Growth regulator requirement for in vitro embryogenic cultures of snowdrop (Galanthus nivalis L.) suitable for germplasm preservation.

In this study, we report on the production of bulb scale-derived tissue cultures capable of efficient shoot and plant regeneration in three genotypes of snowdrop (Galanthus nivalis L., Amaryllidaceae), a protected ornamental plant. For culture line A, high auxin and low cytokinin concentration is required for callus production and plant regeneration. The type of auxin is of key importance: α-naphthaleneacetic acid (NAA) in combination with indole-3-acetic acid (IAA) at concentrations of 2 mg L-1 or 2-10 mg L-1 NAA with 1 mg L-1 N6-benzyladenine (BA), a cytokinin on full-strength media are required for regeneration. Cultures showing regeneration were embryogenic. When lines B and C were induced and maintained with 2 mg L-1 NAA and 1 mg L-1 BA, they produced mature bulblets with shoots, without roots. Line A produced immature bulblets with shoots under the above culture condition. Amplified Fragment Length Polymorphism (AFLP) analysis showed that (i) genetic differences between line A and its bulb explants were not significant, therefore these tissue cultures are suitable for germplasm preservation, and (ii) different morphogenetic responses of lines A, B and C originated from genetic differences. Culture line A is suitable for field-growing, cultivation and germplasm preservation of G. nivalis and for the production of Amaryllidaceae alkaloids.

Variability of microcystins and its synthetase gene cluster in Microcystis and Planktothrix waterblooms in shallow lakes of Hungary.

Waterbloom samples of Microcystis aeruginosa and Planktothrix agardhii were collected from a variety of ponds, lakes and reservoirs in Hungary. Samples were tested with matrix-assisted laser desorption/ionization - time-of-flight mass spectrometry (MALDI-TOF MS) to identify the microcystin forms. The concentration of the microcystins was measured with capillary electrophoresis and the toxicity was tested by sinapis test. DNA was extracted from the samples and tested using a range of primers linked to the biosynthesis of microcystin. All of the fourteen collected samples gave positive results for the presence of the mcy genes with PCR products with sizes between of 425 and 955 bp, respectively, indicating the presence of the genes implicated in the production of microcystins. The results showed that a wide range of microcystin (MC) forms were detected in the Microcystis containing samples, among which MC-LR, -RR, and -YR were the most common. The highest MC concentration was 15,701 mg g-1, which was detected in an angling pond. The samples containing Planktothrix agardhii were less toxic, and the most common form in this species was the Asp3-MC-LR.

Histological, cytological and biochemical alterations induced by microcystin-LR and cylindrospermopsin in white mustard (Sinapis alba L.) seedlings.

This study compares the histological, cytological and biochemical effects of the cyanobacterial toxins microcystin-LR (MCY-LR) and cylindrospermopsin (CYN) in white mustard (Sinapis alba L.) seedlings, with special regard to the developing root system. Cyanotoxins induced different alterations, indicating their different specific biochemical activities. MCY-LR stimulated mitosis of root tip meristematic cells at lower concentrations (1 µg ml-1) and inhibited it at higher concentrations, while CYN had only inhibitory effects. Low CYN concentrations (0.01 µg ml-1) stimulated lateral root formation, whereas low MCY-LR concentrations increased only the number of lateral root primordia. Both inhibited lateral root development at higher concentrations. They induced lignifications, abnormal cell swelling and inhibited xylem differentiation in roots and shoots. MCY-LR and CYN induced the disruption of metaphase and anaphase spindles, causing altered cell divisions. Similar alterations could be related to decreased protein phosphatase (PP1 and PP2A) activities in shoots and roots. However, in vitro phosphatase assay with purified PP1 catalytic subunit proved that CYN in contrast to MCY-LR, decreased phosphatase activities of mustard in a non-specific way. This study intends to contribute to the understanding of the mechanisms of toxic effects of a protein phosphatase (MCY-LR) and a protein synthesis (CYN) inhibitory cyanotoxin in vascular plants.

