Pluronic F-68 Stimulates Growth of Solanum dulcamara in Culture

The effects have been studied of the non-ionic surfactant, PluronicF-68, on the growth of transformed roots, callus and protoplastsof Solanum dulcamara L. Root growth was stimulated by additionof 0001–005% (w/v) of freshly-prepared, commercial gradePluronic to culture medium, with maximum increases in root freshand dry weights at 001%. Higher concentrations (05–10%w/v) of freshly-prepared Pluronic inhibited growth. A Pluronicfraction, prepared by passage through silica-Amberlite resin,retarded root growth even at concentrations that were stimulatorywith the commercial preparation. Similarly, commercial gradePluronic solutions stored at 4C or 22C for 5 d (‘aged’)also inhibited root growth. Roots grew faster on Pluronic F-68-treatedmembrane rafts compared with growth on commercially-availablerafts; such growth enhancement was comparable to that seen inmedium supplemented with 001% (w/v) freshly-prepared commercialPluronic. Callus growth was also stimulated by the addition of freshly-prepared,commercial grade Pluronic F-68 to medium, with maximum increasesat 01% (w/v); in contrast, 10% (w/v) Pluronic was inhibitoryto callus growth. The mean plating efficiency (15 d after plating)of protoplasts cultured at densities of 01–2010⁵ cm_–3was increased up to 26% by 01% (w/v) Pluronic, while 10% wasinhibitory. Both root and callus soluble carbohydrates and proteinswere increased by exposure to freshly-prepared, commercial Pluronic.Similarly, the specific activities of malate dehydrogenase andacid phosphatase were increased in Pluronic F-68-treated callusand roots. The biotechnological implications of these resultsare discussed in relation to the potential value of non-ionicsurfactants as growth-stimulating additives to plant culturemedia. Key words: Solanum dulcamara, Pluronic F-68, surfactant, transformed roots, callus, protoplasts, malate dehydrogenase, acid phosphatase     

Optimization of cryopreservation of Artemisia annua L. callus

Cryopreservation of callus tissue of Artimisia annua L. was optimized. Two lines of calli were precultured on MS medium with 5% (v/v) dimethyl sulfoxide, and protected by a cryoprotectant containing 15% (v/v) ethylene glycol, 15% (v/v) dimethyl sulfoxide, 30% (v/v) glycerol and 13.6% (w/v) sucrose. The highest survival rate of callus A201 reached 87% after it was pretreated at 25°C, cryopreserved by liquid nitrogen, recovered in water bath at 25°C and reloaded at 25°C with 34% (w/v) sucrose solution, and that of callus A202 reached 78% after it was treated as callus A201, except pretreated at 35°C, recovered at 35°C and reloaded with 47.8% (w/v) sucrose solution.     

Improvement of Kefir yeast by mutation with N-methyl-N- nitrosoguanidine

A commercial culture of Kefir grew well at 30°C and 37°C with NaCl up to 4% (w/v) but at 42°C and 4% (w/v) NaCl it grew very slowly. These conditions were selected for the mutation with N-methyl-N-nitrosoguanidine in order to improve cell yield at high temperatures and salt concentration. The mutated cells had higher biomass and growth rate compared with the original culture. The improvement of Kefir at high temperatures and salt concentrations offers advantages for uses in single cell protein production and alcohol production from whey containing high salt.     

Spatial distribution of Pleuragramma antarcticum (Pisces: Nototheniidae) near the Filchner- and Larsen ice shelves (Weddell sea/antarctica)

Summary In the zooplankton collected during three German Antarctic expeditions to the southern Weddell Sea in 1979/80, 1980/81 and 1983, the post-larvae of the nototheniid fish Pleuragramma antarcticum were found in 71–94% of the samples and represented 85–98% in numbers of all fish caught. In 1983, the abundance of post-larvae was up to 88 ind./m² (corresponding to 3 ind./m³). Highest concentrations were generally found over the continental slope and innershelf depressions. More than 70% of the post-larvae were caught in the upper 50–100 m water layer, in the well stratified Summer Water of -1.3 to -0.5°C. The Summer Water is shifted towards the outer edge of the shelf by Ekman transport and accumulation of post-larvae in the slope front and eddies can be explained by this drift. Older Pleuragramma antarcticum of age 1 resemble in their vertical distribution the juveniles and adults, 40–60% of which were caught on the shallower parts of the shelf in cold Ice Shelf Water of -1.8 to -2.1°C in depths below 200 m. Mean abundances of the yearclasses varied by a factor of 16 in age 0 fish and by factor of 10 in age 1. The size of a yearclass may be related to the varying appearance and persistence of an ice free coastal polynya.     

