Surface Layer Of Cells Research Articles

Evidence for substantial maintenance of membrane integrity and cell viability in normally developing grape (Vitis vinifera L.) berries throughout development

Fluorescein diacetate (FDA) was used as a vital stain to assay membrane integrity (cell viability) in mesocarp tissue of the developing grape (Vitis vinifera L.) berry in order to test the hypothesis that there is a substantial loss of compartmentation in these cells during ripening. This technique was also used to determine whether loss of viability was associated with symptoms of a ripening disorder known as berry shrivel. FDA fluorescence of berry cells was rapid, bright, and stable for over 1 h at room temperature. Confocal microscopy detected FDA staining through two to three intact surface cell layers (300-400 mum) of bisected berries, and showed that the fluorescence was confined to the cytoplasm, indicating the maintenance of integrity in both cytoplasmic as well as vacuolar membranes, and the presence of active cytoplasmic esterases. FDA clearly discriminated between living cells and freeze-killed cells, and exhibited little, if any, non-specific staining. Propidium iodide and DAPI, both widely used to assess cell viability, were unable to discriminate between living and freeze-killed cells, and did not specifically stain the nuclei of dead cells. For normally developing berries under field conditions there was no evidence of viability loss until about 40 d after veraison, and the majority (80%) of mesocarp cells remained viable past commercial harvest (26 degrees Brix). These results are inconsistent with current models of grape berry development which hypothesize that veraison is associated with a general loss of compartmentation in mesocarp cells. The observed viability loss was primarily in the locule area around the seeds, suggesting that a localized loss of viability and compartmentation may occur as part of normal fruit development. The cell viability of berry shrivel-affected berries was similar to that of normally developing berries until the onset of visible symptoms (i.e. shrivelling), at which time viability declined in visibly shrivelled berries. Berries with extensive shrivelling exhibited very low cell viability (15%).

The thrombogenicity of human umbilical vein endothelial cell seeded collagen modules

Modular tissue-engineered constructs are assembled from sub-mm sized cylindrical collagen gel modules which are covered with a surface layer of human umbilical vein endothelial cells (HUVEC). The resulting construct is permeated by a network of interconnected endothelial cell lined channels to facilitate blood perfusion and nutrient delivery. This design strategy relies critically on the endothelial cells' layer behaving in a non-thrombogenic manner on the module surface and the objective here was to characterize this thrombogenicity. HUVEC prolonged clotting times in whole blood-module mixtures, and enabled slightly heparinized whole blood perfusion of an assembled modular construct in vitro with no increase in platelet loss compared to background levels. Flow cytometry and scanning electron microscopy indicated that HUVEC seeded modules reduced platelet activation and deposition but not leukocyte activation, compared to collagen only modules. Plasma recalcification times on non-stimulated HUVEC were longer compared to stimulated HUVEC but not different than that on collagen only module films and were not prolonged by incubation with a tissue factor blocking antibody. Together these data suggest that a functional non-thrombogenic layer of EC was generated on the module surface and that this layer should be sufficient to maintain continuous blood flow through an engineered modular tissue. In/ex vivo studies are warranted to confirm this conclusion.

Genotypic variation of iron partitioning in rice grain

AbstractThe present study investigates the variation of Fe concentration and partitioning in different grain tissues, particularly in the endosperm (white rice) among different rice cultivars from diverse genetic backgrounds. Iron concentration ranged from 10 to 20 mg kg−1 in brown rice and 3–11 mg kg−1 in white rice. Iron concentration in white rice was not correlated with that in brown rice. Polishing removed 25–84% of Fe from the brown rice, resulting in a low Fe concentration in the white rice and changed the ranking order for Fe concentration among the cultivars tested. Total Fe content in the whole grain including husk (r = 0.28, NS) and total Fe content in brown rice (r = 0.09, NS) was not significantly correlated with Fe concentration in white rice. Iron content in white rice was found to vary between 0.05 and 0.2 µg grain−1 among cultivars tested, with the highest Fe content in cv. Sakha. Iron concentration in white rice was negatively correlated with total Fe content in the bran fraction, which is lost during the polishing process to produce white rice from brown rice (r = − 0.82**). The partitioning of Fe between the endosperm and bran seemed to play a key role in achieving high Fe concentration in the white rice. The surface cell layers of the caryopsis appeared to be the main resistance of Fe partitioning into the endosperm (white rice) as there was a significantly negative correlation between Fe concentration in the white rice and Fe content in the bran, which is lost during the polishing process. Copyright © 2007 Society of Chemical Industry

