Enhanced plumbagin accumulation in embryogenic cell suspension cultures of Plumbago rosea L. following elicitation

This study focused on enhancing the production of plumbagin, an anticancer compound, in embryogenic cell suspension cultures of Plumbago rosea. Elicitation techniques have been reported to enhance plumbagin production. Cell suspension cultures raised from embryogenic calli induced from in vitro leaf explants were exposed to different concentrations of jasmonic acid, yeast extract and different auxin combinations. Influence of these on cell growth, biomass and plumbagin production was studied. To our knowledge this is the first report on elicitation of embryogenic cell suspension cultures of P. rosea for enhanced plumbagin production. Elicitor treated suspension cultures exhibited decreased culture viability and increased plumbagin synthesis. A maximum of 5.59-fold enhancement of plumbagin production was observed in cultures added with 1 mg L^?1 naphthalene acetic acid after 6 days of incubation. Viability of cultures decreased with increased concentration of elicitors and prolonged incubation period. Application of elicitors in cell suspension cultures induces defense related responses which lead to increased secondary metabolite production for making the cells adapt to the situation. If the stressed condition persists or is in intolerable level this will eventually lead to programmed cell death and loss of culture viability.     

Surface characterization of two amoebae relative to cell adhesion

This is a comparative study of the surfaces of two Acanthamoeba castellanii strains, one of which clumps during exponential growth. Proteins from the isolated surface membranes of the strains were compared by disc electrophoresis. The number and similarity of the proteins between the two strains depended on the method of solubilization and electrophoresis. Periodic acid Schiff staining indicated that most of the proteins were glycoproteins. The molecular weights of the proteins ranged between 50 000–70 000 with those of the clumping strain being slightly larger. The non-clumping strain's surface membrane exhibited glucosyltransferase activity while the clumping strain did not. Galactosyltransferase activity was present in surface membranes of both strains but the specific activity of the clumping strain was greater. The non-clumping strain agglutinated in the presence of concanavalin A, soybean agglutinin and wheat germ agglutinin while the clumping strain was affected by soybean agglutinin only. Whole cell electrophoresis and enzymic treatments further indicated differences in the surfaces of the two strains. The data is discussed with reference to differences between the two amoebae strains and their possible role in cell-cell adhesion.     

Cytophysiological characteristics of Arabidopsis thaliana cultivated cells with disable perception of ethylene signal by the ETR1 receptor

Contradictory data about ethylene influence on cell growth and division prompted us to investigate cytophysiological characteristics of suspension cultures of Arabidopsis thaliana of wild type Col-0 and ert1-1 mutant carrying a point mutation in the site of ethylene binding by the ETR1 receptor. Some cytophysiological characteristics of the etr1-1 cultivated cells differed from those of Col-0: the growth rate of mutant cells was less and cell sizes were smaller, the culture was committed to the formation of tracheary elements (TE), had a pronounced modal class of nuclei (54%) with the amount of DNA 8C and a tendency to expand the ploidy toward 32C. Despite the absence of ethylene perception by the ETR1 receptor, the cell culture of mutant responded to treatment with ethylene by growth acceleration, an increase in cell viability and in the number of cells in the S-phase of the cell cycle. The inhibitor of ethylene binding to receptors, 1-methylcyclopropene, suppressed growth and viability of the cells of both genotypes. In the etr1-1 cell culture, the inhibitor reduced the number of S-phase nuclei and activated TE formation. All data obtained indicate that ethylene perception and transduction of ethylene signal are required for the maintenance of cell viability and active in vitro growth. It is supposed that the functional activity of the ETR1 receptor is necessary for optimal cell expansion, whereas other receptors are responsible for cell proliferation.     