Partial characterization of β-glucosidase activity produced by Kluyveromyces marxianus IMB3 during growth on cellobiose-containing media at 45°C

Summary We report- the partial characterization of a -glucosidase produced during growth of the thermotolerant yeast, K. marxianus IMB3 on lactose-containing media at 45°C. The enzyme had Km values of 1.1mM and 14.8mM for the substrates p-nitrophenyl--D-glucoside and cellobiose, respectively. The enzyme had a pH optimum of 5.5 and was optimally active at 50°C. It was stable up to 125 hours at 25°C and 35°, with half-lives of 45 hours and 2 hours at 45°C and 50°C, respectively. The enzyme was inhibited to varying degrees in the presence of metal ions and was completely inactivated by Hg²⁺. Ethanol concentrations [1–10% (v/v)] had little effect on activity. Glucose (20mM) caused inhibition when p-nitrophenyl--D-glucoside was used as substrate, whereas lactose at similar concentrations had no effect.     

Purification and characterization of aspartate aminotransferase from developing embryos of the camel tick Hyalomma dromedarii

Aspartate transaminase (AST) activity in the camel tick Hyalomma dromedarii was followed throughout embryogenesis. During purification of AST to homogeneity, ion exchange chromatography lead to four separate forms (termed I, II, III and IV). AST II with the highest specific activity was pure after chromatography on Sephacryl S-300. The molecular mass of AST II was 52KDa for the native enzyme, composed of one subunit of 50KDa. AST II had a Km value of 0.67mM for -ketoglutarate and 15.1mM for aspartate. AST II had a pH optimum of 7.5 with heat stability up to 50°C for 15min. The enzyme was activated by MnCl₂, and inhibited by CaCl₂, MgCl₂, NiCl₂, and ZnCl₂.     

Activation of protein kinase C alpha is required for TPA-triggered ERK (MAPK) signaling and growth inhibition of human hepatoma cell HepG2

The signaling mechanisms for most of the antiproliferative processes are not fully understood. We have demonstrated that ERK(MAPK) signaling was involved in the induction of both p15^INK4band p16^INK4a CDK inhibitors and growth inhibition of hepatoma cell HepG2 triggered by the tumor promoter tetradecanoyl phorbol acetate (TPA). In this study, the upstream signal mechanism for TPA-induced ERK(MAPK) activation was investigated. In HepG2 cells only one of the cPKC isozymes, PKC, but not cPKCII, nPKC or aPKC was activated by TPA as demonstrated by its membrane translocation within 10–30 min and down-regulation at 24 h after TPA treatment. Pretreatment of 0.2–2.0 M Bisindolylmaleimides, an inhibitor of PKC, attenuated the TPA-induced phosphorylation of ERK, gene expressions of p15^INK4band p16^INK4a, and growth inhibition of HepG2 cell in a dose-dependent manner. Consistently, transfection of HepG2 with 1.0–3.0 M antisense (AS) PKC, but not (AS) PKCII, or nPKC oligonucleotides (ODN), for 36 h prior to TPA treatment also prevented the TPA-induced molecular and cellular effects described above. Taken together, we concluded that PKC is specifically required for TPA-induced ERK(MAPK) signaling to trigger gene expressions of p15^INK4band p16^INK4a leading to HepG2 growth inhibition.     

