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PubMed1993

The carboxy terminus of the beta amyloid protein is critical for the seeding of amyloid formation: implications for the pathogenesis of Alzheimer's disease.

Several variants of the beta amyloid protein, differing only at their carboxy terminus (beta 1-39, beta 1-40, beta 1-42, and beta 1-43), have been identified as the major components of the cerebral amyloid deposits which are characteristic of Alzheimer's disease. Kinetic studies of aggregation by three naturally occurring beta protein variants (beta 1-39, beta 1-40, beta 1-42) and four model peptides (beta 26-39, beta 26-40, beta 26-42, beta 26-43) demonstrate that amyloid formation, like crystallization, is a nucleation-dependent phenomenon. This discovery has practical consequences for studies of the beta amyloid protein. The length of the C-terminus is a critical determinant of the rate of amyloid formation ("kinetic solubility") but has only a minor effect on the thermodynamic solubility. Amyloid formation by the kinetically soluble peptides (e.g., beta 1-39, beta 1-40, beta 26-39, beta 26-40) can be nucleated, or "seeded", by peptides which include the critical C-terminal residues (beta 1-42, beta 26-42, beta 26-43, beta 34-42). These results suggest that nucleation may be the rate-determining step of in vivo amyloidogenesis and that beta 1-42 and/or beta 1-43, rather than beta 1-40, may be the pathogenic protein(s) in AD.

Alzheimer Disease↗
PubMed1988

Effect of seeding density on stability of the differentiated phenotype of pig articular chondrocytes in culture.

Articular chondrocytes are known to be phenotypically unstable in culture. One condition that has been reported to suppress dedifferentiation is cultivation at high density on tissue-culture plastic. The aim of the experiments described here was to study the effect of seeding density on chondrocyte proliferation and 35SO4 incorporation, and on the types of collagen and proteoglycan synthesized. I found that cells seeded at low or high density reached the same final density at confluence, and that 35SO4 incorporation, while initially higher (per cell) in high-density cultures, fell under both conditions, reaching the same low level after 3 weeks. The proportion of cells expressing keratan sulphate fell in low- but not high-density cultures and the decline was not prevented by inhibition of cell division. In all the cultures cells expressing keratan sulphate tended to have a rounded morphology. After 21 days in culture, chondrocytes grown at high density expressed predominantly large proteoglycans that aggregated with hyaluronic acid, whereas in low-density cultures a smaller, non-aggregating form was also present. By 21 days in culture cells at both high and low density were expressing type I collagen, although the high-density cells also had an extensive extracellular matrix of type II collagen. These observations support the conclusion that high seeding density stabilizes the chondrocyte phenotype to a greater extent than low seeding density. They also suggest that enhanced dedifferentiation at low density may be due to cell spreading, rather than to selective proliferation of a phenotypically unstable subpopulation of cells.

Animals↗
PubMed1987

Mutational analysis of the embryo-specific urease locus of soybean.

By a non-destructive urease screen of M2 soybean (Glycine max [L.] Merr. cv. Williams) seeds, four true-breeding mutants (n4, n6, n7 and n8) were recovered which lack most (n6, n8) or all (n4, n7) embryo-specific urease activity. This trait was due to a single, recessive lesion at the Sun (seed urease-null) locus identified earlier in an exotic germplasm (PI 229324, Itachi). All sun mutants produced normal ubiquitous urease, the low abundance isozyme found in all soybean tissues examined. Tight mutants n4 and n7 accumulated no detectable embryo-specific urease protein or mRNA; n6 and n8 accumulated normal or near normal levels of urease mRNA but had seed urease protein levels approximately 5% and .05%, respectively, of the progenitor. Mutant n8 appeared to produce a low level of fully active urease (approximately 0.7% activity level, approximately 0.5% protein level) while n6 produced a higher level of an altered, nearly inactive urease (.0.09% activity level, approximately 5% protein level). Urease alterations in n6 were manifested by its increased temperature sensitivity and variation in aggregation state and pH preference. Thus, mutations in the Sun locus affected both the level and the nature of the embryo-specific urease gene products indicating that Sun encodes the embryo-specific urease. We reported earlier that the Eul locus, which controls the aggregation state of the embryo-specific urease, is on mep unit from Sun and that the Eul allele is cis to sun is not expressed (Kloth et al. 1987). That the level of urease gene product, its aggregation state and other enzyme properties can be affected by induced sun mutations, suggests that the Eul and sun alleles are at the same locus.