Properties of stem cells isolated from subcutaneous and subepicardial adipose tissues

Stem cells (SCs) vary in morphological, immunophenotypic, proliferative, and differentiation characteristics depending on their tissue source. Comparative analysis of their biological properties is essential for making an optimal SC choice for regenerative therapy. Using immunocytochemistry, flow cytometry, histochemistry, and RT-PCR, we have investigated SCs obtained from human subepicardial (SEC-AT) and subcutaneous (SC-AT) adipose tissues and cultured under similar conditions without any differentiation-promoting factors. The cultures were similar in having a high proportion of proliferating cells positive for nuclear antigen (PCNA). In both cultures, immunophenotyping has revealed high expression of mesenchymal stem-cell surface markers CD29, CD44, CD73, and CD105; low expression of CD31, CD34, and CD45; and variability in CD117, CD146, and CD309 expression. The only difference in the CD marker profile was the significantly lower expression of CD90 in the culture of SCs from SC-AT than from SEC-AT. Histochemical analysis showed a lack of Oil Red O-positive cells in both cultures and an about ten times higher number of alkaline phosphatase-positive cells among SCs from SC-AT. In both cultures, immunocytochemistry detected low expression of the slow myosin heavy chain marker MAB1628 and smooth muscle actin marker α-hSMA. Expression of the gap junction protein connexin-43 was markedly higher in cells from SC-AT cultures. Only the cells of these cultures expressed the epithelial cell marker cytokeratin-19. GATA4 mRNA expression detected with RT-PCR was identified in SEC-AT rather than in SC-AT cells. Our results suggest that SC-AT is enriched compared to SEC-AT with epithelial cell and osteogenic progenitors. In turn, SEC-AT possesses cardiomyogenic SCs and can be considered an alternative source for cell cardiotherapy.     

Influence of humus substances on the toxic effect of 2-methyl-4-chlorphenoxyacetic acid

Effect of humus fractions on the toxicity of 2-methyl-4-chlorphenoxyacetic acid (MCPA) was examined in water cultures. Humus fractions and different MCPA quantities were added to Richter's nutrient solution used for maize (Zea mays L., cv. Ko?ovská raná) cultivation. The results show that MCPA and humus substances in the applied concentrations intensively influence the maize root system, especially the rhizodermis and apical meristem cells. MCPA reduces ion absorption, induces phosphorus excretion from root, reduces tissue hydratation and relatively quickly kills these cells, at the end stage and/or in the highest concentration. Humus fractions have an unambiguously positive effect on these processes, they increase the cell vitality. When humus substances are applied, the MCPA effect does not appear in potassium and nitrogen absorption, the inhibitory effect is maintained in phosphorus absorption and in tissue hydratation even with the most effective fraction—with fulvo acids. But all fractions increased the cell vitality of sorption tissue and meristems in the presence of MCPA.     

Chromosomal instabilities in callus tissue from haploid barley (Hordeum vulgare L.)

Callus culture was derived from haploid barley embryos after crossing withHordeum bulbosum. The callus tissue is cytologically heterogeneous, containing haploid, diploid and polyploid cells. Aneuploidy and karyokinetic irregularities were also observed. Some problems of chromosomal instabilities in plant tissue cultures are discussed.     

Ubiquinone 10 formation in Rhodospirillum rubrum under different culture conditions

The formation of ubiquinone 10 and bacteriochlorophyll (bchl) was determined in Rhodospirillum rubrum grown under different culture conditions. Transfer of chemotrophically grown cultures to photosynthetic conditions leads to the formation of the pigments until cells reach the stationary phase of growth. Bchl-synthesis initially exceeds quinone synthesis. On a cellular protein basis quinone levels first decrease by about a factor of two and subsequently increase by a factor of four. Bchl levels per protein increase until cells reach the stationary phase of growth. Quinone levels per bchl decrease rather rapidly and become constant in the growing culture. When cells were transferred under continuous phototrophic conditions to new culture medium, both pigments are formed concomitantly. While protein synthesis starts immediately, bchl and ubiquinone formation is slightly delayed. This causes a short time decrease in the amount of both pigments per cellular protein followed by an increase to a constant level. Ratios of ubiquinone per bchl are constant. The transfer of phototrophically grown cultures to chemotrophic conditions results in a complete inhibition of bchl formation while quinone synthesis resumes. Quinone cellular levels decrease slightly and then remain constant. Quinone values increase per bchl which is eventually diluted out of the cells.     

