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PubMed2000

Spatial and temporal distribution of chloroplast DNA polymorphism in a tropical tree species.

The level and the spatial organization of chloroplast DNA polymorphism were investigated in Dicorynia guianensis Hamshoff (Caesalpiniaceae) at different spatial and temporal scales. D. guianensis is a canopy tree of the rain forest that is distributed throughout the Guiana plateau in small aggregates. Twelve different haplotypes were identified using restriction analysis of polymerase chain reaction (PCR) amplified fragments of the chloroplast genome. When populations from different areas of French Guiana were compared, a clear geographical pattern of haplotype frequencies was identified along the Atlantic coast. This pattern is most likely the result of the restriction-expansion dynamics of the tropical forest during the Quaternary. At the local level, D. guianensis was characterized by a high level of within population diversity. Maintenance of within population diversity results from the dynamics of the aggregates; stochastic demography associated with the turnover of aggregates generates genetic differentiation among them. At the stand level, a strong spatial aggregation of haplotypes persisted from the adult to the seedling cohort indicating limited seed flow. There was also a strong difference in levels of diversity between the cohorts which suggested that recruitment over several years is needed in order to maintain genetic diversity during regeneration.

DNA, Chloroplast↗
PubMedDate not supplied

Intercellular junctions, apical differentiation, and infiltrative features in colon cancer: an ultrastructural study. The Colon Cancer Team at IMAS.

Changes in the structure and number of cell junctions have been related to the infiltrative and metastatic potential of tumor cells. Apparently, the loss of cell adhesion should be coordinated with significant changes in the apical and basal cell domains. The authors have performed a sequential ultrastructural study of cells in the superficial, middle, and deep regions of well- and moderately differentiated colon adenocarcinomas. This was to investigate the differences in the organization of different membrane domains among tumor cells in the in situ areas, the advancing, infiltrative edge of the tumors, and the infiltrating zones between these two extreme zones. The results of the study suggest that the organization of these domains is not strictly coordinated, and that, for each infiltration level, both a settling and an infiltrating cell population can be found. These findings could explain the fact that apparently well-differentiated tumors are able to seed distant tissues with individual cells, rather than with well-differentiated glandular aggregates that would hardly be able to reach the vessel lumina without significantly modifying their organization.

Actin Cytoskeleton↗
PubMed2005

Formation of embryoid bodies by mouse embryonic stem cells on plastic surfaces.

Mouse embryonic stem (ES) cells were cultured on artificial polymeric biomembranes with a phospholipid polymer (phosphatidylcholine, PC) surface. ES cells aggregated to form an embryoid body (EB) on the PC surface immediately after seeding. Single EBs formed on the PC surface after 3 d, and their size was depended on the initial number of cells that were seeded. In contrast, many small EBs with a nonuniform shape formed on a conventional hydrophobic nontreated polystyrene surface. RT-PCR assays of the EBs indicated that cell-cell interactions were enhanced in EBs that formed on the PC surface compared with EBs that formed on the polystyrene surface. The transcription factor Pax6, which is a marker of the differentiation of ES cells to neurons, was not expressed in EBs that formed on the PC surface; however, EBs that formed on the polystyrene surface did express Pax6, indicating that they were undergoing differentiation into neurons. When stimulated with retinoic acid (an inducer of differentiation into neurons), EBs on the PC surface expressed Pax6. We also observed that the adhesion of ES cells to the PC surface was reduced. Thus, the formation of large EBs on the PC surface was due to enhanced cell-cell interaction and inhibition of nonspecific differentiation to neurons.

Animals↗
PubMed1991

[Effects of urinary pH and acid-base balance on the formation of calcium oxalate stone].

The effects of urinary pH and acid-base balance on the calcium oxalate stone formation was investigated by two experiments. 24-hr urine samples were collected from 15 recurrent CaOx stone formers, 9 single stone formers and 6 age-matched controls. Inhibitory effect of 1% urine in various pH (4.0-9.0) were calculated by a seed crystal method. In the seed crystal system, there were no significant differences in the inhibitory activity of aggregation (Ia) and in the inhibitory activity of size (Is) for each pH of metastable solution between the stone former group and the control group. However, the value of Ia and Is showed a tendency of rise in proportion to a rise in pH. Rats model for calcium oxalate urolithiasis were fed with three different diets (1% NH4Cl, 5% NaHCO3 and 8% NaHCO3 diet) for three weeks. On the fourth week, 24-hr urine samples were collected. In the animal experiment, calcium oxalate stone formations were predominantly recognized in the kidney of the 1% NH4Cl diet group. The biochemical data showed an increase of urinary calcium and oxalate, and a decrease of urinary citrate. These results suggest that low urinary pH and metabolic acidosis are promoters of the calcium oxalate stone formation.

