Inhibition of Cell Growth by Overexpression of Manganese Superoxide Dismutase (MnSOD) in Human Pancreatic Carcinoma

Manganese superoxide dismutase (MnSOD) levels have been found to be low in human pancreatic cancer [Pancreas26, (2003), 23] and human pancreatic cancer cell lines [Cancer Res.63, (2003), 1297] when compared to normal human pancreas. We hypothesized that stable overexpression of pancreatic cancer cells with MnSOD cDNA would alter the malignant phenotype. MIA PaCa-2 cells were stably transfected with a pcDNA3 plasmid containing sense human MnSOD cDNA or containing no MnSOD insert by using the lipofectAMINE method. G418-resistant colonies were isolated, grown and maintained. Overexpression of MnSOD was confirmed in two selected clones with a 2-4-fold increase in MnSOD immunoreactive protein. Compared with the parental and neo control cells, the MnSOD-overexpressing clones had decreased growth rates, growth in soft agar and plating efficiency in vitro, while in vivo, the MnSOD-overexpressing clones had slower growth in nude mice. These results suggest that MnSOD may be a tumor suppressor gene in human pancreatic cancer.     

A protoplast-to-plant system in chrysanthemum: differential responses among several commercial clones

Summary Plant regeneration has been obtained from Chrysanthemum mesophyll protoplasts. Of the twenty-nine clones studied, division was observed for eighteen clones, and coupled with colony formation for sixteen of them. Elimination of NH₄NO₃ from the culture medium greatly improved colony survival in culture. Calli were obtained from colonies of five clones, and for clone n^o 42 buds were produced. Regeneration ability, for such protoplast-derived calli of clone n^o 42, was retained over a prolonged culture period. The regenerated plants were successfully transferred to the glasshouse (4 to 5 months from protoplast isolation).Abbreviations BAP 6-benzylaminopurine - F.P.E. final plating efficiency - f.wt. fresh weight - GA₃ gibberellic acid - IAA indole-3-acetic acid - I.P.E. initial plating efficiency - MS Murashige and Skoog 1962 - NAA 1-naphthaleneacetic acid - 2,4-D 2,4-dichloro-phenoxyacetic acid     

Regeneration of Mesophyll Protoplasts Isolated from Dihaploid Clones of Solanum tuberosum

Protoplasts have been isolated from leaves of shoot cultures of six dihaploid clones of Solanum tuberosum L. (2n = 2x = 24). In the KM medium (Kao and Michayluk 1975), sustained cell divisions were obtained in up to 50% of the plated protoplasts of four clones, whereas only a few divisions occurred in the other two clones. The first mitosis appeared 2–8 days after plating, dependent on the clones. In the clones showing sustained cell divisions, a protoplast titre of about 5 × 10³ per ml turned out to be optimal. The culture conditions for protoplasts of one of the poorly growing clones, clone H² 140, have been improved using modified KM media, plating at a concentration of as high as 5 × 10⁴ cells per ml, and subsequent diluting at intervals 5 days. The dilutions were carried out with media containing 0.25% agar. Up to 60% of the plated protoplasts underwent divisions within 10 days under these conditions. After about 15 days, the regenerants were transferred onto media inducing organogenesis. Shoots and roots were formed on modified media MS (Murashige and Skoog 1962) and B5 (Gamborg et al. 1968). Plants have been regenerated in four of the investigated clones. Countings of chromosomes revealed a satisfactory stability of the karyotype in shoot culture and protoplast regeneration.     

A relaxed mutant with an altered ribosomal protein L11

Summary A replica plating method for isolating ts amoebal mutants of Physarum polycephalum has been devised. Temperature-sensitive mutations occur at a frequency after nitrosoguanidine mutagenesis of 10^-3 per survivor, are stable but are not usually expressed in the plasmodia formed from these amoebae in clones. Some of these mutants appear to be cell-cycle stage specific.     