A Laboratory Case Study of Efficient Polyhydoxyalkonates Production by Bacillus cereus,a Contaminant in Saccharophagus degradans ATCC 43961 in Minimal Sea Salt Media

A contaminating bacterium growing along with the stock culture of Saccharophagus degradans ATCC 43961 (Sde 2-40) on marine agar plate was isolated and investigated for its ability to produce polyhydoxyalkonates (PHA). Preliminary screening by Sudan black B and Nile blue A staining indicated positive characteristic of the isolate to produce PHA. The isolate was able to grow and produce PHA in minimal sea salt medium broth. PHA quantification studies with gas chromatographic analyses of the dry cells derived from culture broths revealed accumulation of PHA in bacterial cells. PHA production started after 20 h and increased with cell growth and attained maximum values of 61 % of dry cell weight at 70 h of cultivation. After 70 h, a slight decrease in the level of PHA content was observed. The nature/type of PHA was found to be poly(3-hydroxybutyraye) by Fourier transform-infrared spectroscopy. Microbiological and 16S rRNA gene sequencing analyses suggested that the PHA producing bacterial isolate belongs to Bacillus genera and shows 100 % nucleotide sequence similarity with Bacillus cereus species in GenBank. This study is a first report for ability of Bacillus species to grow in marine sea salt media and produce PHA. The media used for the polymer production was novel in the context of the genus Bacillus and the production of PHA was three-fold higher than Sde 2-40 using same growth medium. This study shows that the contaminant bacteria once properly investigated can be used for advantageous characteristic of metabolites production in place of original cultures.     

Cooperation,clumping and the evolution of multicellularity

The evolution of multicellular organisms represents one of the major evolutionary transitions in the history of life. A potential advantage of forming multicellular clumps is that it provides an efficiency benefit to pre-existing cooperation, such as the production of extracellular ‘public goods’. However, this is complicated by the fact that cooperation could jointly evolve with clumping, and clumping could have multiple consequences for the evolution of cooperation. We model the evolution of clumping and a cooperative public good, showing that (i) when considered separately, both clumping and public goods production gradually increase with increasing genetic relatedness; (ii) in contrast, when the traits evolve jointly, a small increase in relatedness can lead to a major shift in evolutionary outcome—from a non-clumping state with low public goods production to a cooperative clumping state with high values of both traits; (iii) high relatedness makes it easier to get to the cooperative clumping state and (iv) clumping can be inhibited when it increases the number of cells that the benefits of cooperation must be shared with, but promoted when it increases relatedness between those cells. Overall, our results suggest that public goods sharing can facilitate the formation of well-integrated cooperative clumps as a first step in the evolution of multicellularity.     

The effect of parenteral alimentation fluid,undiluted or diluted with saline or frseh sera,on the growth of Candida albicans in vitro at 37 °C

Parenteral alimentation is often complicated by Candida albicans infection which may be fatal. This study investigated the effect of alimentation fluid (Aminosol) on C. albicans' growth in vitro. It was found that concentrated Aminosol (1400 milliosmoles) maintained C. albicans in a viable state but inhibited replication. Dilution of alimentation fluid to physiological concentrations (300 milliosmoles) with either saline or aged pooled normal sera promoted in vitro growth of C. albicans which was equivalent to that obtained in BHI broth and was slightly less than that obtained in Sabouraud's broth. The effects of fresh sera with full complement activity were also investigated. In fresh sera appropriately diluted with physiological saline, some clumping of the yeasts was observed and all formed germ tubes. Growth as defined by budding or the formation of hyphae was inhibited. When Aminosol was diluted to 300 milliosmoles with fresh sera, all yeasts were noted to be in clumps with germ tubes as well as continually growing hyphae. Growth was approximately equal to that seen in Aminosol similarly diluted with saline.     

Clumping behavior as a strategy against drilling predation: Implications for the fossil record