Genes, Plant↗
PubMed1987

Agglomeration to nodules modulates human arterial smooth muscle cells to distinct postinjury phenotype via foam cell transition.

Cultures of arterial smooth muscle cells (SMCs) tend to form loci with multilayered growth as "hills" or "nodules," which is unusual for normal but common for transformed cells. Earlier it was shown that such nodules were composed of SMCs with the distinctive properties of small cell size, low adhesivity, and scarce or no fibronectin and filamentous actin, features which may also characterize tumor cells. Similar properties could be induced by cultivation of SMCs in aggregates, indicating modulation of SMCs to a distinct "multilayered" phenotype, rather than selection of variant SMCs with preference for multilayered growth. Transfer of SMCs to a three-dimensional arrangement by agglomeration to nodules, "spheroids," by seeding of SMCs on low-adhesive substratum, like agarose, was followed by signs of SMC injury with focal autodigestion and with loss of material from the cells, which to some extent was deposited extracellularly, transition to foam cells with cholesterol accumulation mainly as cholesteryl esters, and eventually decrease in cell size. Identically treated fibroblasts showed similar, but much less pronounced, changes and were largely protected by whole blood serum, in contrast to SMCs. The results indicate that the "multilayered" SMC type can be conceived of as a postinjury phenotypic state which is preceded by overt cellular injury and transition to foam cells in conjunction with sudden transfer to three-dimensional arrangement in spheroids. It is suggested that similar modulation may be important in atherosclerosis, in which foam cell transition and deposition of debris are prominent changes.

Cell Adhesion↗
PubMed1985

The growth and morphology of FRTL-5 thyroid epithelial cells grown as multicellular spheroids in vitro.

FRTL-5 cells, a diploid line of differentiated rat thyroid epithelial cells, have been grown as multicellular spheroids in spinner culture. Spheroids were initiated by seeding FRTL-5 cells either into Lab-Tek dishes or culture flasks with a 0.5% agar base. Thyroid stimulating hormone (TSH, greater than 1.0 mU/ml) was required for initial cell aggregation and spheroid growth. After 1 wk cellular aggregates were transferred to suspension culture in spinner flasks. As with FRTL-5 monolayer cultures, continued spheroid growth required the addition of TSH to the culture medium. The most unique characteristic of the FRTL-5 spheroids was the development of central lumina similar to thyroid follicles in vivo. Follicular structures were absent from spheroids not stimulated with TSH. In the presence of TSH epithelial cells seem metabolically active with morphological evidence of biosynthesis of thyroglobulin-like material and basal laminar-like components. In contrast, all evidence of cellular metabolic activity is absent from cells in spheroids maintained in the absence of TSH. Thus, nontransformed FRTL-5 cells grown as three-dimensional multicellular spheroids responded to hormonal manipulation in a manner comparable to follicular epithelial cells in vivo. This spheroid model might therefore prove to be a very effective tool for investigating aspects of thyroid physiology and pathology in vitro.

Animals↗
PubMed2005

A kinetic modeling of chondrocyte culture for manufacture of tissue-engineered cartilage.

For repairing articular cartilage defects, innovative techniques based on tissue engineering have been developed and are now entering into the practical stage of clinical application by means of grafting in vitro cultured products. A variety of natural and artificial materials available for scaffolds, which permit chondrocyte cells to aggregate, have been designed for their ability to promote cell growth and differentiation. From the viewpoint of the manufacturing process for tissue-engineered cartilage, the diverse nature of raw materials (seeding cells) and end products (cultured cartilage) oblige us to design a tailor-made process with less reproducibility, which is an obstacle to establishing a production doctrine based on bioengineering knowledge concerning growth kinetics and modeling as well as designs of bioreactors and culture operations for certification of high product quality. In this article, we review the recent advances in the manufacturing of tissue-engineered cartilage. After outlining the manufacturing processes for tissue-engineered cartilage in the first section, the second and third sections, respectively, describe the three-dimensional culture of chondrocytes with Aterocollagen gel and kinetic model consideration as a tool for evaluating this culture process. In the final section, culture strategy is discussed in terms of the combined processes of monolayer growth (ex vivo chondrocyte cell expansion) and three-dimensional growth (construction of cultured cartilage in the gel).