Acid-Base Equilibrium↗
PubMed2002

Influence of seeding density and dynamic deformational loading on the developing structure/function relationships of chondrocyte-seeded agarose hydrogels.

Chondrocytes cultured in agarose hydrogels develop a functional extracellular matrix. Application of dynamic strain at physiologic levels to these constructs over time can increase their mechanical properties. In this study, the effect of seeding density (20 and 60 x 10(6) cells/ml) on tissue elaboration was investigated. Higher seeding densities increased tissue properties in free-swelling culture, with constructs seeded at 20 and 60 x 10(6) cells/ml reaching maximum values over the 63 day culture period of aggregate modulus HA: 43 +/- 15 kPa, Young's modulus EY: 39 +/- 3 kPa, and glycosaminglycan content [GAG]: 0.96% +/- 0.13% wet weight; and HA: 58 +/- 12 kPa, EY: 60 +/- 5 kPa, and [GAG]: 1.49% +/- 0.26% wet weight, respectively. It was further observed that the application of daily dynamic deformational loading to constructs seeded at 20 x 10(6) cells/ml enhanced biochemical content (approximately 150%) and mechanical properties (approximately threefold) compared to free-swelling controls by day 28. However, at a concentration of 60 x 10(6) cells/ml, no difference in mechanical properties was found in loaded samples versus their free-swelling controls. Multiple regression analysis showed that the mechanical properties of the tissue constructs depend more strongly on collagen content than GAG content; a finding that is more pronounced with the application of daily dynamic deformational loading. Our findings provide evidence for initial cell seeding density and nutrient accessibility as important parameters in modulating tissue development of engineered constructs, and their ability to respond to a defined mechanical stimulus.

Animals↗
PubMed2002

Inhibition of endogenous carcinoembryonic antigen (CEA) increases the apoptotic rate of colon cancer cells and inhibits metastatic tumor growth.

It has been suggested that carcinoembryonic antigen (CEA) enhances metastatic seeding of colon cancer cells due to its homo- and heterophilic binding properties. Our recent finding that endogenous CEA protects colon cancer cells against apoptosis suggests a more complex role of CEA in cancer progression. In this study we compared the in vitro effects of endogenous CEA on tumor cell aggregation and cell cycle regulation of human HT29 colon cancer cells with the corresponding in vivo effects, i.e. tumor cell seeding and formation of metastatic lesions. Stable expression of CEA targeted ribozymes (Rz) under control of a tet-off promoter system allowed regulation of CEA levels on the mRNA and protein level by 50%. Downregulation of CEA levels inhibited tumor cell aggregation by 70%. In accordance with previous studies, reduction of CEA levels increased in vitro the apoptotic rate and reduced colony formation by 30% to 50%. To determine the in vivo effect of CEA-dependent aggregate formation and its growth regulating role under apoptotic stress, HT29 cells with high and low CEA levels, respectively, were injected into nude mice. Immunostaining of lung microsections revealed similar numbers of tumor cells one hour after injection. 24 h later virtually all cells were removed from the lung in both groups. However, after 6 weeks all doxycycline treated mice (Rz off = CEA high) showed 14.5 +/- 4.6 metastatic lung lesions/mouse while 0.2 +/- 0.2 lesions/mouse appeared in the untreated group (Rz on = CEA low) (P < 0.001). Our study demonstrates a multifunctional role of CEA and indicates a prometastatic role of CEA independent of its adhesive function possibly due to its anti-apoptotic function.

Animals↗
PubMed1988

Precipitation of calcium palmitate from bile salt-containing dispersions.