Femoral artery complications associated with the Mynx closure device.

BACKGROUND AND PURPOSE: Devices to close a femoral arteriotomy are frequently used after catheterization for interventional radiology and cardiac procedures to decrease the time to hemostasis and ambulation and, potentially, to decrease local complications. The Mynx vascular closure device uses a sealant designed to occlude the access tract, resulting in hemostasis. MATERIALS AND METHODS: We retrospectively reviewed all cases in which the Mynx device was used and for which follow-up angiography was available. A total of 146 devices were deployed in 135 patients. A follow-up vascular study visualizing the femoral artery was performed in 26 patients (27 studies). RESULTS: There were 5 (5/27, 18%) cases of intravascular Mynx sealant on follow-up vascular imaging. Three pseudoaneurysms (3/27, 11%) were identified. CONCLUSIONS: In this small study, intravascular sealant and pseudoaneurysms were found frequently after femoral arterial closure with the Mynx vascular closure device.

Screening of common Plantago species in Hungary for bioactive molecules and antioxidant activity.

Five species of Plantago genus, namely P. lanceolata, P. major, P. media, P. altissima and P. maritima were screened for iridoid content (CE-MEKC), total caffeoyl phenylethanoid glycoside (CPG) content and antioxidant activity (CUPRAC assay). The five species could be distinguished by TLC pattern analysis in a single run in a system commonly used for quality management of P. lanceolata leaves, as shown by cluster analysis of major bands; with the exception, that P. altissima and P. lanceolata did not show enough pattern difference to be fully separated. P. maritima was shown to have the highest antioxidant capacity (0.42 µmol ascorbic acid equivalent (AAE)/g DW), and the highest level of CPGs (4.29%). P. altissima was shown to be chemically indistinguishable from P. lanceolata with repsect to iridoid content (aucubin 0.55 ± 0.04%, 0.68 ± 0.23%, catalpol 0.66 ± 0.13% and 0.89 ± 0.22%, respectively), CPG content (2.40 ± 0.38% and 2.54 ± 0.56%, respectively) and antioxidant capacity (0.2206 ± 0.0290 and 0.2428 ± 0.0191 µmol AAEAC/g DW). The presented data show the potency of medicinal use of Hungarian wild populations of the studied five species, especially in the case of P. maritima, and that P. altissima can be a potential replacement of P. lanceolata in herbal mixtures.

Cylindrospermopsin inhibits growth and modulates protease activity in the aquatic plants Lemna minor L. and Wolffia arrhiza (L.) Horkel.

The toxic effects of cylindrospermopsin (cyanobacterial toxin) on animals have been examined extensively, but little research has focused on their effects on plants. In this study cylindrospermopsin (CYN) caused alterations of growth, soluble protein content and protease enzyme activity were studied on two aquatic plants Lemna minor and Wolffia arrhiza in short-term (5 days) experiments. For the treatments we used CYN containing crude extracts of Aphanizomenon ovalisporum (BGSD-423) and purified CYN as well. The maximal inhibitory effects on fresh weight of L. minor and W. arrhiza caused by crude extract were 60% and 54%, respectively, while the maximum inhibitory effects were 30% and 43% in the case of purified CYN at 20 µg ml(-1) CYN content of culture medium. In CYN-treated plants the concentration of soluble protein showed mild increases, especially in W. arrhiza. Protease isoenzyme activity gels showed significant alterations of enzyme activities under the influence of CYN. Several isoenzymes were far more active and new ones appeared in CYN-treated plants. Treatments with cyanobacterial crude extract caused stronger effects than the purified cyanobacterial toxins used in equivalent CYN concentrations.

Chromosome aberrations induced by dual exposure of protons and iron ions.