Animals↗
PubMed1986

Different effects of aspirin, dipyridamole and UD-CG 115 on platelet activation in a model of vascular injury: studies with extracellular matrix covered with endothelial cells.

Cultured endothelial cells produce an extracellular matrix (ECM) which activates platelets, similarly to deendothelialized vascular segments. Platelet-rich plasma (PRP) was incubated with endothelial cells cultures seeded in various densities on ECM. The interaction of the platelets with this artificial intima was evaluated by phase microscopy and by thromboxane A2 (TXA2) and prostacyclin (PGI2) measurement. Large platelet aggregates were formed on exposed ECM. Platelets aggregation but not adhesion on the ECM was markedly inhibited by the presence of endothelial cells. Pretreatment of the endothelial cells with 0.1 mM aspirin reduced their PGI2 synthesis and was associated with platelet aggregation on the ECM. 10 microM dipyridamole markedly inhibited platelet activation by ECM when the drug was added to citrated whole blood before PRP preparation. UD-CG 115 which elevates cyclic AMP in cardiac muscle, inhibited platelet aggregation and TXA2 production induced by ECM, in the presence as well as in the absence of endothelial cells, without any effect on endothelial PGI2 production.

6-Ketoprostaglandin F1 alpha↗
PubMed2001

Enhancing the start-up of a UASB reactor treating domestic wastewater by adding a water extract of Moringa oleifera seeds.

Water extract of Moringa oleifera seeds (WEMOS) was used to enhance the start-up of a self-inoculated upflow anaerobic sludge blanket (UASB) reactor treating raw domestic wastewater. Two reactors labelled control (RC) and WEMOS addition (RM) were started without special inoculum. Both reactors were fed continuously for 22 weeks with domestic wastewater containing an average total chemical oxygen demand (COD) of 320 mg O2/l and suspended solid (SS) of 165 mg/l. The reactors operated during the entire experimental period at 29 degrees C and at a hydraulic retention time (HRT) of 4 h. The RM reactor received 2 ml WEMOS per litre of influent. WEMOS solution was prepared on the basis of 2.5% (w/v) ground M. oleifera seeds in water. The results of 22 weeks' operation showed an improvement in the performance of the RM compared to that of the RC. The dosage of WEMOS in the feed (1) shortened the biological start-up period by 20%, (2) increased acidogenic and methanogenic activity by a factor of 2.4 and 2.2 respectively, (3) increased the specific biogas production by a factor of 1.6, (4) favoured fast growth of the sludge bed, and (5) allowed the aggregation of coccoid bacteria and growth of microbial nuclei, which are precursors of anaerobic granulation.

Anaerobiosis↗
PubMed2000

Cytodifferentiation and transformation of embryogenic callus lines derived from anther culture of wheat.

Three types of callus tissues established from anther culture of eleven doubled haploid (DH) lines of wheat (Triticum aestivum L.) were evaluated for their ability in enhancing friable embryogenic (Type II) culture differentiation and genetic transformation. Differences between types of callus inocula were highly significant (P < 0.001), suggesting that the quality of the initial callus explant is of profound importance in encouraging the proliferation of Type II cultures. Other factors found to be crucial included weekly subculture of friable embryogenic callus tissues on a maintenance medium containing 30 microM dicamba and a predominance of amino-acid nitrogen supplement. Transfer and integration of the beta-glucuronidase gene was also affected by the type of inoculum when suitable embryogenic cell cultures were transformed using silicon carbide whiskers and high velocity microprojectiles. Expression of the hygromycin phosphotransferase selectable marker gene sequence was confirmed in all the stably transformed cell lines maintained on selection media containing lethal levels of hygromycin. Comparatively, there were differences in the frequency of regenerable, transgenic clonal segments between whisker-treated and microprojectile bombarded tissues mainly as a result of the fact that cultures vortexed with whiskers were more capable of post-treatment cell proliferation and embryo differentiation than those bombarded with cDNA-coated microprojectiles. Conditions for obtaining these results are outlined and discussed in relation to the suitability of the two transformation strategies for producing transgenic cell aggregates of wheat.