Addition of calcium chloride to mixed micellar systems composed of sodium salts of palmitic acid and high concentrations of different bile acids results in precipitation of Ca(palmitate)2 only when the palmitate concentration exceeds a critical value, which is dependent on the concentrations of Ca2+, Na+ and bile salt, and on the type of bile salt used. All these dependencies, as well as the complex and interrelated effects of the various parameters on the kinetics of Ca(palmitate)2 precipitation are consistent with the following mechanism: (i) calcium binds to palmitate-bile salt mixed micelles and promotes their aggregation, at a rate governed by the concentration ratio between bound calcium and micelles (here denoted "binding ratio"). (ii) Ca(palmitate)2 precipitation occurs within the aggregate of micelles only if those micelles include sufficient amounts of Ca2+ and palmitate to allow for the formation of large enough crystal units of Ca(palmitate)2 which can serve as nucleation "seeds". Both the concentrations of micelles and Na+ have dual effects on the rate of precipitation. Increasing micelle concentration, by itself, accelerates aggregation but at the same time leads to a decrease of the binding ratio, thus reducing the rate of precipitation. Na+ which reduces the binding ratio through competitive binding also reduces the surface charge, thus assisting micelle aggregation. Our model also explains the facilitation of precipitation observed when phosphatidylcholine is contained in the palmitate-bile salt mixed micelles and the inhibitory effect of the water soluble bovine serum albumin.

Bile Acids and Salts↗
PubMed2001

Fine-scale genetic structure in Pinus clausa (Pinaceae) populations: effects of disturbance history.

Spatial autocorrelation analyses of 12 allozyme loci were used to compare genetic structure within populations of two varieties of Pinus clausa. P. clausa var. immuginata populations tend to be uneven-aged, with continuous recruitment in small gaps created by wind damage, whereas P. clausa var. clausa populations are more even-aged, with recruitment postdating periodic canopy fires. Three var. immuginata populations and three matched pairs of var. clausa populations, including both a mature and a nearby recently burned population, were examined. Aggregation of multilocus genotypes at small distances was evident in all young var. clausa populations. Little inbreeding was apparent among juveniles or adults in these populations; their genetic structure is likely to have resulted from limited seed dispersal. Genotypes were not significantly spatially structured in nearby matched mature populations. Genetic structure was less evident in var. immuginata populations. Aggregated genotypes were only apparent in the population where patches included juveniles of similar ages; dense juvenile clumps in the other two var. immuginata populations comprised a variety of ages. Interannual variability in allele frequencies of surviving seedlings may account for the absence of genetic structure in these populations.

Alleles↗
PubMed1992

Evidence for a novel route of wheat storage proteins to vacuoles.

Wheat seed storage proteins are deposited in protein bodies (PB) inside vacuoles, but their subcellular site of aggregation and their route to vacuoles are still controversial. In the present work, an ultra structural analysis of developing wheat endosperm at early to mid maturation was performed to address these issues. Golgi complexes were rarely detected, indicating that their role in wheat storage protein transport is limited. In contrast, a considerable amount of PB was detected in the cytoplasm. Many of these PB were surrounded by RER membranes and were enlarged by fusion of smaller PB. Small, electron lucent vesicles were detected around the surfaces of the PB in the cytoplasm, or attached to them, suggesting that such attachments and subsequent fusion of the vesicles with each other lead to the formation of small vacuoles containing PB inclusions. Immunogold labeling with serum raised against yeast-BiP, an ER-localized protein, demonstrated that the wheat BiP homolog was present within the PB in the cytoplasm as well as inside vacuoles. This confirmed that the PB were formed within the RER and that the Golgi complex was not involved in their transport to vacuoles. It is concluded that a considerable part of the wheat storage proteins aggregate into PB within the RER and are then transported as intact PB to the vacuoles by a novel route that does not utilize the Golgi complex.

Biological Transport↗
PubMed1993

DNA- and PCR-fingerprinting in fungi.

DNA-fingerprinting has been successfully used to detect hypervariable, repetitive DNA sequences (minisatellites and microsatellites) in fungi. Combined with methods used to identify random amplified polymorphic DNA (RAPD), conventional DNA-fingerprinting hybridization probes can also be used as single primers to detect DNA polymorphisms among fungal species and strains. The oligonucleotides (CA)8, (CT)8, (CAC)5, (GTG)5, (GACA)4 and (GATA)4, as well as the phage M13 and its core sequence, have been used as specific probes in hybridization experiments and as primers for PCR analysis. Both methods have enabled the differentiation of all the fungal species and strains that were examined, including species of Penicillium, Trichoderma, Leptosphaeria, Saccharomyces, Candida and Cryptococcus. These methods have been used 1) to clarify the taxonomic relationships among relevant species of the Trichoderma aggregate, 2) to discriminate between aggressive and non-aggressive isolates of the rape seed phytopathogen, Leptosphaeria maculans, and 3) to identify strains of the pathogenic yeasts, Cryptococcus neoformans and Candida albicans. PCR-fingerprinting allowed serotypes of C. neoformans to be distinguished. The application of DNA- and PCR-fingerprinting to fungal DNA should aid in clarification of their taxonomy and improved diagnosis of mycotic disease.