Immunolocalization of Aquaporins in Vocal Fold Epithelia

To investigate the presence of aquaporin (AQP) water channels 1, 2, and 3 in stratified squamous vocal fold epithelium. Immunolocalization analysis of excised ovine vocal fold epithelia. Sheep. Ovine vocal fold epithelia were prepared for immunoelectron microscopy using primary antibodies directed against AQP-1, AQP-2, and AQP-3. Photographic profiles of epithelium exposed to each antibody were used to calculate the immunogold labeling density of the plasma membrane and cytoplasm. Density of immunolabeling was compared across 3 regions that represent cell layers closest to the glottal lumen for the plasma membrane and cytoplasm, respectively. Labeling densities of AQP-1 and AQP-2 were significantly greater for the plasma membrane region of the luminal cells than for deeper cell layers. Cytoplasmic labeling and labeling of circular structures was greatest for cell layers 2 through 5 beneath the vocal fold surface compared with the surface cell layer. Immunogold labeling of AQP-3, an aquaglyceroporin, in vocal fold epithelium was inconclusive. Aquaporins 1 and 2, associated with the plasma membrane region of ovine vocal fold epithelial cells, demonstrate the presence of an intrinsic mechanism to permit transcellular water flux in response to osmotic gradients.

Modular tissue engineering: fabrication of a gelatin-based construct

Modular tissue-engineered constructs are assembled from sub-millimetre-sized cylindrical modules which are seeded with a surface layer of endothelial cells. The resulting construct is permeated by a network of interconnected, endothelial cell-lined channels to facilitate blood perfusion and nutrient delivery. To provide adequate stiffness, yet consist of a substrate suitable for endothelial cells, modular constructs were fabricated from gelatin, the denatured form of collagen. Gelatin modules containing HepG2 cells or spheroids were fabricated using a sieving process. A surface layer of bovine aortic endothelial cells completely covering the modules was generated within 1 week of seeding. Modules were assembled into constructs within a flow circuit and flow rate-pressure difference profiles measured. Fluid perfusion resulted in negligible construct compaction, even at high flow rates. It was necessary, however, to crosslink the gelatin modules with glutaraldehyde to prevent dissolution at 37 degrees C. This resulted in a significant loss of cell viability within the modules. A strategy to enable non-toxic crosslinking of the gelatin modules is required to fabricate constructs containing viable cells.

Coating performance of finishes on wood modified with an N-methylol compound

This study describes the effects of wood modification with modified 1,3-dimethylol-4,5-dihydroxyethyleneurea (mDMDHEU) on the coating performance of solvent- and water-borne finishes. Although the basic colour of wood was slightly changed due to the modification, modified and unmodified samples did not display any colour differences after coating with translucent stains. Blocking of coated wooden substrates (degree of adhesiveness) was dependant on the type of finish system and, in a few cases, on the wood substrate. For all wood substrates, both treated and untreated, solvent-borne alkyd finish penetrated into the surface layers of the wood cells, while water-borne acrylic finishes spread only on the wood surfaces. The solvent-borne alkyd finish dried much more slowly on wood substrates than did the water-borne acrylics. Modification with mDMDHEU did not affect the drying rates of the finishes tested. The wet adhesion depended on the finish systems tested and on the pre-treatment of the wooden substrate. With regard to the pre-treatment, wet adhesion of one water-borne and the solvent-borne finish systems was significantly enhanced through precedent modification with mDMDHEU. The other two water-borne finish systems showed a higher degree of wet adhesion. This study demonstrates that modification of the wood substrate with mDMDHEU is compatible with both water-borne and solvent-borne finish systems and improves their wet adhesion on the wood surface.