Isolation in vitro of naphthaleneacetic acid-tolerant mutants of Nicotiana tabacum, which are impaired in root morphogenesis

Summary Mutants resistant to the toxic effect of the auxin analogue naphthaleneacetic acid have been selected in vitro in mutagenized populations of haploid mesophyll protoplasts of Nicotiana tabacum. Among the regenerated clones obtained, two clones impaired in root morphogenesis were further characterized. The parental mutant clones isolated were found to be heterozygotes for the mutations conferring the inability to root. These mutations were transmissible to the progeny as single nuclear dominant traits and cosegregated with resistance to naphthaleneacetic acid at the cellular level. Apart from the inability to root and a slightly modified leaf morphology, no obvious modification of stem structure, apical dominance, and flower fertility was detected in the development of homozygotes obtained in the progeny of one of the parental mutant clones when they were grafted onto normal plants.Abbreviations NAA 1-Naphthaleneacetic acid - PE plating efficiency - p-cells protoplast-derived dividing cells - MNNG N-methyl-N'-nitro-N-nitrosoguanidine - UV Ultraviolet light - 2,4-D 2,4-dichlorophenoxyacetic acid Dedicated to Professor Georg Melchers to celebrate his 50-year association with the journal     

Sonication-assisted Agrobacterium rhizogenes-mediated transformation of Verbascum xanthophoeniceum Griseb. for bioactive metabolite accumulation

An efficient protocol for the establishment of transformed root culture of Verbascum xanthophoeniceum using sonication-assisted Agrobacterium rhizogenes-mediated transformation is reported. Only 10 days after the inoculation with A. rhizogenes ATCC 15834 and 45 s ultrasound exposure, hairy roots appeared on 75% of the Verbascum leaves. Ten hairy root lines were isolated, although only half of them were free of bacterial contamination and started growing when excised from mother explants. The transgenic nature of the most vigorously growing hairy root clones (VX1 and VX6) was confirmed by polymerase chain reaction. Under submerged cultivation both hairy root clones accumulated high biomass amounts (12.8 and 14.3 g L⁻¹, respectively) and significant amounts of bioactive phenylethanoid glycoside verbascoside (over 6-times more than in mother plant leaves). LC-APCI-MS analyses confirmed verbascoside accumulation in hairy root clones along with three other phenylethanoid glycosides (forsythoside B, leucosceptoside B and martynoside) and an iridoid glycoside aucubin. This is the first report on the induction of hairy roots of Verbascum plants.     

PEG-tolerant cell clones of chili pepper: Growth,osmotic potentials and solute accumulation

Cell cultures of chili pepper (Capsicum annuum L.) were established from callus tissue inoculated in MS liquid medium supplemented with 6.25 M 2,4-d and 0.44 M BA. Cell clones were isolated by plating the cell suspension on filter paper discs supported by polyurethane foam that were bathed with culture medium containing 15% PEG. The cell clones T6 and T7 were chosen based on their characteristics of growth and friability. These cell clones were established as cell suspensions in the presence of 15% PEG and subsequently subcultured in increasing concentrations of osmoticum. By this approach the cell clones T7 and T6 were capable of growing in the presence of 20 and 25% PEG, respectively. The cell clone T7 was found to grow better in the presence of 5–10% PEG after a period of subculturing in the absence of osmoticum indicating that the tolerance trait was stable. The tolerant cell clones exhibited a 3 to 3.5-fold decrease in the osmotic potentials in comparison with the nonselected cells suggesting that osmotic adjustment occurred. K⁺ was the major contributing solute to the osmotic potential in all the cell cultures among those tested and was found to be higher in concentration in the PEG-tolerant clones (1.3–3 times higher than nonselected cells). Proline and glycine betaine levels showed a positive correlation with the degree of tolerance to water deficit in the PEG-tolerant cell clones. The levels of proline in the cell clone T7 subcultured in the absence of PEG in the culture medium decreased to values similar to those of nonselected cells, whereas the contents of glycine betaine in the same conditions were maintained at high levels.Abbreviations BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog medium - PEG polyethylene glycol     