Cell Differentiation↗
PubMed2004

Bacteroid proline catabolism affects N(2) fixation rate of drought-stressed soybeans.

In prior work, we observed that soybean (Glycine max L. cv Merr.) seeds inoculated with a mutant Bradyrhizobium japonicum strain unable to catabolize Pro (Pro dehydrogenase(-) [ProDH(-)]) resulted in plants that, when forced to depend on N(2) fixation as the sole source of nitrogen and subjected to mild drought stress, suffered twice as large a loss in seed yield as did plants inoculated with the parental strain. Here, we used a continuous gas flow system to measure H(2) evolution as a function of time and leaf water potential (Psi(L)). Since one H(2) is produced for every N(2) fixed as an obligate part of the mechanism of N(2) fixation, these measurements serve as the basis for continuous monitoring of the N(2) fixation rate. In five replicate experiments, the slope of the decline in N(2) fixation rate in response to water stress was always greater for plants inoculated with the mutant strain unable to catabolize Pro or take up H(2) (ProDH(-), hup(-)) than it was for plants inoculated with the parental strain (ProDH(+), hup(-)). In aggregate, the probability that this difference occurred by chance alone was 0.005. In combination with the earlier result, this is consistent with bacteroid catabolism of Pro synthesized in response to mild drought stress having a positive impact on N(2) fixation rate and seed yield.

Bradyrhizobium↗
PubMed1999

Fractal Approach to Adsorption Isotherms of Water Vapor on Active Carbons.

Adsorption isotherms of water vapor on active carbons are interpreted in terms of the fractal approach. The linear range of plots in the steep rise of the adsorption branch is the process of the cluster formation of water on the seed water molecules adsorbed on the primary adsorption centers, mainly the oxygen-surface complexes, with the fractal dimension D = 1.64-1.67 which is equivalent to that for the diffusion-limited aggregation (DLA) model with D approximately 1.66. Also, it is shown that the two-dimensional characteristics of micropore structures are reflected in the adsorption branch and the three-dimensional ones are in the desorption branch, respectively, by the analyses of the D and the average micropore width L based on the characteristic adsorption amounts at the beginning and the ending points of the steeply rising and falling linear ranges on both branches. Copyright 1999 Academic Press.

Journal Article↗
PubMed2001

Porous silicon: an effective nucleation-inducing material for protein crystallization.

Protein crystals play a pivotal part in structural genomics, hence there is an urgent requirement for new and improved methodology to aid crystal growth. Considerable effort has been invested in the search of substances (nucleants) that will induce efficient nucleation of protein crystals in a controlled manner. To date, nucleation has been facilitated mainly by seeding, epitaxy, charged surfaces or mechanical means. A different approach is introduced here, involving the use of a mesoporous material that is likely to constrain protein molecules and thereby encourage them to aggregate in crystalline order. Large single crystals were obtained using porous silicon at conditions that are not sufficient for spontaneous nucleation, for five out of six proteins that were investigated. We propose that this success is due to the size distribution of pores in the specially designed porous silicon.

Chemical Precipitation↗
PubMed2001

Immunization with a nontoxic/nonfibrillar amyloid-beta homologous peptide reduces Alzheimer's disease-associated pathology in transgenic mice.