Developmental changes in antioxidant metabolites, enzymes, and pigments in fruit exocarp of four tomato ( Lycopersicon esculentum Mill.) genotypes: β-carotene, high pigment-1, ripening inhibitor, and ‘Rutgers’

In surface cell layers of fleshy fruit, antioxidants must limit photooxidative reactions that generate reactive oxygen species (ROS) in high light. Our objective was to measure changes in the concentrations of antioxidant metabolites and pigments, and the activities of enzymes of the Mehler-peroxidase, ascorbate-glutathione cycle in fruit exocarp tissue under non-stress conditions of the following fruit-specific tomato ( Lycopersicon esculentum Mill. = Solanum lycopersicum) mutants and their parent: (1) β-carotene ( B), (2) high pigment ( hp-1), (3) ripening inhibitor ( rin), and (4) the nearly isogenic wild-type ‘Rutgers’. Developmental variables included days after anthesis (DAA) and fruit surface color. The highest total ascorbic acid (AsA) concentration was in the exocarp of immature green fruit of hp-1, being 32% higher than ‘Rutgers’. The hp-1 mutant also had the highest chlorophyll and total carotenoid concentrations, comprised mostly of lycopene in red ripe fruit; whereas, β-carotene comprised 90% of the carotenoids in B. Although enzyme activities varied within genotype, they generally increased with development, then decreased as fruit maturity was reached, being coupled with AsA and glutathione (GSH) concentrations. In all mutants, dark-green (DG) exocarp had more chlorophyll and protein, higher concentrations of reduced AsA and GSH, and usually lower enzyme activities than light-green (LG) exocarp taken from the same fruit.

Surface Position, Not Signaling from Surrounding Maternal Tissues, Specifies Aleurone Epidermal Cell Fate in Maize

Maize (Zea mays) endosperm consists of an epidermal-like surface layer of aleurone cells, an underlying body of starchy endosperm cells, and a basal layer of transfer cells. To determine whether surrounding maternal tissues perform a role in specifying endosperm cell fates, a maize endosperm organ culture technique was established whereby the developing endosperm is completely removed from surrounding maternal tissues. Using cell type-specific fluorescence markers, we show that aleurone cell fate specification occurs exclusively in response to surface position and does not require specific, continued maternal signal input. The starchy endosperm and aleurone cell fates are freely interchangeable throughout the lifespan of the endosperm, with internalized aleurone cells converting to starchy endosperm cells and with starchy endosperm cells that become positioned at the surface converting to aleurone cells. In contrast to aleurone and starchy endosperm cells, transfer cells fail to develop in in vitro-grown endosperm, supporting earlier indications that maternal tissue interaction is required to fully differentiate this cell type. Several parameters confirm that the maize endosperm organ cultures described herein retain the main developmental features of in planta endosperm, including fidelity of aleurone mutant phenotypes, temporal and spatial control of cell type-specific fluorescent markers, specificity of cell type transcripts, and control of mitotic cell divisions.

Role of fluid dynamics on the healing of an in vivo tissue engineered vascular graft

A polyester (PET) reinforced fibrin-FN-VEGF-TGFbeta vascular graft, formed by a four-step preclotting technique of a porous PET arterial graft, shows the overlapping inflammation, proliferation, and remodeling steps of normal wound healing when implanted in the descending thoracic aorta (DTA) position in the dog, forming a surface layer of endothelial cells. While the DTA grafts readily healed (i.e., endothelialized), similar grafts implanted in the carotid-femoral artery position did not fully heal. Since the initial phases of healing were shown to be dependent upon the transport of blood-borne constituents to the graft surface, the extent of healing appears to be dependent on the fluid dynamics present in the artery-graft-artery construct. The length of the noncompliant graft, the construction of the anastomoses, bends in the construct, graft diameter, and graft compliance can affect the fluid dynamics in the implant, and thus the healing of the graft. This has clinical relevance for the testing and development of new vascular graft materials.