AN IMPROVED METHOD FOR OBTAINING AXENIC CLONES OF PLANKTONIC BLUE-GREEN ALGAE1,2

Axenic clones from 5 isolates of Anabaena flosaquae, 1 isolate of Microcystis acruginosa, and 1 isolate of Aphanizomenon flos-aquae were obtained by a combination of steps that provided a 1000-fold reduction in the bacteria-algae ratio and permitted bacteria-free filaments or cells to be isolated and grown from agar pour plates. The first step consisted of the addition of phenol to a dark-treated culture to selectively reduce the numbers of actively growing bacteria while leaving the resting algal cells viable. The next steps involved washing the treated algal suspension on a Millipore filter pad or membrane followed by plating in washed agar containing buffered mineral medium plus vitamins and soil extract. The final steps consisted of incubating the agar pour plates, coring bacteria-free filaments or cells, culturing the agar cores in a buffered mineral medium, and rigorously testing the resulting cultures for bacteriological contamination. Between 50 and 90% of the cores grew, and of these about 50% were judged axenic. The method, with appropriate adaptations, should be suitable for obtaining axenic clones of other freshwater and marine algae.     

Factors promoting sustained divisions of mesophyll protoplasts isolated from dihaploid clones of potato (Solanum tuberosum L.) and a cytological analysis of regenerated plants

A culture protocol has been developed for mesophyll protoplasts isolated from various dihaploid clones of potato. A special effort was made to promote the growth of initially dividing cells to form cell colonies and calli. An increase in plating efficiency in 3 different dihaploid clones and one doubled dihaploid clone was obtained after serial dilution of cultures with a suitable amount and type of medium at different stages of cell colony development. Plating on a refined semi-solid medium after 14 days of culture further improved both the yield and the quality of calli obtained. The refined plating medium also enhanced shoot regeneration ability from 67 to 90% in one of the dihaploid clones (67:9). The refined culture protocol could also be used without causing a decrease in plating efficiency at a low population density adjusted after 3 days of culture. The ploidy level of plants regenerated from dihaploid protoplasts were determined by chromosome counting and DNA analysis by flow cytometry. Most of the plants were aneuploid or tetraploid although, some dihaploid plants were obtained after protoplast culture of 2 dihaploid clones derived from the same cultivar (cv. Stina).Abbreviations BA benzyladenine - 2,4-D dichlorophenoxyacetic acid - GA₃ gibberellic acid - NAA naphthaleneacetic acid     

Diversity of bacterial community in freshwater of Woopo wetland

Diversity of bacterial community in water layer of Woopo wetland was investigated. Cultivable bacterial strains were isolated by the standard dilution plating technique and culture-independent 16S rRNA gene clones were obtained directly from DNA extracts of a water sample. Amplified rDNA restriction analysis (ARDRA) was applied onto both of the isolates and 16S rRNA gene clones. Rarefaction curves, coverage rate and diversity indices of ARDRA patterns were calculated. Representative isolates and clones of all the single isolate/clone phylotype were partially sequenced and analyzed phylogenetically. Sixty-four and 125 phylotypes were obtained from 203 bacterial isolates and 235 culture-independent 16S rRNA gene clones, respectively. Bacterial isolates were composed of 4 phyla, of which Firmicutes (49.8%) and Actinobacteria (32.0%) were predominant. Isolates were affiliated with 58 species. Culture-independent 16S rRNA gene clones were composed of 8 phyla, of which Proteobacteria (62.2%), Actinobacteria (15.5%), and Bacteroidetes (13.7%) were predominant. Diversity of 16S rRNA gene clones originated from cultivation-independent DNA extracts was higher than that of isolated bacteria.     