Transgenic mice with brain amyloid-beta (Abeta) plaques immunized with aggregated Abeta1-42 have reduced cerebral amyloid burden. However, the use of Abeta1-42 in humans may not be appropriate because it crosses the blood brain barrier, forms toxic fibrils, and can seed fibril formation. We report that immunization in transgenic APP mice (Tg2576) for 7 months with a soluble nonamyloidogenic, nontoxic Abeta homologous peptide reduced cortical and hippocampal brain amyloid burden by 89% (P = 0.0002) and 81% (P = 0.0001), respectively. Concurrently, brain levels of soluble Abeta1-42 were reduced by 57% (P = 0.0019). Ramified microglia expressing interleukin-1beta associated with the Abeta plaques were absent in the immunized mice indicating reduced inflammation in these animals. These promising findings suggest that immunization with nonamyloidogenic Abeta derivatives represents a potentially safer therapeutic approach to reduce amyloid burden in Alzheimer's disease, instead of using toxic Abeta fibrils.

Alzheimer Disease↗
PubMed1994

Enhanced proteolytic activity is responsible for the aberrant morphogenetic development of SV40-immortalized normal human salivary gland cells grown on basement membrane components.

BACKGROUND: A reconstituted basement membrane extract, Matrigel, is a potent inducer of cell growth and differentiation in vitro in a number of cell types. In this study, we examined the effect of Matrigel on the morphogenesis of cultured normal human salivary gland cells. EXPERIMENTAL DESIGN: SV40-immortalized normal human salivary gland-cell clones with duct- (NS-SV-DC) or myoepithelial phenotype (NS-SV-MC), which had been established in our laboratory (Azuma M, et al., Lab Invest, 1993;69:24-42), were cultured on Matrigel in serum-free culture conditions, and then morphogenetic behavior of cell clones was examined. RESULTS: When cell clones were seeded on Matrigel, they formed round or zonal clusters on day 1; however, they failed to develop into a salivary gland morphogenesis. Semithin sections of cell clones cultured on Matrigel exhibited multicellular aggregates on day 1, whereas on days 2 and 3, these cells lost both cell-Matrigel and cell-cell interactions and eventually entered crisis. In an attempt to understand the mechanism involved in this phenomenon, we investigated proteolytic enzymes and their inhibitors secreted by cell clones. Although cell clones produced almost identical levels of gelatinases, they released increased amounts of plasminogen activators as compared with a neoplastic human salivary gland cell line, which had already been demonstrated to differentiate into acinar cells when cultured on Matrigel. Obvious difference of expression level of tissue inhibitor of metalloproteinases-1 was not observed in these cells, however, NS-SV-DC produced a relatively small amount of endothelial-type plasminogen activator inhibitor as compared with NS-SV-MC. Neutralization of excess plasminogen activators by exogenously added serine protease inhibitors corrected the aberrant in vitro morphogenesis of NS-SV-DC, but not of NS-SV-MC, and allowed NS-SV-DC to form glandular-like structures.

Animals↗
PubMed2000

Mechanisms for the formation and maintenance of traditional night roost aggregations in a territorial damselfly.

Communal roosting has been studied extensively in birds, but the mechanisms and functions of this taxonomically widespread behaviour pattern remain poorly understood. We studied the roosting behaviour of rubyspot damselflies, Hetaerina americana, in relation to sex and territorial status, and conducted field experiments to test for specific mechanisms of roost formation and maintenance. Both sexes tended to return close to their previous night's roost, but only males were significantly more roost site faithful than chance expectations based on individual day ranges. Males were more roost site faithful when they held mating territories. After acquiring a territory, males usually began roosting closer to the territory after a delay of a few days. Roosts were not located at sites that reduced the daily commuting distance between hunting areas and territories; males generally hunted closer to their territories than to their roosts. In field experiments, sites 'seeded' with synthetic models of male rubyspots attracted more recruits than vacant control sites and control sites seeded with nonrubyspot (clear-winged) damselfly models. Sites seeded repeatedly with rubyspot models often remained popular for roosting after the models were removed, suggesting that the models established new traditional roosts. These results indicate that conspecific attraction and individual spatial memory together may be sufficient to explain, at a proximate level, the traditional night roost aggregations of this species. We discuss these results in relation to functional hypotheses for roost site choice and fidelity. Copyright 2000 The Association for the Study of Animal Behaviour.

Journal Article↗
PubMed2000

Sugar levels altered by ectopic expression of a yeast-derived invertase affect cellular differentiation of developing cotyledons of Vicia narbonensis L.