RECOGNITION AND SELECTION OF SETTLEMENT SUBSTRATA DETERMINE POST-SETTLEMENT SURVIVAL IN CORALS

Habitat recognition and selective settlement by dispersive propagules greatly increases the post-settlement survival chances of sessile organisms. To better understand the key role some species can play in the structure of highly complex coral reef ecosystems, we compare the role of two independent, but sequential, processes: settlement choice and post-settlement survival. This study describes the chemical and physical recognition and ranking of specific settlement substrata by coral larvae. Several species of crustose coralline algae (CCA) are known to induce coral settlement; however they also employ physical and biological anti-settlement defense strategies that vary greatly in effectiveness. We examine the interactions between settling larvae of two common reef building coral species (Acropora tenuis and A. millepora) and five species of CCA (Neogoniolithon fosliei, Porolithon onkodes, Hydrolithon reinboldii, Titanoderma prototypum, and Lithoporella melobesioides) that co-occur on reef crests and slopes of the Great Barrier Reef, Australia. Distinct settlement patterns were observed when coral larvae were provided with a choice of settlement substrata. Settlement on the most preferred substratum, the CCA species T. prototypum, was 15 times higher than on N. fosliei, the least preferred substratum. The rates of post-settlement survival of the corals also varied between CCA species in response to their anti-settlement strategies (shedding of surface cell layers, overgrowth, and potential chemical deterrents). Rates of larval settlement, post-settlement survival, and the sensitivity of larvae to chemical extracts of CCA were all positively correlated across the five species of CCA. Nonliving settlement substrata on coral reefs is sparse; consequently the fact that only a few CCA species (notably T. prototypum) facilitate coral recruitment, has important implications for structuring the reef ecosystem.

Biochemical Background and Compartmentalized Functions of Cytosolic Glutamine Synthetase for Active Ammonium Assimilation in Rice Roots

Rice plants in paddy fields prefer to utilize ammonium as a major nitrogen source. Glutamine synthetase (GS) serves for assimilation of ammonium in rice root, and ameliorates the toxic effect of ammonium excess. Among the three isoenzymes of the cytosolic GS1 gene family in rice, OsGLN1;1 and OsGLN1;2 were abundantly expressed in roots. Analysis of the purified enzymes showed that OsGLN1;1 and OsGLN1;2 can be classified into high-affinity subtypes with relatively high V(max) values, as compared with the major high-affinity isoenzyme, GLN1;1, in Arabidopsis. Low-affinity forms of GS1 comparable to those in Arabidopsis (GLN1;2 and GLN1;3) were absent in rice roots. The OsGLN1;1 and OsGLN1;2 transcripts showed reciprocal responses to ammonium supply in the surface cell layers of roots. OsGLN1;1 accumulated in dermatogen, epidermis and exodermis under nitrogen-limited condition. By contrast, OsGLN1;2 was abundantly expressed in the same cell layers under nitrogen-sufficient conditions, replenishing the loss of OsGLN1;1 following ammonium treatment. Within the central cylinder of elongating zone, OsGLN1;1 and OsGLN1;2 were both induced by ammonium, which was distinguishable from the response observed in the surface cell layers. The high-capacity Gln synthetic activities of OsGLN1;1 and OsGLN1;2 facilitate active ammonium assimilation in specific cell types in rice roots.

Bi-directional cell contact-dependent regulation of gene expression between endothelial cells and osteoblasts in a three-dimensional spheroidal coculture model

Multiple cell–cell interactions control bone morphogenesis and vascularization. We have employed a spheroidal coculture system of endothelial cells (EC) and osteoblasts (OB) to study cell contact-dependent gene regulation between these two cell types that may play a role in regulating OB differentiation and EC angiogenic properties. Coculture spheroids differentiate spontaneously to organize into a core of OB and a surface layer of endothelial cells. Individual spheroid culture of EC or OB leads to significant alterations in gene expression compared to standard monolayer culture (upregulation of Tie-2 in EC; upregulation of angiopoietin-2 in osteoblasts). More importantly, spheroidal coculture of endothelial cells and osteoblasts leads to significant changes of gene expression in both cell populations (upregulation of VEGFR-2 in EC; downregulation of VEGF, and upregulation of alkaline phosphatase in osteoblasts). These changes are dependent on cell–cell contact and are not seen in stimulation experiments with conditioned supernatants. Collectively, the data demonstrate complex bi-directional gene regulation mechanisms between EC and OB that are likely to play a critical role during OB differentiation and in controlling the properties of angiogenic EC.