Life cycles of marine nematodes

Summary Monhystera denticulata Timm, a free-living nematode present in the aufwuchs assemblages of several marine macrophytes located in North Sea Harbor, Southampton, New York, was isolated from Zostera marina and established in laboratory culture in order to study the influences of temperature and salinity on its life history. Under experimental conditions, M. denticulata has a generation time (Measured as the time elapsing between the first egg depositions of consecutive generations) of 10–12 days at 25° C and 26 S, which represent optimal growth conditions in the laboratory. The organism has a generation time of 20 days at 25° C and 13, 17 days at 25° C and 39, 18 days at 15° C and 26, 36 days at 15° C and 13 and 34 days at 15° C and 39. As conditions vary from the optimum of 25° C and 26 S, a decrease in temperature of 10° C and an increase or decrease in salinity of 13 results in a doubling of the generation time. At 5° C the generation time is about 180–197 days.Assuming optimum conditions and average generation time, about 15 generations of M. denticulata could occur in North Sea Harbor during the year. The number of generations occurring in reality is probably less, however, due to the fact that the females deposit their eggs over a period of several days.This work was supported by National Science Foundation Grant GB-19245.Contribution No. 04 from the Institute of Oceanography, City University of New York.     

Selenium modulates the activities of antioxidant enzymes,osmotic homeostasis and promotes the growth of sorrel seedlings under salt stress

Using physiological assays coupled with ultrathin tissue sections, we investigated the impacts of exogenous selenium (Se) on the growth, antioxidant enzymes, osmotic regulation and ultrastructural modifications of leaf mesophyll and root tip cells of 100 mM NaCl-stressed sorrel (Rumex patientia × R. tianshanicus) seedlings. At low concentrations (1–5 M), Se tended to stimulate the growth, the activities of superoxide dismutase and peroxidase enzymes, as well as the accumulation of water-soluble sugar in leaves of sorrel seedlings. At higher concentrations (10–30 M), Se exerted diminished beneficial effects on growth and enzyme activities. CAT activity did not change with Se addition (1–30 M). Electrolyte leakage of leaf cells declined, and K⁺ and Na⁺ ions increased in leaves with Se treatment, notably at 5 M of Se. TEM observations revealed that treatment with 5 M of Se positively promoted the integrity of membrane systems and cellular organelles, such as chloroplasts and mitochondria in leaf mesophyll and root tip cells. These results strongly suggest that an appropriate concentration of exogenous Se functions positively to promote the antioxidative and osmoregulatory capacity, and enhance the salt-resistance in sorrel seedlings.     

Abscisic acid, temperature and salinity interactions on growth and some mineral elements in Carthamus plants

Growth and contents of sodium (Na), potassium (K), calcium (Ca), magnesium (Mg), chloride (Cl), phosphorus (P) and sulphur (S) in shoot and root tissues of Carthamus tinctorius plants were measured at combinations of four nutrient solution osmotic potentials (_s=0, -0.3, -0.6 and -0.9 MPa) induced by NaCl and CaCl treatments, three constant temperatures (T) ranging from 15 to 35°C and four abscisic acid (ABA) concentrations (0,10,50 and 100 mg L^–1). Unstressed and stressed plants grown in optimal temperature conditions (25°C) maintained higher growth rates (dry mass production) than plants grown under low and high temperatures (15 and 35°C respectively). Shoot and root growth (dry mass production) were largely inhibited by salinity but the magnitude of growth inhibition was temperature dependent. Safflower plants respond to salinity stress by increases in Ca, Cl and to a lesser extent Na in their shoots and roots and by a decrease in the ratio of fresh to dry weight. The ratio of K/Na was decreased progressively on salinization. With stressed plants, ABA application reduced the toxicity of salt treatment, improved K uptake under salinity, effectively increased K/Na ratio and helped the plants to avoid Na toxicity and sometimes enhanced growth. The effect of ABA on the growth was more pronounced at optimum temperature (25°C). The association between the internal mineral element concentrations was largely affected by ABA application and temperature change but a wide fluctuation in response was noticed. The effects of single factors (_s, T and ABA) on the growth and mineral contents were statistically significant. Also, bifactorial (_s× T, _s × ABA and T × ABA) and three factorial (_s × T × ABA) interactions significantly affected the parameters. Further statistical treatment of the data (coefficient of determination ²) led to four important findings: (1) Salinity (_s) was dominant in affecting Ca and Cl contents in both shoot and root as well as root Na content. (2) Temperature (T) had a dominant effect on growth, shoot K, Mg, P, S and root P, and S contents (3) The share of _s × T × ABA interaction was dominant for root Na and Mg contents. (4) The single factors and their interactions had a dual role in their subsidiary effects.Abbreviations ABA abscisic acid - _s osmotic potential - ² coefficient of determination - F.wt fresh weight - d.m. dry matter - T temperature - MPa mega pascal - SAR sodium adsorption ratio - P phosphorus - S sulphur     