Drilling gastropod predators are of particular interest to paleontologists, because predatory drill-holes in marine invertebrates serve as one of the rare sources of data for the study of ancient predator-prey interactions. Modern laboratory studies are an important part of predation research providing valuable ecological insight and constraining fossil evidence and interpretations. Previous studies have shown that mussels use clumping behavior against durophagous predation [Okamura, B., 1986. Group living and the effects of spatial position in aggregations of Mylitus edulis. Oecologia 69, 341-347.; Lin, J., 1991. Predator-prey interactions between blue crabs and ribbed mussels living in clumps. Estuar. Coast. Shelf Sci. 32, 61-69.], but its role against drilling predation had not been explored. In this study, we explore the effect of clumping on predator success (drill-hole frequency) and prey handling (drill-hole position) using the mussel, Mytilus trossulus, as prey and the gastropod, Nucella lamellosa, as drilling predator. We assigned mussels to two groups: in one, mussels were allowed to clump together with their byssal threads, and in the other, they were kept separate. We observed a significant decrease in the drilling frequency within the group containing clumped mussels, confirming that clumping acts as a successful anti-predatory strategy against drilling predators. The use of clumping as an effective strategy against multiple types of predators may relax the trade-offs associated with aggregated lifestyles [Bertness, M.D., Grosholz, E., 1985. Population dynamics of the ribbed mussel, Geukensia demissa: the costs and benefits of an aggregated distribution. Oecologia 67, 192-204.]. The increased benefit and unchanged metabolic cost of clumped living alters estimates of individual fitness with evolutionarily significant implications (e.g., eliminating the need to invoke group or species selection to explain the adaptive benefit of an aggregated lifestyle). In spite of potential differences in prey handling and grappling due to clumping, mean drill-hole placement and variation in drill-hole placement showed no significant differences between the two groups. These observations suggest that comparison of predation intensities across clumping and non-clumping taxa must consider the anti-predatory effect of this behavior.     

The development of Porphyridium purpureum (Bory de Saint-Vincent) Drew et Ross, 1965 (Rhodophyta) from Amursky Bay,Sea of Japan,in a laboratory culture

The growth of the microscopic red alga Porphyridium purpureum was experimentally studied in different culture media and at various levels of water salinity. The ability of this species to recover after cryopreservation was analyzed. It was revealed that the growth rate and generation time of P. purpureum during the exponential growth phase did not significantly differ between cultures on f medium and those on Goldberg’s medium. The species has been found to withstand salinity variations ranging from 8 to 32 vol %. Cryopreservation of P. purpureum cells using three-step freezing with trehalose as a cryoprotectant showed that the dynamics of cell density after thawing insignificantly differed from the control ones.     

A new murine osteoblastic cell line immortalized with the SV40 large T antigen

The murine preosteoblastic cell line, MC3T3-E1, is widely used to study bone formation and differentiation in vitro. However, this cell line is unstable in culture. The current study was designed to establish a stable osteoblastic cell line. A mammalian expression vector carrying the SV 40 large T antigen was introduced into a primary culture of cells isolated from the calvaria of newborn mice. Among isolated cell lines, the MN16 cell line was selected for further characterization. The MN16 cell line was cultured for 28 days, and compared with the MC3T3-E1 cell line with or without induction. The expression of bone-related genes was examined using the real-time RT-PCR technique. Alizarin red and von Kossa staining were used to detect mineralization of nodules in the cultures. The cell line showed the characteristics of osteoblastic cells in term of gene expression patterns of various molecular markers and calcium deposition in the cell layer after induction. Furthermore, the MN16 cells showed strong adhesion to the basic domain of collagen, a result that is specific for bone-derived cells. The MN16 cell line was found to be stably differentiated into bone formation cells in vitro and should be useful for studying bone biology.     

Cytogenetic studies in mammalian cells after treatment with insect pathogenic viruses [Baculoviridae]. II.In vitro studies with mammalian cell lines

Mammalian cell-lines from Chinese hamster, Indian muntjac and mouse were inoculated with infectious supernatant ofAutographa californica (Speyer) nuclear polyhedrosis virus (Ac NPV) replicated inMamestra brassicae (L.) cell cultures (IZD-Mb-0503). There was no adverse effect on cell proliferation, nor was a cytopathogenic effect (CPE) induced in such cultures. Cytogenetic data indicate that uptake ofAc-NPVs into the cytoplasm of mammalian cells, as shown by an electron microscopic study, induced neither numerical or structural chromosome aberrations nor sister chromatid exchange (SCE) events.     