Regulation of high-affinity sulphate transporters in plants: towards systematic analysis of sulphur signalling and regulation.

Plants require the function of plasma membrane-bound sulphate transporters for the initial uptake of inorganic sulphate. Part of this fundamental process is the energy-dependent proton/sulphate co-transport systems that are located in the surface cell layers of roots. During sulphur limitation, plants are able to activate the expression of sulphate transporters that facilitate the uptake of sulphate in roots. SULTR1;1 and SULTR1;2 are suggested to be the essential components of the sulphate uptake system in Arabidopsis roots. The physiological importance of SULTR1;1 and SULTR1;2 is supported by characteristics that can cope with sulphur deficiency: they were (i) functional high-affinity sulphate transporters; (ii) induced by sulphur limitation at the mRNA levels; and (iii) predominantly localized in the root hairs, epidermis, and cortex. The expression of high-affinity sulphate transporters was primarily regulated by sulphur in a promoter-dependent manner. Aside from the sulphur-specific regulation, the induction of SULTR1;1 and SULTR1;2 high-affinity sulphate transporters by sulphur limitation was dependent on the supply of carbon and nitrogen. In this review, the application of SULTR promoter-GFP systems for the analysis of regulatory pathways of sulphate acquisition in plants is described.

Adaptive cytoprotection through modulation of nitric oxide in ethanol-evoked gastritis

To assess the mechanisms of protective action by different mild irritants through maintenance of gastric mucosal integrity and modulation of mucosal nitric oxide (NO) in experimental gastritis rats. Either 200 mL/L ethanol, 50 g/L NaCl or 0.3 mol/L HCl was pretreated to normal or 800 mL/L ethanol-induced acute gastritis Sprague-Dawley rats before a subsequent challenge with 500 mL/L ethanol. Both macroscopic lesion areas and histological damage scores were determined in the gastric mucosa of each group of animals. Besides, gastric mucosal activities of NO synthase isoforms and of superoxide dismutase, along with mucosal level of leukotriene (LT)C4 were measured. Macroscopic mucosal damages were protected by 200 mL/L ethanol and 50 g/L NaCl in gastritis rats. However, although 200 mL/L ethanol could protect the surface layers of mucosal cells in normal animals (protection attenuated by NG-nitro-L-arginine methyl ester), no cytoprotection against deeper histological damages was found in gastritis rats. Besides, inducible NO synthase activity was increased in the mucosa of gastritis animals and unaltered by mild irritants. Nevertheless, the elevation in mucosal LTC4 level following 500 mL/L ethanol administration and under gastritis condition was significantly reduced by pretreatment of all three mild irritants in both normal and gastritis animals. These findings suggest that the aggravated 500 mL/L ethanol-evoked mucosal damages under gastritis condition could be due to increased inducible NO and LTC4 production in the gastric mucosa. Only 200 mL/L ethanol is truly "cytoprotective" at the surface glandular level of non-gastritis mucosa. Furthermore, the macroscopic protection of the three mild irritants involves reduction of LTC4 level in both normal and gastritis mucosa, implicating preservation of the vasculature.