During space travel, astronauts will be exposed to protons and heavy charged particles. Since the proton flux is high compared to HZE particles, on average, it is assumed that a cell will be hit by a proton before it is hit by an HZE ion. Although the effects of individual ion species on human cells have been investigated extensively, little is known about the effects of exposure to mixed beam irradiation. To address this, we exposed human epithelial cells to protons followed by HZE particles and analyzed chromosomal damage using the multicolor banding in situ hybridization (mBAND) procedure. With this technique, individually painted chromosomal bands on one chromosome allowed the identification of intra-chromosomal aberrations (inversions and deletions within a single painted chromosome) as well as inter-chromosomal aberrations (translocation to unpainted chromosomes). Our results indicated that chromosome aberration frequencies from exposures to protons followed by Fe ions did not simply decrease as the interval between the two exposures increased, but peak when the interval was 30 min.

Comparison of genotoxic damage in monolayer cell cultures and three-dimensional tissue-like cell assemblies.

Assessing the biological risks associated with exposure to the high-energy charged particles encountered in space is essential for the success of long-term space exploration. Although prokaryotic and eukaryotic cell models developed in our laboratory and others have advanced our understanding of many aspects of genotoxicity, in vitro models are needed to assess the risk to humans from space radiation insults. Such models must be representative of the cellular interactions present in tissues and capable of quantifying genotoxic damage. Toward this overall goal, the objectives of this study were to examine the effect of the localized microenvironment of cells, cultured as either 2-dimensional (2D) monolayers or 3-dimensional (3D) aggregates, on the rate and type of genotoxic damage resulting from exposure to Fe-charged particles, a significant portion of space radiation. We used rodent transgenic cell lines containing 50-70 copies of a LacI transgene to provide the enhanced sensitivity required to quantify mutational frequency and type in the 1100-bp LacI target as well as assessment of DNA damage to the entire 45-kbp construct. Cultured cells were exposed to high energy Fe charged particles at Brookhaven National Laboratory's Alternating Gradient Synchrotron facility for a total dose ranging from 0.1 to 2 Gy and allowed to recover for 0-7 days, after which mutational type and frequency were evaluated. The mutational frequency was found to be higher in 3D samples than in 2D samples at all radiation doses. Mutational frequency also was higher at 7 days after irradiation than immediately after exposure. DNA sequencing of the mutant targets revealed that deletional mutations contributed an increasingly high percentage (up to 27%) of all mutations in cells as the dose was increased from 0.5 to 2 Gy. Several mutants also showed large and complex deletions in multiple locations within the LacI target. However, no differences in mutational type were found between the 2D and the 3D samples. These 3D tissue-like model systems can reduce the uncertainty involved in extrapolating risk between in vitro cellular and in vivo models.

Simultaneous measurement of multiple radiation-induced protein expression profiles using the Luminex(TM) system.

Space flight results in the exposure of astronauts to a mixed field of radiation composed of energetic particles of varying energies, and biological indicators of space radiation exposure provides a better understanding of the associated long-term health risks. Current methods of biodosimetry have employed the use of cytogenetic analysis for biodosimetry, and more recently the advent of technological progression has led to advanced research in the use of genomic and proteomic expression profiling to simultaneously assess biomarkers of radiation exposure. We describe here the technical advantages of the Luminex(TM) 100 system relative to traditional methods and its potential as a tool to simultaneously profile multiple proteins induced by ionizing radiation. The development of such a bioassay would provide more relevant post-translational dynamics of stress response and will impart important implications in the advancement of space and other radiation contact monitoring.

Three-dimensional transgenic cell model to quantify genotoxic effects of space environment.