Structures of asparagine linked oligosaccharides of immunoglobulins (IgY) isolated from egg-yolk of Japanese quail

Structures of the Asn linked oligosaccharides of quail egg-yolk immunoglobulin (IgY) were determined in this study. Asn linked oligosaccharides were cleaved from IgY by hydrazinolysis and labelled withp-aminobenzoic acid ethyl ester (ABEE) afterN-acetylation. The ABEE labelled oligosaccharides were then fractionated by a combination of Concanavalin A-agarose column chromatography and anion exchange, normal phase and reversed phase HPLC before their structures were determined by sequential exoglycosidase digestion, methylation analysis, HPLC, and 500 MHz¹H-NMR spectroscopy. Quail IgY contained only neutral oligosaccharides of the following categories: the glucosylated oligomannose type (0.6%, Glc1-3Glc1-3Man₉GlcNAc₂; 35.6%, Glc1-3Man_7–9GlcNAc₂). oligomannose type (15.0%, with the structure Man_5–9GlcNAc₂) and biantennary complex type with core structures of-Man1-3(-Man1-6)Man1-4GlcNAc1-4GlcNAc (9.9%),-Man1-3(GlcNAc1-4)(-Man1-6)Man1-4GlcNAc1-4GlcNAc (25.1%) and-Man1-3(GlcNAc1-4)(-Man1-6)Man1-4GlcNAc1-4(Fuc1-6)GlcNAc (11.4%). Although never found in mammalian proteins, glucosylated oligosaccharides (Glc₁Man_7–9GlcNAc₂) have been located previously in hen IgY.Abbreviations IgG, IgM, IgA, IgY immunoglobulin G, M, A and Y, respectively - ABEE p-aminobenzoic acid ethyl ester     

Influence of environmental factors on the growth in culture of a New Zealand strain of the fast-spreading algaHydrodictyon reticulatum (water-net)

Hydrodictyon reticulatum (L.) Lagerh. is a recent addition to the New Zealand flora and is expanding its distribution rapidly. Proliferations of the alga now constitute an economic nuisance in waters which have not previously suffered filamentous algal blooms. To better understand the current and likely future spread of the alga and to identify possible management options the alga's growth requirements have been investigated. A strain isolated from New Zealand tolerated temperatures between 5 and 40 °C and salinities from 0 to 5. Optimal growth was at 25 °C, 150 mol photon m^–2s^–1 and in freshwater. Nett photosynthesis was saturated at photon flux densities of 100 and 160 mol m^–2s^–1 at 12 and 20 °C, respectively. Growth rate was linearly related to internal N concentration and hyperbolically to internal P concentration. Minimum cellular nutrient contents, by weight, were 1% N and 0.2% P. Growth was saturated at contents of 5% N and 0.5% P under the conditions of culture (20 °C, 150 mol photon m^–2s^–1). The alga maintained optimal cellular N content at low ambient nitrate concentrations (100 mg m^–3) half optimum content at 18 mg m^–3. Affinity for filtrable reactive phosphorus was not unusually high compared to other filamentous algae. We suggest that this alga is occupying a niche in New Zealand which has been precluded from other filamentous nuisance algae by low N concentration and N:P ratio. The significance of these findings in setting environmental targets for management of this nuisance alga is discussed.Author for correspondence     

Production of cellulase and β-glucosidase activities following growth of Streptomyces hygroscopicus on cellulose containing media

The actinomycete, Streptomyces hygroscopicus was shown to be capable of producing extracellular cellulase and cell associated -glucosidase activity during growth on cellulose containing media. Cell homogenates were shown to contain a -glucosidase fraction which was stable for up to 24h. at 30°C and had half-lives of 480min. and 220min. at 40 and 50°C, respectively. The enzyme fraction was also shown to be optimally active at pH 4.0 suggesting that it might represent a suitable supplement for fungal cellulase systems, deficient in -glucosidase activity.     