Base Sequence↗
PubMed1992

Amyloid fibril formation requires a chemically discriminating nucleation event: studies of an amyloidogenic sequence from the bacterial protein OsmB.

The sequence of the Escherichia coli OsmB protein was found to resemble that of the C-terminal region of the beta amyloid protein of Alzheimer's disease, which seems to be the major determinant of its unusual structural and solubility properties. A peptide corresponding to residues 28-44 of the OsmB protein was synthesized, and its conformational properties and aggregation behavior were analyzed. The peptide OsmB(28-44) was shown to form amyloid fibrils, as did two sequence analogs designed to test the sequence specificity of fibril formation. These fibrils bound Congo red, and two of the peptides showed birefringence. The peptide fibrils were analyzed by electron microscopy and Fourier transform infrared spectroscopy. Subtle differences were observed which were not interpretable at the molecular level. The rate of fibril formation by each peptide was followed by monitoring the turbidity of supersaturated aqueous solutions. The kinetics of aggregation were characterized by a delay period during which the solution remained clear, followed by a nucleation event which led to a growth phase, during which the solution became viscous and turbid due to the presence of insoluble fibrils. The observation of a kinetic barrier to aggregation is typical of a crystallization event. The delay period could be eliminated by seeding the supersaturated solution with previously formed fibrils. Each peptide could be nucleated by fibrils formed from that same peptide, but not by fibrils from closely related sequences, suggesting that fibril growth requires specific hydrophobic interactions. It appears likely that this repeated sequence motif, which comprises most of the OsmB protein sequence, dictates the structure and possibly the function of that protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1984

Differential control of cytokeratins and vimentin synthesis by cell-cell contact and cell spreading in cultured epithelial cells.

The expression of cytokeratins and vimentin was investigated in Madin-Darby bovine epithelial cells (MDBK) in culture under conditions of varied cell spreading and cell-cell contact. When extensive cell-cell contact was achieved by seeding cells at high density in monolayer, or in suspension culture in which multicellular aggregates formed, the cells synthesized high levels of cytokeratins and low levels of vimentin. In contrast, in sparse monolayer and suspension cultures where cell-cell contact was minimal, the cells synthesized very low levels of cytokeratins. The level of vimentin synthesis was high in sparse monolayer culture and was low in both sparse and dense suspension cultures. The ratio of cytokeratin to vimentin synthesis was not affected during the cell cycle, or when cell growth was inhibited by ara C and in serum-starvation-stimulation experiments. The variations in the synthesis of cytokeratins and vimentin under the various culture conditions were also reflected at the level of mRNA activity in a cell-free in vitro translation system and as determined by RNA blot hybridization with cDNA to vimentin and cytokeratins. The results suggest that control of cytokeratin synthesis involves cell-cell contact, characteristic of epithelia in vivo, while vimentin synthesis responds to alterations in cell spreading.

Animals↗
PubMed1981

The Glycoproteins of Plant Seeds : ANALYSIS BY TWO-DIMENSIONAL POLYACRYLAMIDE GEL ELECTROPHORESIS AND BY THEIR LECTIN-BINDING PROPERTIES.

Protein from the jack bean, peanut, soybean and kidney bean seeds were extracted with a solution containing 9.3 molar urea, 5 millimolar K(2)CO(3), 0.5% dithiothreitol and 2% Nonidet P-40 and then subjected to two-dimensional gel electrophoresis. After electrophoresis, the slab gels were stained with a variety of (125)I-labeled lectins and the lectin-binding proteins were identified after autoradiography. Incubation of slab gels of jack bean with concanavalin A, peanut with peanut agglutinin, soybean with soybean agglutinin, and kidney bean with phytohemagglutinin showed that the majority of the polypeptides in each seed type were able to bind to their homologous lectins. Control slab gels in which incubations were carried out with identical amounts of proteins, (125)I-lectin and an appropriate sugar inhibitor showed little or no lectin binding to the polypeptides. Additionally, incubation of slab gels of peanut proteins with (125)I-ricin, (125)I-wheat germ agglutinin, (125)I-concanavalin A, and (125)I-soybean agglutinin each revealed a clearly distinct binding pattern compared to the one observed with the peanut agglutinin. The results demonstrate that a large number of legume seed polypeptides are glycoproteins and that the carbohydrate groups within a seed species are heterogeneous in structure, thus indicating the existence of complex glycosylating enzyme systems in legume seeds. It is suggested that the high degree of binding between seed proteins and their homologous lectins might have some functional significance in maintaining large aggregates of protein in compact, insoluble form.