In order to change the sugar status during seed development a yeast-derived invertase gene was expressed in cotyledons of Vicia narbonensis. As a result, sucrose decreased whereas hexoses accumulated. We analysed cell structure and cellular differentiation in cotyledons expressing the yeast-invertase. Transgenic cells contained large and long-persisting vacuoles apparently serving as storage compartments for hexoses and clusters of storage-protein aggregates. In the wild-type, large vacuoles did not persist but were replaced by smaller protein bodies. During maturation and desiccation, the transgenic cells showed plasmolysis and vesiculation of the endo-membrane system. Immunogold-labelling revealed that the storage proteins vicilin and legumin were present within the cytoplasm and the extraprotoplasmic space and were attached to membranes of the endoplasmic reticulum and the nuclei. Protein storage vacuoles in mature seeds appeared heterogeneous and only partially filled. The data suggest that sugars control the subcellular organisation of the vacuolar system. Transcript levels encoding a tonoplast intrinsic protein, a marker for membranes of protein storage vacuoles, remained unchanged whereas mRNA levels of a hexose and a sucrose transporter increased. Generally, transgenic seeds appeared to be physiologically younger than wild-type seeds of the same age. The data underline the important role of sugars in legume seed development.

Cell Differentiation↗
PubMed1998

Formation of spheroidal aggregates of hepatocytes on biodegradable polymers under continuous-flow bioreactor conditions.

Our laboratory has investigated heterotopic hepatocyte transplantation on biodegradable polymer matrices as an experimental treatment for end-stage liver disease. One of the limitations has been survival of sufficient cell mass after transplantation. We hypothesize that in vitro conditioning of cells within polymer matrices prior to implantation may increase hepatocyte survival and function. In this preliminary study we investigated the effect of continuous flow on hepatocytes and sinusoidal endothelial cells on poly-L-lactic acid (PLLA) discs in vitro. Highly porous PLLA discs were manufactured measuring 18 mm diameter by 1 mm thickness using previously described techniques. Hepatocytes were isolated from adult, male Lewis rats (200-300 g) using a two-step collagenase digestion. Sinusoidal endothelial cells were isolated using a two-step collagenase digestion, differential sedimentation, Percoll gradient centrifugation, and selective adherence. PLLA discs were seeded with hepatocytes alone or with co-cultures of hepatocytes and sinusoidal endothelial cells. Seeded discs were then secured within a flow bioreactor chamber and exposed to continuous flow of culture media at a rate of 20 ml/minute through the chamber. Seeded discs placed in static culture conditions served as controls. Specimens seeded with only hepatocytes were harvested at 24 hours, 48 hours, and 168 hours after seeding. Co-culture specimens were harvested after 168 hours. Specimens were viewed under phase-contrast microscopy and then formalin-fixed and prepared for histologic sectioning. Sections were stained with Hematoxylin and Eosin and then analyzed with light microscopy. Hepatocytes under flow conditions formed spheroidal aggregates of cells of 50 to 200 microns in diameter by 24 hours in culture. Hepatocytes in static conditions showed decreased aggregation of cells and spheroid formation was absent. Co-cultured specimens under flow also showed spheroid formation with endothelial cells lining the outside of hepatocyte spheroids. Co-cultured specimens in static culture showed no spheroid formation and no organization between sinusoidal endothelial cells and hepatocytes. These results suggest that continuous flow increases organization of hepatocytes cultured within biodegradable polymer matrices.

Animals↗
PubMed1965

ELECTROPHORESIS STUDY OF EXTRACTS OF SUBMAXILLARY SALIVARY GLANDS FROM NATURALLY RABID DOGS.

Rabies virus in submaxillary salivary glands from naturally infected dogs was investigated by a paper electrophoresis technique, and the virus activity was quantitated by intracerebral titration in mice. Extracts from these salivary glands were found to contain (a) 10(4) to 10(6) mouse ICLD (50) units of wild rabies seed and (b) a protein complex that migrated electrophoretically in the albumin band and more conspicuously in the beta and gamma bands of normal horse serum. The protein complex was interpreted to comprise aggregates of neutralizing antirabies antibody.

Animals↗