Potentiation of growth suppression and modulation of the antigenic phenotype in human melanoma cells by the combination of recombinant human fibroblast and immune interferons

Summary Administration of interferon as a single therapeutic regimen in cancer patients with various neoplasias has had only limited efficacy in ameliorating the negative clinical course of their disease. In the present study, we have evaluated the effect of recombinant human fibroblast (IFN) and immune (IFN) interferon, alone and in combination, on growth, differentiation and the expression of class I and II histocompatibility locus antigens (HLA) and melanoma-associated antigens on the human melanoma cell line H0-1. The effect of combinations of interferons on the antigenic profile of human melanoma cells displaying different organ colonization and spontaneous metastatic potential in athymic nude mice was also determined. H0-1 cells were more sensitive to the antiproliferative activity of IFN than to IFN and the combination of interferons resulted in a potentiation of growth suppression. The antiproliferative effect of both interferons was greater in later-passage than in earlier-passage H0-1 cells, possibly reflecting alterations in the evolving tumor cell population as a result of long-term in vitro propagation and/or the selective outgrowth of cells with an increased growth rate. The enhanced growth suppression observed in H0-1 cells treated with the combination of IFN plus IFN was not associated with a significant increase in the level of melanin, a marker of melanoma differentiation, above that observed with either interferon used alone. IFN and IFN differentially modulated the expression of class I and II HLA and melanoma-associated antigens in H0-1 cells and a series of melanoma cells with different organ colonization and metastatic potential, including MeWo, MeM 50-10, MeM 50-17, 3S5 and 70W. No consistent potentiation or antagonism in the expression of any specific antigen was observed in any of the melanoma cell lines exposed to the combination of interferons. The present study demonstrates that the combination of IFN plus IFN can potentiate growth suppression in H0-1 human melanoma cells and that this effect is not associated with an increase in differentiation or a potentiation in antigenic modulation. In addition, no direct correlation between the expression of any specific antigen or its modulation by IFN or IFN, alone or in combination, and organ colonization and metastatic potential in nude mice was observed in the different melanoma cell lines.     

Production of active human interferon-β inE. coli I. Preferential production by lower culture temperature

Summary Unusually low culture temperature, such as 20°C, was shown to be preferable for the synthesis of active human interferon- (IFN-) inE. coli harboring a recombinant plasmid. TheE. coli cells cultured at 20°C gave 8.6-fold higher IFN- activity than those cultured at 37°C. However, almost the equal amounts of IFN- protein were accumulated in both cells cultured at 20°C and at temperature higher than 20°C, suggesting that IFN- might exist as an active form in the cells cultured at 20°C, while as a rather denatured form in the cells cultured at higher temperature.     

Effect of orchard and postharvest application of daminozide on ethylene synthesis by apple fruit

The effects of daminozide (butanedioic acid-2,2-dimethylhydrazide) on ethylene synthesis by apple fruits were investigated. The objective was to determine the effects of postharvest applications as compared to the standard application of diaminozide in the orchard. Immersion in a solution containing 4.25 g L^–1 active ingredient for 5 min delayed the rise in ethylene production in individual Cox apples at 15°C by about 2 days, whereas orchard application of 0.85 g L^–1 caused delays of about 3 days. Both modes of application depressed the maximal rate of ethylene production attained by ripe apples by about 30%. Daminozide did not affect the stimulation of respiration by ethylene treatment of Gloster apples, but it delayed the increase in ethylene synthesis. Daminozide applied immediately after harvest delayed the rise in ethylene synthesis in Golden Delicious held at 15°C, but it was less effective when applied 48 h after harvest or when apples were held at 5°C. Exposure to 1–2 l L^–1 ethylene for 48 h was less effective in promoting the rise in ethylene in daminozidetreated Cox and Gloster apples than in untreated fruit. High (100–1000 l L^–1) concentrations of ethylene more or less overcame the daminozide effect. Apples absorbed about 40% of surface-applied [¹⁴C]daminozide in 48 h, but more than 90% of the radioactivity in the fruit was recovered from the peel and outer 1 cm of the cortex. Daminozide was partly converted to carbon dioxide and other metabolites.     