Coenzyme A-transferase-independent butyrate re-assimilation in Clostridium acetobutylicum—evidence from a mathematical model

The hetero-dimeric CoA-transferase CtfA/B is believed to be crucial for the metabolic transition from acidogenesis to solventogenesis in Clostridium acetobutylicum as part of the industrial-relevant acetone-butanol-ethanol (ABE) fermentation. Here, the enzyme is assumed to mediate re-assimilation of acetate and butyrate during a pH-induced metabolic shift and to faciliate the first step of acetone formation from acetoacetyl-CoA. However, recent investigations using phosphate-limited continuous cultures have questioned this common dogma. To address the emerging experimental discrepancies, we investigated the mutant strain Cac-ctfA398s::CT using chemostat cultures. As a consequence of this mutation, the cells are unable to express functional ctfA and are thus lacking CoA-transferase activity. A mathematical model of the pH-induced metabolic shift, which was recently developed for the wild type, is used to analyse the observed behaviour of the mutant strain with a focus on re-assimilation activities for the two produced acids. Our theoretical analysis reveals that the ctfA mutant still re-assimilates butyrate, but not acetate. Based upon this finding, we conclude that C. acetobutylicum possesses a CoA-tranferase-independent butyrate uptake mechanism that is activated by decreasing pH levels. Furthermore, we observe that butanol formation is not inhibited under our experimental conditions, as suggested by previous batch culture experiments. In concordance with recent batch experiments, acetone formation is abolished in chemostat cultures using the ctfa mutant.     

Seasonal density,distribution and interactions of predatory and scavenger arthropods in accumulating poultry wastes in coastal and interior Southern California

Seasonal predatory and scavenger arthropod densities were studied at interior and coastal southern California poultry ranches. Though some seasonal population clumping occurred with some species, the distribution of predators and scavengers was fairly uniform within each ranch. Correlation analyses of key predators in theHisteridae, Staphylinidae, Hydrophylidae andDermaptera with the potential hosts,Musca domestica L.,Tinea fuscipunctella Haworth, andFannia spp. suggested that predator activity was seasonally influenced. Possible periodic avoidance of a particular host's habitat was detected as significant negative correlations. The data tend to support the importance of different species of predators in different seasons and the need for natural enemy complexes rather than single species for biological control.     

Promotion of respiration by auxin in the induction of cell division in suspension culture

Auxins (IAA, NAA, p-CPA) caused 20–30% promotion of respiration of auxin-dependent cell cultures ofNicotiana tabacum, Glycine max, Taraxacum mongolica, andAtriplex sp. and had small if any effect on respiration of auxin-independent cell cultures ofRubus sp. andScorzonera hispanica. Antiauxin (p-CPIBA) did not affect the respiration. The auxin effect on the respiration of tobacco cells was revealed 10 min after its addition to the suspension and reached a maximum value in 60 min. This stimulation preceded the induction of cell division by auxin. Mitochondria isolated from auxin-treated tobacco cells had greater oxidative and phosphorylative activity than mitochondria from untreated cells. However, isolated mitochondria did not respond to auxin. The inhibitors of respiration (cyanide, monoiodoacetate, malonate, and 2,4-dinitrophenol) eliminated auxin effect on the respiration and cell division. It is concluded that the promotion of respiration is a common event for the auxin effects both on cell extension and on cell division. This promotion is necessary for the induction of cell division and is exerted via direct activation of mitochondriain situ.     

A novel approach for efficient plant regeneration from long-term suspension culture of wheat

Summary Hexaploid wheat plants were easily regenerated from young embryo-derived callus for twelve genotypes tested. After a 2.5 years culture period, however, most of the callus cells lost their ability to regenerate into shoots, but not into roots.A novel approach was used to regenerate shoots from the long-term suspension cultured cells. In general, instead of selecting embryogenic callus as source material, this approach requires the inoculation of unselected callus into liquid medium followed by removing the free floating cell portion, selecting out non-root forming cell clumps from the root forming primary suspension culture, and growing the putative shoot-competent clumps in liquid medium with reduced auxin concentrations. We have successfully established shoot-competent wheat suspension cultures for cv. Mustang. High (>80%) frequencies of plant regeneration were observed from plating of 2.5 year suspension cultures. The suspension cultures established by this approach have been utilized to select for heat tolerant variants and will be an ideal source material for protoplast culture and transformation studies. This approach can also be applied to other cereal crops which form roots easily but are unstable in maintaining long term regenerable cultures and which are not easily adaptable to suspension culture.