Examination of Optical Depth Effects on Fluorescence Imaging of Cardiac Propagation

Optical mapping with voltage-sensitive dyes provides a high-resolution technique to observe cardiac electrodynamic behavior. Although most studies assume that the fluorescent signal is emitted from the surface layer of cells, the effects of signal attenuation with depth on signal interpretation are still unclear. This simulation study examines the effects of a depth-weighted signal on epicardial activation patterns and filament localization. We simulated filament behavior using a detailed cardiac model, and compared the signal obtained from the top (epicardial) layer of the spatial domain with the calculated weighted signal. General observations included a prolongation of the action upstroke duration, early upstroke initiation, and reduction in signal amplitude in the weighted signal. A shallow filament was found to produce a dual-humped action potential morphology consistent with previously reported observations. Simulated scroll wave breakup exhibited effects such as the false appearance of graded potentials, apparent supramaximal conduction velocities, and a spatially blurred signal with the local amplitude dependent upon the immediate subepicardial activity; the combination of these effects produced a corresponding change in the accuracy of filament localization. Our results indicate that the depth-dependent optical signal has significant consequences on the interpretation of epicardial activation dynamics.

Effect of flow perfusion on the osteogenic differentiation of bone marrow stromal cells cultured on starch-based three-dimensional scaffolds.

This study aims to investigate the effect of culturing conditions (static and flow perfusion) on the proliferation and osteogenic differentiation of rat bone marrow stromal cells seeded on two novel scaffolds exhibiting distinct porous structures. Specifically, scaffolds based on SEVA-C (a blend of starch with ethylene vinyl alcohol) and SPCL (a blend of starch with polycaprolactone) were examined in static and flow perfusion culture. SEVA-C scaffolds were formed using an extrusion process, whereas SPCL scaffolds were obtained by a fiber bonding process. For this purpose, these scaffolds were seeded with marrow stromal cells harvested from femoras and tibias of Wistar rats and cultured in a flow perfusion bioreactor and in 6-well plates for 3, 7, and 15 days. The proliferation and alkaline phosphatase activity patterns were similar for both types of scaffolds and for both culture conditions. However, calcium content analysis revealed a significant enhancement of calcium deposition on both scaffold types cultured under flow perfusion. This observation was confirmed by Von Kossa-stained sections and tetracycline fluorescence. Histological analysis and confocal images of the cultured scaffolds showed a much better distribution of cells within the SPCL scaffolds than the SEVA-C scaffolds, which had limited pore interconnectivity, under flow perfusion conditions. In the scaffolds cultured under static conditions, only a surface layer of cells was observed. These results suggest that flow perfusion culture enhances the osteogenic differentiation of marrow stromal cells and improves their distribution in three-dimensional, starch-based scaffolds. They also indicate that scaffold architecture and especially pore interconnectivity affect the homogeneity of the formed tissue.

ULTRASTRUCTURE AND EARLY EMBRYONIC SHELL FORMATION IN THE TERRESTRIAL PULMONATE SNAIL, EUHADRA HICKONIS

The distribution of calcium ion (Ca-ion) was demonstrated in the mantle epithelial cells by means of two-step chemical precipitation of Ca-ion in the pulmonate snail, Euhadra hickonis. K-oxalate/ K-antimonate chemical replacement was done using a simultaneous computerized microwavestimulated fixation method. These precipitates in the mantle epithelial cells were studied by electronprobe X-ray microanalysis (EDX) and electron energy-filtered imaging (EFI). The calculated values of the essential-elemental X-ray pulse ratios, which were obtained by computerized EDX analysis, agreed with those of the standard samples and theoretical values of Ca-antimonate. Typical EFI images and spectra energy of Ca and Sb were obtained from the surface mantle epithelial cells in the same tissue block as that used for EDX analysis where the elemental distribution of Ca and Sb was the same. The Ca-antimonate precipitates were formed in the intercellular space, vesicles and microvilli of mantle cells. In day 13 embryos, the columella and peripheral area were almost calcified, but in the dorsal area, Ca was still in the ionic state; the binding ratio of the former case was Ca:Sb = 1:0, that of the latter was Ca:Sb = 1:2, and that of the small intermediate area was Ca:Sb = 1:1.4. After day 17, the embryonal periostracum was almost calcified. Between days 15 and 20, strong Ca 2+ -ATPase and carbonic anhydrase (CAHase) enzymatic activities were observed at the sites where Ca-ion was observed; intercellular spaces, vacuoles in the mantle cells, periostracum and homogeneous layer between the periostracum and mantle cell, and in the mitochondria. Ca 2+ -ATPase may play important roles in the transportation of Ca-ion from the basal cell layer to the surface layer of the mantle cells and CAHase may play important roles in the chemical binding of Ca-ion and carbonate ion to produce calcium carbonate crystals in the newly developing embryonal shell.

