachment assay. It showed that the critical shear stress for cell detachment was relatively low on PHB, thus cells tend to aggregate and detach from surfaces. On the other hand, cells tend to spread and formed monolayer on poly-lysine coated PHB surface. When the PHB foams scaffolds were coated by poly-lysine, the seeding percentage of cells was also greatly enhanced.

3T3 Cells↗
PubMed1998

The effects of purified eicosapentaenoic and docosahexaenoic acids on arterial thrombosis tendency and platelet function in rats.

Dietary eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were compared for their effects on arterial thrombus formation in vivo using a well validated rat model. Platelet aggregation (triggered by collagen or adenosine diphosphate in whole hirudinized blood), thromboxane formation (TxB2) and platelet phospholipid fatty acid composition were measured also. Animals fed diets containing hydrogenated coconut oil or sunflower seed oil served as pro- and anti-thrombotic controls, respectively. In a first study, rats were fed a mixture of EPA and DHA ethyl esters (MIX) in increasing amounts and results indicated that 4% of n-3 fatty acids had an optimum reducing effect on thrombosis tendency. Dietary administration of MIX further resulted in a dose-dependent promotion of disaggregation after collagen-induced aggregation, which significantly correlated with the reduction in platelet TxB2 formation. In a subsequent comparative study, both EPA and DHA ethyl esters affected thrombosis tendency, platelet aggregation and TxB2 formation to a similar extent. In addition, both polyenes increased the apparent thromboxane A2-sensitivity of platelets, which appeared negatively related to arterial thrombosis tendency. We conclude that EPA and DHA have similar reducing effects on arterial thrombogenesis in vivo in rats and have comparable effects on the selected platelet functions in vitro.

Animals↗
PubMed2005

Aggregation and mortality of Agriotes obscurus (Coleoptera: Elateridae) at insecticide-treated trap crops of wheat.

Agriotes obscurus (L.) wireworms assembled in increasing numbers at rows of treated (Agrox DL Plus seed treatment) and untreated wheat, Triticum aestivum L., planted at increasing densities (0, 0.15, 0.30, and 0.60 seeds/cm). In treated wheat plots at all planting densities, no wireworm damage to seedlings was apparent, and total wireworms taken in core samples in wheat rows increased according to the asymptotic equation y = B0(1 -e(-Blx)), where B0 is the asymptote, B1 is the slope of the initial rise, and x is the seeding density. The number of dead wireworms in treated plots increased linearly and intercepted the asymptotic models (theoretical point at which 100% mortality of assembled population occurs) at 0.95 seeds/cm on 11 June and 1.14 seeds/cm on 18 June 1996. Untreated wheat at all densities planted had severe wireworm damage and significantly reduced stand. Populations that had assembled at the surviving untreated wheat were fewer than in the treated wheat plots, and although increasing with seeding density, did not follow the asymptotic model. The data suggest that A. obscurus populations can be assembled and killed in fallowed fields in large numbers at treated trap crops of wheat over a 19-d period when planted in rows spaced 1 m apart at a linear seeding density of 1.5 seeds/cm.

Animals↗
PubMed1992

Procedures for the efficient purification of pea seed-borne mosaic virus and its genomic RNA.

An efficient protocol for the purification of pea seed-borne mosaic potyvirus (PSbMV) particles was developed. This led to the purification of 10 PSbMV isolates by a single procedure. Virus aggregation during purification did not occur and consequently, high virus yield was consistently obtained. The virus thus purified was suitable for preparing viral genomic RNA, although conventional methods for RNA extraction resulted in RNA degradation. An alternative method was adopted which yielded reproducibly full length and infectious RNA. This was applied to three isolates of PSbMV and the RNA used to direct complementary DNA synthesis which in turn yielded nearly full length cDNA products.

Fabaceae↗
PubMed2005

Platelet-activating factor (PAF) receptor-binding antagonist activity of Malaysian medicinal plants.

Forty-nine methanol extracts of 37 species of Malaysian medicinal plants were investigated for their inhibitory effects on platelet-activating factor (PAF) binding to rabbit platelets, using 3H-PAF as a ligand. Among them, the extracts of six Zingiberaceae species (Alpinia galanga Swartz., Boesenbergia pandurata Roxb., Curcuma ochorrhiza Val., C. aeruginosa Roxb., Zingiber officinale Rosc. and Z. zerumbet Koenig.), two Cinnamomum species (C. altissimum Kosterm. and C. pubescens Kochummen.), Goniothalamus malayanus Hook. f. Momordica charantia Linn. and Piper aduncum L. are potential sources of new PAF antagonists, as they showed significant inhibitory effects with IC50 values ranging from 1.2 to 18.4 microg ml(-1).