Functional requirements of (Phe,G)-A--L-specific T-cell clones of (H-2 b x H-2 kF1 origin

T-cell clones specific for the synthetic polypeptide antigen poly(LPhe, LGlu)-poly(DLAla)--poly(LLys) of (C57BL/6 x C3H/HeJ)F₁ origin were tested for their biological activities. One group of clones was restricted in its proliferative response to the H-2 ^b haplotype, the second to the H-2 ^k haplotype, and the third to the F₁ unique Ia determinants. All the clones which proliferated in response to antigen secreted interleukin-2 (IL-2) following stimulation. The H-2 restriction of the IL-2 secretion was the same as that of the proliferation. Two of the clones tested, C.6 and C.10, could provide help to B cells in antibody production. However, the genetic restriction profile of the helper activity was less stringent than that for the proliferative response. Thus, C.6, which proliferated in the presence of F₁ antigen-presenting cells only, could help B cells and accessory cells of C3H/HeJ. C.10, which was restricted in its proliferative response to the H-2 ^b haplotype, could collaborate with B cells and accessory cells of the H-2 ^k haplotype as well. The antibody response of both clones was restricted to the parental or F₁ strains.Abbreviations used in this paper (T, G)-A-L poly-(LTyr, LGlu)poly(DLAla)--poly(LLys) - (Phe, G)-A--L poly(LPhe, LGlu)-poly(DLAla)--poly(LLys) - APC antigen-presenting cells - Con A concanavalin A - FCS fetal calf serum - IL-2 interleukin-2     

Neighbor species differentially alter resistance phenotypes in Plantago

In this study, we investigated how neighbors (i.e., competitors) altered resistance phenotypes, namely plant size and levels of secondary compounds (iridoid glycosides), of individual plants and specifically tested whether neighbor identity mattered. We conducted a greenhouse experiment with Plantago lanceolata and Plantago major (Plantaginaceae) in which each species served as focal plants as well as neighbors in a factorial design. In addition, we harvested plants six and nine weeks after transplantation to test whether effects changed as plants grew. In both species, competition reduced plant size, and this effect increased over time. Plantago lanceolata neighbors suppressed growth of both focal plant species more than P. major neighbors. Effects of competition on levels of secondary compounds were more complex. Concentrations of iridoid glycosides were increased by competition in both species at harvest one. By the second harvest, an effect of competition on iridoid glycosides was found only in P. major. Neighbor identity influenced levels of iridoid glycosides in P. lanceolata at harvest one; concentrations were higher in plants grown with P. lanceolata neighbors than in plants grown with P. major neighbors. We also tested whether there was a trade-off between growth (biomass) and defense (levels of iridoid glycosides). Biomass and iridoid glycoside content were significantly correlated only in plants grown with competition and harvested at nine weeks, and this relationship was positive in both species, indicating that there was no trade-off between growth and defense. This study suggests that neighbor identity could play an important role in interspecific interactions, including the interactions of plants with other trophic levels.     

One-step transformation of the dimorphic yeast Yarrowia lipolytica

An efficient one-step transformation method for the dimorphic yeast Yarrowia lipolytica is described. Using cells grown overnight on agar plates, the whole process is carried out within 1 h. The transformant clones could be recovered on selective plates as early as 36–48 h after plating. The efficiency was better than 10⁵ transformants/μg replicative plasmid DNA. Effects of cell density, dithiothreitol, heat shock, poly(ethylene glycol) 4000 concentration and the wetness of selective plates were investigated. Received: 17 February 1997 / Received revision: 4 April 1997 / Accepted: 19 April 1997     

Simple methods for plating single vegetative cells of,and for replica-plating,filamentous blue-green algae

Summary Heterocyst-forming, filamentous blue-green algae may be broken into fragments having an average length as close to one cell as desired, by cavitation in an ultrasonic cleaner. The resulting fragments from six algae give rise to clones, with high plating efficiency. Sporulated clones of Anabeana flos-aquae may be replica-plated with velveteen.     