Variations in skin dose using 6MV or 18MV x-ray beams

This research aimed to quantitatively evaluate the differences in percentage dose of maximum for 6MV and 18MV x-ray beams within the first 1 cm of interactions. Thus provide quantitative information regarding the basal, dermal and subcutaneous dose differences achievable with these two types of high-energy x-ray beams. Percentage dose of maximum build up curves are measured for most clinical field sizes using 6MV and 18MV x-ray beams. Calculations are performed to produce quantitative results highlighting the percentage dose of maximum differences delivered to various depths within the skin and subcutaneous tissue region by these two beams. Results have shown that basal cell layer doses are not significantly different for 6MV and 18MV x-ray beams. At depths beyond the surface and basal cell layer there is a measurable and significant difference in delivered dose. This variation increases to 20% of maximum and 22% of maximum at 1 mm and 1 cm depths respectively. The percentage variations are larger for smaller field sizes where the photon in phantom component of the delivered dose is the most significant contributor to dose. By producing graphs or tables of % dose differences in the build up region we can provide quantitative information to the oncologist for consideration (if skin and subcutaneous tissue doses are of importance) during the beam energy selection process for treatment.

Macroscopic cartilage formation with embryonic stem-cell-derived mesodermal progenitor cells.

The totipotent embryonic stem cell generates various mesodermal cells when stimulated with BMP4. Among the resulting cells, those expressing flk-1 and/or PDGFRalpha displayed chondrogenic activity in the presence of TGFbeta3 and expressed cartilage-specific genes in 7 to 16 day pellet cultures. Depositions of cartilage matrix and type II collagen were detected by day 14. TGFbeta-stimulated chondrogenesis was synergistically enhanced by PDGF-BB, resulting in a larger cartilage particle filled with a cartilaginous area containing type II collagen, with a surface cell layer expressing type I collagen. In contrast, noggin inhibited both the TGFbeta- and TGFbeta+PDGF-stimulated cartilage formation, suggesting that a BMP-dependent pathway is involved. In fact, replacement of TGFbeta3 with BMP4 on days 10 to 12 markedly elevated the cartilage matrix deposition during the following 7 to 8 days. Moreover, culture with TGFbeta3 and PDGF-BB, followed by the incubation with BMP4 alone, resulted in a cartilage particle lacking type I collagen in the matrix and the surface layer, which suggests hyaline cartilage formation. Furthermore, such hyaline cartilage particles were mineralized. These studies indicate that the PDGFRalpha+ and/or flk-1+ cells derived from embryonic stem cells possess the full developmental potential toward chondrocytes, in common with embryonic mesenchymal cells.

Expression of nitric oxide synthase-3 in porcine oocytes obtained at different follicular development.

The present study was designed to determine the localization of nitric oxide synthase-3 (NOS-3) in porcine follicles during follicular development. A 130-kDa NOS-3 protein was found with greater frequency much in the oocytes than in the cumulus cells, as revealed by Western blotting analysis. The content of NOS-3 in the oocyte was higher in large follicles (> 7-mm diameter) than in small follicles (< 2-mm). The data by Western blotting showed the same pattern as the observations obtained from the immunohistochemical studies, in which the periphery of the oocyte stained strong positive. The inner surface cell layer of granulosa cells and cumulus cells were positive staining, especially in large antral follicles. In the primordial follicles, NOS-3 was restricted to the cytoplasm of oocytes, and no stained product was observed in the nucleus of oocytes or granulosa cells. A significant synthesis of NO by oocytes was observed in the presence of ionomycin, but not in the absence of ionomycin, indicating that oocyte NOS-3 functions in response to transient elevations in the intracellular calcium level. We concluded that NOS-3 is expressed in the oocyte from the primordial follicular stage to antral follicular stage, and that it is functional at least in the antral follicles.