Animals↗
PubMed2005

Half-sib family structure of Fagus crenata saplings in an old-growth beech-dwarf bamboo forest.

Half-sib family structure of Fagus crenata saplings was examined in an old-growth beech-dwarf bamboo (Sasa spp.) forest using microsatellite genotypes in parentage analysis to identify the half-sib families in two 50 x 50 m plots: one with 36 adults, 641 saplings and no Sasa cover, the other with 21 adults, 61 saplings and Sasa cover. For large proportions of the saplings (44.6% and 75.4%, respectively) both of their parents were found within the same plot, indicating that pollination events frequently involved short-range pollen dispersal, probably because of the high density of adults in the study population. Although almost all of the adults had half-sib families, the number of offspring in the families was highly variable. In the plot with no Sasa cover, the variation in the number of offspring was significantly explained by the size of parents, i.e. the reproductive success is higher for large adults than for small adults. The half-sibs were aggregately distributed around their parents and the distribution overlapped among different half-sib families, which may be due to the limited seed dispersal and overlapping seed shadows of this species. As expected, there was weak genetic structure in the plot. By contrast, in the plot with Sasa cover, the half-sibs were distributed sparsely with a low density, and the degree of genetic structure was very weak. The difference in the half-sib family structure and genetic structure among saplings presumably reflects the difference in density that should be affected by regeneration dynamics associated with environmental conditions.

Demography↗
PubMed2000

Fibronectin as a potent inhibitor of calcium oxalate urolithiasis.

PURPOSE: Fibronectin (230 kD.) is a multifunctional alpha2-glycoprotein distributed throughout the extracellular matrix and body fluids. Many investigators have demonstrated that fibronectin, because of its cell adhesive action, is related to biological processes such as morphogenesis, wound healing and metastasis. Recent studies have shown that a variety of molecules, including fibronectin, inhibit endocytosis of calcium oxalate crystals in vitro. We investigated other roles of fibronectin in calcium oxalate stone formation. MATERIALS AND METHODS: Immunoblotting of the crystal surface binding substance obtained from pooled healthy male urine samples was used to analyze whether fibronectin was adsorbed onto the surface of calcium oxalate crystals. To clarify the relationship between fibronectin and calcium oxalate crystals, we performed 6 experiments. Experiment 1 was immunohistochemical examination of fibronectin expression in stone forming rat model kidneys, and experiment 2 examined the fibronectin content of stone forming rat kidney models with the enzyme-linked immunosorbent assay. Experiment 3 was designed to determine fibronectin content of Madin-Darby canine kidney (MDCK) cells stimulated by addition of calcium oxalate crystals and experiment 4 identified the inhibitory effect of fibronectin on calcium oxalate crystal growth by the seed crystal method. For experiment 5 we used an aggregometer system to clarify the inhibitory effect of fibronectin on calcium oxalate crystal aggregation and experiment 6 examined the inhibitory effect of fibronectin on the adhesion of calcium oxalate crystals to MDCK cells. RESULTS: In the crystal surface binding substance immunoreactive bands at 230 kD., which correspond to the molecular weight of fibronectin, were detected by Western blot analysis. In stone forming rat kidneys strong expression of fibronectin was found on the renal tubules to which the crystals were attached. The fibronectin content of these kidneys was significantly greater than that of kidneys without calcium oxalate crystals. The fibronectin content of MDCK cells tended to increase in proportion to the concentration of calcium oxalate crystals added to the culture medium. The growth inhibition assay showed that the inhibitory effect of fibronectin on calcium oxalate crystal growth was small in relation to the quantity of fibronectin excreted. However, fibronectin had inhibitory effects on calcium oxalate crystal aggregation and adhesion of the crystals to MDCK cells. CONCLUSIONS: Fibronectin secretion can be stimulated by calcium oxalate crystals, and this protein, which is excreted from the tubular cells, may inhibit calcium oxalate crystal aggregation and attachment to cells.

Animals↗