Direct plasmid transfer from replica-plated E. coli colonies to competent B. subtilis cells. Identification of an E. coli clone carrying the hisH and tyrA genes of B. subtilis

Summary We have cloned the hisH tyrA wild-type genes of Bacillus subtilis with the aid of the chimeric plasmid pBJ194, which replicates both in B. subtilis and Escherichia coli. Primary cloning was done in E. coli. The original E. coli clone, carrying the recombinant plasmid (pGR1) which complements hisH tyrA mutants of B. subtilis, was selected directly from a mixture of plated E. coli clones by replicaplating these clones onto minimal agar plates without tyrosine spread just before with competent B. subtilis cells. After overnight incubation clusters of small colonies had developed exclusively in the E. coli [pGR1] colony prints.The Tyr⁺ minicolonies were shown to be B. subtilis carrying pGR1 because (i) their appearance depended linearly on the number of B. subtilis cells plated, (ii) they produced extracellular protease and amylase and (iii) plasmids could be reisolated from the minicolonies and used to transform B. subtilis recE4 tyrA1 both to Cm^r and Tyr⁺.Plasmid pGR1 transfer through replica plating was compared with plasmid transfer in liquid. Both systems depended on transformable B. subtilis strains and were sensitive to DNAseI. However, whereas integration of the tyrA ⁺ gene into the chromosome and concomittant loss of plasmids occurred frequently during regular plasmid transformation of Rec⁺ B. subtilis, this was a rare event during plasmid transfer through replica plating.     

Low rates of iridoid glycoside hydrolysis in two Longitarsus leaf beetles with different feeding specialization confer tolerance to iridoid glycoside containing host plants

Iridoid glycosides are plant defence compounds that are deterrent and/or toxic for unadapted herbivores but are readily sequestered by dietary specialists of different insect orders. Hydrolysis of iridoid glycosides by β‐glucosidase leads to protein denaturation. Insect digestive β‐glucosidases thus have the potential to mediate plant–insect interactions. In the present study, mechanisms associated with iridoid glycoside tolerance are investigated in two closely‐related leaf beetle species (Coleoptera: Chrysomelidae) that feed on iridoid glycoside containing host plants. The polyphagous Longitarsus luridus Scopoli does not sequester iridoid glycosides, whereas the specialist Longitarsus tabidus Fabricius sequesters these compounds from its host plants. To study whether the biochemical properties of their β‐glucosidases correspond to the differences in feeding specialization, the number of β‐glucosidase isoforms and their kinetic properties are compared between the two beetle species. To examine the impact of iridoid glycosides on the β‐glucosidase activity of the generalist, L. luridus beetles are kept on host plants with or without iridoid glycosides. Furthermore, β‐glucosidase activities of both species are examined using an artificial β‐glucosidase substrate and the iridoid glycoside aucubin present in their host plants. Both species have one or two β‐glucosidases with different substrate affinities. Interestingly, host plant use does not influence the specific β‐glucosidase activities of the generalist. Both species hydrolyse aucubin with a much lower affinity than the standard substrate. The neutral pH reduces the β‐glucosidase activity of the specialist beetles by approximately 60% relative to its pH optimum. These low rates of aucubin hydrolysis suggest that the ability to sequester iridoid glycosides has evolved as a key to potentially preventing iridoid glycoside hydrolysis by plant‐derived β‐glucosidases.     

An improved procedure for plant regeneration from indica and japonica rice protoplasts