In this paper we describe a three-dimensional, multicellular tissue-equivalent model, produced in NASA-designed, rotating wall bioreactors using mammalian cells engineered for genomic containment of multiple copies of defined target genes for genotoxic assessment. Rat 2 lambda fibroblasts, genetically engineered to contain high-density target genes for mutagenesis (Stratagene, Inc., Austin, TX), were cocultured with human epithelial cells on Cytodex beads in the High Aspect Ratio Bioreactor (Synthecon, Inc, Houston, TX). Multi-bead aggregates were formed by day 5 following the complete covering of the beads by fibroblasts. Cellular retraction occurred 8-14 days after coculture initiation culminating in spheroids retaining few or no beads. Analysis of the resulting tissue assemblies revealed: multicellular spheroids, fibroblasts synthesized collagen, and cell viability was retained for the 30-day test period after removal from the bioreactor. Quantification of mutation at the LacI gene in Rat 2 lambda fibroblasts in spheroids exposed to 0-2 Gy neon using the Big Blue color assay (Stratagene, Inc.), revealed a linear dose-response for mutation induction. Limited sequencing analysis of mutant clones from 0.25 or 1 Gy exposures revealed a higher frequency of deletions and multiple base sequencing changes with increasing dose. These results suggest that the three-dimensional, multicellular tissue assembly model produced in NASA bioreactors are applicable to a wide variety of studies involving the quantification and identification of genotoxicity including measurement of the inherent damage incurred in Space.

Cardiac organogenesis in vitro: reestablishment of three-dimensional tissue architecture by dissociated neonatal rat ventricular cells.

The mammalian heart does not regenerate in vivo. The heart is, therefore, an excellent candidate for tissue engineering approaches and for the use of biosynthetic devices in the replacement or augmentation of defective tissue. Unfortunately, little is known about the capacity of isolated heart cells to re-establish tissue architectures in vitro. In this study, we examined the possibility that cardiac cells possess a latent organizational potential that is unrealized within the mechanically active tissue but that can be accessed in quiescent environments in culture. In the series of experiments presented here, total cell populations were isolated from neonatal rat ventricles and recombined in rotating bioreactors containing a serum-free medium and surfaces for cell attachment. The extent to which tissue-like structure and contractile function were established was assessed using a combination of morphological, physiological, and biochemical techniques. We found that mixed populations of ventricular cells formed extensive three-dimensional aggregates that were spontaneously and rhythmically contractile and that large aggregates of structurally-organized cells contracted in unison. The cells were differentially distributed in these aggregates and formed architectures that were indistinguishable from those of intact tissue. These architectures arose in the absence of three-dimensional cues from the matrix, and the formation of organotypic structures was apparently driven by the cells themselves. Our observations suggest that cardiac cells possess an innate capacity to re-establish complex, three-dimensional, cardiac organization in vitro. Understanding the basis of this capacity, and harnessing the organizational potential of heart cells, will be critical in the development of tissue homologues for use in basic research and in the engineering of biosynthetic implants for the treatment of cardiac disease.

Neonatal rat heart cells cultured in simulated microgravity.

In vitro characteristics of cardiac cells cultured in simulated microgravity are reported. Tissue culture methods performed at unit gravity constrain cells to propagate, differentiate, and interact in a two-dimensional (2D) plane. Neonatal rat cardiac cells in 2D culture organize predominantly as bundles of cardiomyocytes with the intervening areas filled by nonmyocyte cell types. Such cardiac cell cultures respond predictably to the addition of exogenous compounds, and in many ways they represent an excellent in vitro model system. The gravity-induced 2D organization of the cells, however, does not accurately reflect the distribution of cells in the intact tissue. We have begun characterizations of a three-dimensional (3D) culturing system designed to mimic microgravity. The NASA-designed High-Aspect Ratio Vessel (HARV) bioreactors provide a low shear environment that allows cells to be cultured in static suspension. HARV-3D cultures were prepared on microcarrier beads and compared to control-2D cultures using a combination of microscopic and biochemical techniques. Both systems were uniformly inoculated and medium exchanged at standard intervals. Cells in control cultures adhered to the polystyrene surface of the tissue culture dishes and exhibited typical 2D organization. Cells cultured in HARVs adhered to microcarrier beads, the beads aggregated into defined clusters containing 8 to 15 beads per cluster, and the clusters exhibited distinct 3D layers: myocytes and fibroblasts appeared attached to the surfaces of beads and were overlaid by an outer cell type. In addition, cultures prepared in HARVs using alternative support matrices also displayed morphological formations not seen in control cultures. Generally, the cells prepared in HARV and control cultures were similar; however, the dramatic alterations in 3D organization recommend the HARV as an ideal vessel for the generation of tissuelike organization of cardiac cells in vitro.