An improved micropropagation system for Chrysanthemum based on Pluronic F-68-supplemented media

Supplementation of MS-based medium containing 4.4 M 6-benzyladenine and 2.7 M -naphthaleneacetic acid with 0.001–0.1% (w/v) of the non-ionic, co-polymer surfactant, Pluronic F-68, significantly (p<0.05) increased the mean fresh weight gain of cultured leaf explants of chrysanthemum (Dendranthema grandiflora) Tone Maid and Early Charm by a maximum of 74% and 34%, respectively. The percentage of individual explants giving adventive shoots was also stimulated by Pluronic F-68; for cv. Early Charm, 0.001% (w/v) Pluronic F-68 induced the maximum response, whereas for cv. Tone Maid, the maximum response occurred with 0.01% surfactant. Shoot regeneration from explants was also enhanced at these concentrations of surfactant, compared to explants cultured in the absence of Pluronic F-68.Abbreviations BA 6-benzyladenine - MS Murashige & Skoog 1962 - NAA -naphthaleneacetic acid     

The use of psyllium (isubgol) as an alternative gelling agent for microbial culture media

Psyllium (Isubgol), the mucilage husk of Plantago ovata, was successfully used as an alternative gelling agent (5% w/v as ground husk for pouring medium and 4% w/v as ground husk in combination with 0.5% w/v agar for slant) for microbial culture. Most of the undesirable features of psyllium-gelled media (slant as well as pouring) were removed by adding the u.v.-treated (20min) or oven-sterilized (120^°C for 1h) psyllium as ground husk to autoclaved medium at 50–60^°C under aseptic condition just before pouring.     

Mode of uptake of sterol by Arthrobacter simplex

The mechanism of uptake of water-insoluble -sitosterol by a newly isolated strain of Arthrobacter simplex SS-7 was studied. The production of an extracellular sterol-pseudosolubilizing protein during growth of A. simplex on -sitosterol was demonstrated by isolating the factor from the cell-free supernatant and its subsequent purification by Sephadex G-150 column chromatography. The M _r of the purified sterol-pseudosolubilizing protein determined by SDS–PAGE was 19kDa. The rate of sterol pseudosolubilization (5.2×10^–3g l^–1h^–1) could not adequately account for the rate of sterol uptake (72×10^–3g l^–1h^–1) and the specific growth rate (56×10^–3 h^–1). However in the unfavourable growth condition, when the cells were treated with sodium azide at the level of 30–60% of MIC, the sterol pseudosolubilization accounted for nearly 74% of the total growth containing 96% free cells. Cellular adherence to substrate particles was found to play an active role in the normal growth of the strain on -sitosterol. Unlike sodium acetate-grown cells, whose surface activity was negligible (60mNm^–1), the sterol-grown cells had strong surface activity (40mNm^–1). The high lipid content and long chain fatty acids in the cell-wall of -sitosterol-grown cells probably contribute to the high sterol adherence activity of the cells.     

Production and potential applications of a xylanase from a new strain of Streptomyces cuspidosporus

Enzyme production by a new mesophilic Streptomyces isolate was investigated which grew optimally on 1% (w/v) xylan and 10% (w/v) wheat bran at pH 7 and 37 °C. Xylan induced only CMCase (0.29 U/ml) besides xylanase (22–35 U/ml, 40–49 U/mg protein). Wheat bran induced xylanase (105 U/ml, 17.5 U/mg protein), CMCase (0.74 U/ml), -xylosidase (0.009 U/ml), -glucosidase (0.026 U/ml), -L-arabinofuranosidase (0.049 U/ml), amylase (1.6 U/ml) and phytase (0.432 U/ml). The isolate was amenable to solid state cultivation and produced increased levels of xylanase (146 U/ml, 28 U/mg protein). The pH and temperature optima of the crude xylanase activity were 5.5 and 65 °C respectively. The pI was 6.0 as determined by PEG precipitation. The crude enzyme was applied in treatment of paper pulp and predigestion of poultry feed and was found to be effective in releasing sugars from both and soluble phosphorus from the latter.