Plant regeneration from protoplasts of two commercially cultivated Indian indica rice varieties, Pusa Basmati 1 and Java, has been accomplished by plating embryogenic cell suspension-derived protoplasts on the surface of filter membranes overlying agarose-embedded feeder cells of Lolium multltiflorum and Oryza ridleyi, combined with the use of a maltose-containing shoot regeneration medium. Embryogenic cell suspension cultures of Pusa Basmati 1 and Jaya were initiated from mature seed scutellum-derived calli in liquid R₂ medium modified by the addition of 560 mg l^–1 of proline and 1.0 % (w/v) maltose. In both varieties, protoplast plating efficiencies up to 0.4 % were obtained, depending on the nature of the feeder cells. L. multiflorum feeder cells induced a 6-fold higher plating efficiency than feeder cells of O. ridleyi. In combination, O. ridleyi and L. multiflorum feedercells further enhanced protoplast plating efficiency. Protoplast-derived cell colonies were not obtained from protoplasts of either indica varieties in the absence of feeder cells. MS-based medium containing kinetin (2.0 mg l^–1) and -naphthaleneacetic acid (0.5 mg 1^–1), together with sucrose and maltose both at 1.5 % (w/v), induced green shoot regeneration in 44 % of protoplast-derived tissues, depending on the feeder cells used for protoplast culture. In both varieties, tissues obtained using O. ridleyi feeder cells were more morphogenic than tissues obtained using L. multiflorum feeder cells, either alone or in combination with cells of O. ridleyi. In the japonica rice variety Taipei 309, this new procedure resulted in a 30-fold increase in plant regeneration from protoplasts compared to previous published procedures.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - GPFs growth promoting factors - NAA -naphthaleneacetic acid On leave from Department of Genetics, Haryana Agricultural University, Hisar, IndiaOn leave from Biotechnology Centre, Punjab Agricultural University, Ludhiana, India     

An unusual root tip formation in hairy root culture of Hyoscyamus muticus

Summary Hairy root cultures of Hyoscyamus muticus were established using Agrobacterium rhizogenes ATCC 15834. In one out of 8 clones established, an unusual root tip formation was observed after transfer of cultures from half-strength Murashige and Skoog (1962) to White's medium (1939). This phenomenon was associated with the production of a fine brownish cell suspension culture. Hairy root development resumed after transfer of the root tips from White to half-strength Murashige and Skoog medium. After plating the isolated brownish cells on hormone-free half-strength Murashige and Skoog or White solid medium, callus proliferation was observed, and then redifferentiation of hairy roots occurred. The polymerase chain reaction analysis of the H. muticus hairy root (clone Z2) revealed that only the tl region of the T-DNA was integrated. The growth and the production of five tropane alkaloids by this clone were examined.Abbreviations PCR Polymerase Chain Reaction - MS medium Murashige and Skoog Medium - 1/2 MS medium half-strength MS medium - WP medium Woody Plant medium - RC medium Root Culture medium - WH medium White medium - HPLC High Performance Liquid Chromatography - wt. weight     

Iridoid Glycosides from Tabebuia avellanedae

Five novel iridoid glycosides, avellanedaesides A‐E ( 1  –  5 ) were isolated from the H₂O extract of Tabebuia avellanedae. Their structures were determined on the basis of NMR and MS analysis. Isolated compounds suppressed inflammatory cytokine, tumor‐necrosis factor‐α and interleukin‐1β production in cultured human myeloma THP‐1 cells co‐stimulated with lipopolysaccharide (LPS). In addition, the study revealed iridoid glycosides inhibited the activity of cytochrome CYP3A4 enzyme.     

An efficient plating system for rapid isolation of mutants from plant cell suspensions

Summary A plating system for cell suspensions of soybean, SB-1, (Glycine max L. cv. Mandarin) and Datura innoxia D.I. (Mill) was developed using feeder cells. The characteristics of the system are: a) the efficiency of plating (EOP) is high (0.5–0.6), b) over a range of 10–300 plated clumps the EOP is constant, c) the growth rate of plated cells resembles that of suspension cultures (generation time 24 hr.). Clumps with few or with many cells have similar plating efficiencies.Employing the plating system, a mutant resistant to 8 azaguanine (8AG) was isolated from SB-1 in 7 days and purified and tested within an additional 3 weeks. Feeder plates were used to selectively re-isolate 8 AG resistant and maltose utilizing mutants from a 1000-fold excess of wild type cells.The plating technique also can be utilized to isolate auxotrophic mutants since free amino acids are not produced by the feeder suspension. Other applications of this plating technique are discussed.Abbreviations 8AG 8 Azaguanine - 6TG 6 Thioguanine - EMS Ethyl methanesulfonate - EOP Efficiency of plating - HGPRT Hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8)