Skeletal muscle satellite cells cultured in simulated microgravity.

Satellite cells are postnatal myoblasts responsible for providing additional nuclei to growing or regenerating muscle cells. Satellite cells retain the capacity to proliferate and differentiate in vitro and, therefore, provide a useful model to study postnatal muscle development. Most culture systems used to study postnatal muscle development are limited by the two-dimensional (2-D) confines of the culture dish. Limiting proliferation and differentiation of satellite cells in 2-D could potentially limit cell-cell contacts important for developing the level of organization in skeletal muscle obtained in vivo. Culturing satellite cells on microcarrier beads suspended in the High-Aspect-Ratio-Vessel (HARV) designed by NASA provides a low shear, three-dimensional (3-D) environment to study muscle development. Primary cultures established from anterior tibialis muscles of growing rats (approximately 200 gm) were used for all studies and were composed of greater than 75% satellite cells. Different inoculation densities did not affect the proliferative potential of satellite cells in the HARV. Plating efficiency, proliferation, and glucose utilization were compared between 2-D culture and 3-D HARV culture. Plating efficiency (cells attached divided by cells plated x 100) was similar between the two culture systems. Proliferation was reduced in HARV cultures and this reduction was apparent for both satellite cells and nonsatellite cells. Furthermore, reduction in proliferation within the HARV could not be attributed to reduced substrate availability because glucose levels in medium from HARV and 2-D cell culture were similar. Morphologically, microcarrier beads within the HARV were joined together by cells into 3-D aggregates composed of greater than 10 beads/aggregate. Aggregation of beads did not occur in the absence of cells. Myotubes were often seen on individual beads or spanning the surface of two beads. In summary, proliferation and differentiation of satellite cells on microcarrier beads within the HARV bioreactor results in a 3-D level of organization that could provide a more suitable model to study postnatal muscle development than is currently available with standard culture methods.

Electron microscopy of platelet interactions with heme-octapeptide-labeled fibrinogen.

Binding of fibrinogen to ADP-activated platelets was visualized by labeling the molecule with heme-octapeptide (microperoxidase) for direct cytochemical staining. Transmission electron microscopy of the platelet aggregates showed most of the fibrinogen distributed widely over the platelet surface in nonbridging rims of 7- to 9-nm thickness. Short peroxidase-positive bridges (less than 25 nm) were found in clusters in regions of close contact between the platelets, but 50-nm bridging corresponding to the length of the molecule was not seen by this method. Thus the fibrinogen appeared to be binding in a predominantly prone rather then upright orientation on the platelets. Abundant 50-nm bridging seen by nonspecific staining appeared unrelated to the length of the fibrinogen molecule because the bridging did not change when the length of the fibrinogen was more than doubled by end-to-end cross-linking with factor XIIIa. It is suggested that the observed binding and bridging of fibrinogen in a prone orientation is promoted by the existence of multiple platelet-binding domains on the molecule.

The determination of pharmacokinetic parameters of ketotifen in steady state in young children.

Ketotifen kinetics at steady state in small children compared to adults show very good absorption for a dose of 2 X daily 1 mg. Relative to adult kinetics, a higher dose in relation to body weight is justified, since a more rapid kinetic pattern was proven for the substance in the volunteer collective tested.

Thromboembolic complications in children with nephrotic syndrome. Risk and incidence.