Journal Article↗
PubMed1993

Rapid formation of multicellular spheroids of adult rat hepatocytes by rotation culture and their immobilization within calcium alginate.

Tyrosine aminotransferase (TAT) induction and albumin secretion abilities were examined in rat hepatocytes immobilized within calcium alginate; the immobilized hepatocytes lost these abilities within a week. An attempt was then made to immobilize multicellular spheroids of hepatocytes for the purpose of stabilizing the liver functions. Although it takes at least 4 days to form spheroids in the conventional method using monolayer-cultured cells, in this study we developed a new method for rapid spheroid formation. Isolated hepatocytes were seeded into a polystyrene dish and incubated on a rotary shaker. Hepatocytes started to aggregate after 6 h of the rotation culture, and spheroids approximately 100 microns in diameter formed within 24 h. The immobilized spheroids had higher TAT induction and albumin secretion abilities, which were maintained for a longer time, than the immobilized nonaggregated cells. Further stabilization was observed in immobilized heterospheroids formed in the presence of nonparenchymal liver cells. This method for the rapid formation of spheroids consisting of hepatocytes and nonparenchymal liver cells could be utilized in the construction of a bioartificial liver support system.

Albumins↗
PubMed1993

The C-terminus of the beta protein is critical in amyloidogenesis.

The beta amyloid protein found in extracellular deposits in Alzheimer's disease (AD) is heterogeneous at its C-terminus; proteins ending at residues 40, 42, and 43 have been identified in neuritic deposits, while protein in vascular amyloid appears to end at residue 39 or 40. Studies of synthetic beta proteins (beta 1-39, beta 1-40, beta 1-42), and model peptides (beta 26-39, beta 26-40, beta 26-42, beta 26-43) demonstrate that amyloid formation is a nucleation-dependent phenomenon. Peptides ending at residues 39 or 40 were kinetically soluble for hours to days, while peptides ending at residues 42 or 43 aggregated immediately; all eventually reached similar thermodynamic solubility. The kinetically soluble variants could be seeded with the kinetically insoluble variants. The secondary structure of beta 26-39 fibrils was different from that of beta 26-42 fibrils, however, seeding beta 26-39 with beta 26-42 produces mixed fibrils with structure similar to beta 26-42. These results suggest that neuritic plaques may be seeded by their minor component; this may determine the structure and properties of amyloid in AD.

Alzheimer Disease↗
PubMedDate not supplied

A possible route to large-scale production of SWNTs through a combination of the substrate and floating catalyst methods.

We describe a possible route for large-scale synthesis of single wall carbon nanotubes (SWNTs) by a combination of the substrate and floating methods. The template prohibits metal particle aggregation, resulting in high-purity SWNT growth, and the three-dimensional floating seeded state of the template induces improved yields of SWNTs in a semi-continuous system. The SWNTs obtained by this method exhibit large variations of texture (isolated tubes and bundles) and a wide range of diameters (0.4-4 nm).

Catalysis↗
PubMed2006

Safety and efficacy of ginkgo (Ginkgo biloba) during pregnancy and lactation.

BACKGROUND: There is a lack of basic knowledge on the part of both clinicians and patients as to the indications for use and safety of herbal medicines used in pregnancy and lactation. This is one article in a series that systematically reviews the evidence for commonly used herbs during pregnancy and lactation. OBJECTIVES: To systematically review the literature for evidence on the use, safety, and pharmacology of ginkgo focusing on issues pertaining to pregnancy and lactation. METHODS: We searched 7 electronic databases and compiled data according to the grade of evidence found. RESULTS: There is some very weak scientific evidence from animal and in vitro studies that ginkgo leaf has antiplatelet activity, which may be of concern during labour as ginkgo use could prolong bleeding time. Low-level evidence based on expert opinion shows that ginkgo leaf may be an emmenagogue and have hormonal properties. The safety of ginkgo leaf during lactation is unknown. Patients and clinicians should be aware of past reports of ginkgo products being adulterated with colchicine. CONCLUSIONS: Ginkgo should be used with caution during pregnancy, particularly around labour where its anti-platelet properties could prolong bleeding time. During lactation the safety of ginkgo leaf is unknown and should be avoided until high quality human studies are conducted to prove its safety.

Animals↗