Coagulation studies were performed in 16 children with steroid responsive minimal change nephrotic syndrome in order to elucidate the incidence of thromboembolic complications. Fibrinogen and alpha 2-macroglobulin concentrations were inversely correlated with serum albumin concentrations, antithrombin III correlated positively (p less than 0.001). Factor VIII:R:AG concentration was elevated. Coagulation disturbances in children are not less severe than in adults with nephrotic syndrome. Combined scintigraphic pulmonary ventilation and perfusion studies were employed in 26 children to detect noninvasively events of pulmonary embolism, respectively their residual changes. The lung scintigraphic investigation demonstrated a pattern consistent with pulmonary embolism in 7 patients (27.9%), residual changes in 10 (38.5%) and normal findings in 9 (34.9%). The incidence of thromboembolic complications in children with severe nephrotic syndrome is as high as reported for adults. Pulmonary symptoms may well be due to pulmonary embolism.

Largomycin FII chromophore component 4, a new pluramycin antibiotic.

The chromophore of the antitumor chromoprotein largomycin FII is a mixture of components belonging to the pluramycin class of antitumor antibiotics. Against most organisms tested, component 4 exhibited activity equal to or greater than the major chromophore components pluramycin A and deacetylpluramycin A. Data obtained from UV, IR, 1H and 13C NMR, and from fast atom bombardment mass spectrometry were used to determine the structure of component 4 as epoxykidamycin, a new member of the pluramycin class.

Structure and properties of major largomycin FII chromophore components.

Largomycin FII, a protein antitumor antibiotic of molecular weight 29,300 daltons, contains a chromophore that is separable under mild denaturing conditions. The chromophore complex was found to be considerably less stable than the holoprotein towards light and heat, suggesting a protective effect of the protein on the chromophore. Separation of the chromophore into several components was achieved using high performance liquid chromatography, and the biological activity of the isolated components was determined. Data gathered from UV, IR, proton and carbon NMR, and fast atom bombardment mass spectrometry indicated that all the chromophore components belong to the pluramycin class of antitumor agents. Pluramycin A and deacetylpluramycin A were found to be the two major components.

Activation of antitumor agent gilvocarcins by visible light.

Gilvocarcins that are antitumor agents are activated by low doses of visible light to induce bacteriophage lambda in Escherichia coli. This result is dependent on interaction with DNA. Gilvocarcin M, an analog without antitumor activity, failed to induce the prophage after light exposure, thus demonstrating a correlation between photosensitizing and antitumor activities. These results raise several possibilities regarding the mode of action of gilvocarcins as antitumor agents in vivo, involving light or enzymatic activating systems, which could be exploited in human cancer therapy.

Genetic and biochemical factors affecting the induction of bacteriophage lambda by N-nitroso compounds.

As part of our effort to validate a biochemical (prophage) induction assay (BIA) as a screening test for carcinogens, we have tested more than 100 N-nitroso compounds. An enzyme, beta-galactosidase, is induced as an indirect consequence of DNA damage to the host, as part of the 'SOS' response. Besides the obvious practical importance of detecting this class of carcinogen, there is the question of the mechanism by which these compounds work. Mutagenesis by one compound, N-methyl-N'-nitro-N-nitrosoguanidine, is known to proceed by both SOS (recA)-dependent and SOS-independent pathways. Mispairing due to O6 alkylation of guanine is thought to be responsible for the SOS-independent pathway; however, there has been little consideration of recA-dependent functions, of which phage induction is one. Although nitrosamides could be detected as phage inducers in our assay, N-nitrosamines in the presence of rat liver 9 000 X g supernatant usually gave no response. We found that the use of several mutant strains, particularly a lexA mutant, in combination with hamster liver 9 000 X g supernatant, allowed us to detect most N-nitrosamines reasonably well, in either a spot test or a quantitative tube assay. Induction in a lexA strain was most unexpected, since this mutation usually diminishes the expression of SOS functions induced by ultra-violet light. Because the genetic and biochemical conditions that favour phage induction are different from those that favour mutagenesis, it seems likely that the lesions in DNA leading to the two biological end-points are different.