Inhibitory effect of curcumin on the invasion of rat ascites hepatoma cells in vitro and ex vivo

Curcumin, a yellow pigment in turmeric, is a food factor withantioxidative activity. The effect of curcumin on the proliferation and invasion of the rat ascites hepatoma AH109Acells was studied in vitro and ex vivo assay systems. Especially, a co-culture system of the hepatoma cellswith mesothelial cells derived from rat mesentery was employed to investigate the invasive motility. Curcumin suppressed thehepatoma slipping motility in a dose-dependent manner up to 5 M and thereafter maintained the effect up to 20 M, whereas this substance exerted little influence on the proliferation of the hepatoma cells at the same concentrations. Sera obtained from rats orally given curcumin also inhibited the AH109A cellular invasive movement when added to the culturemedium. Hepatoma cells previously cultured with hypoxanthineand xanthine oxidase showed a highly invasive activity. Curcumin and curcumin-loaded rat sera suppressed this reactive oxygen species-potentiated invasive capacity by simultaneously treating AH109A cells with hypoxanthine, xanthine oxidase and either of curcumin samples. These resultssuggest that the antioxidative property of curcumin may beinvolved in its anti-invasive action.     

Al-induced inhibition of root elongation in corn,Zea mays L. is overcome by Si addition

The effect of silicic acid on Al-induced inhibition of root elongation was investigated in corn roots (Zea mays L. cv. golden cross bantam) in 100 t M CaCl2 solution at pH 4.3. Twenty t M Al inhibited root elongation (20 h) about 70%, however, inhibition was alleviated by addition of silicic acid. The alleviative effect increased with higher silicic acid concentrations. The concentration of Al3+, the toxic species, in solution was decreased to about 15, 10, and 5 t M, respectively, from the initial concentration of 20 t M by addition of silicic acid at 500, 1000, and 2000 t M Si. Under the same concentration of Al3+, Al-induced inhibition of root elongation showed the same extent regardless of the addition of silicic acid or not by comparing 5 t M Al treatment with 20 t M Al + 2000 t M Si treatment, and 10 t M Al treatment with 20 t M Al +1000 t M Si treatment. Viability of cells on the root tip surface was decreased by Al addition. Cell viability was not improved by addition of silicic acid under the same concentration of Al3+. All these facts suggest that the alleviative effect of silicic acid on Al toxicity resulted from decreasing toxic Al3+ concentration by forming Al-Si complexes rather than from other physiological effects of silicic acid in corn roots.     

Clonal propagation of Callistemon by tissue culture techniques

Callus development in Callistemon viminalis was readily achieved when axillary buds derived from nodal tissue were placed in a medium containing macro- and micro-nutrients, sucrose (0.06 M), inositol (300 M), nicotinic acid (20 M), pyridoxine hydrochloride (3 M), thiamine hydrochloride (2 M), riboflavin (10 M), cytokinins (5 M) and auxins (0.1 M). The presence of benzylaminopurine (5 M) and p-chlorophenoxyacetic acid (0.1 M) promoted the most vigorous callus development and sprout formation. Rooting of nodal material was rare but occurred readily following the transference of sprouts developed on callus to a basal medium containing sucrose and salts. Root initiation was stimulated, however, by the presence of auxins. Chlorophenoxyacetic acid while stimulating root initiation repressed root growth. Indole butyric acid stimulated both root initiation and shoot growth at concentrations of 0.005 to 0.1 M. The treatment of choice for rooting and shoot growth was the addition of indole butyric acid at a concentration of 0.01 M.     

Influence of phenolic acids on phosphorus mobilisation in acidic and calcareous soils

Phenolic acids and phenols are abundant in soils. However, little information is available on the role of these compounds in mobilisation of soil phosphorus (P). The present study examined the effects of three phenolics on P mobilisation in comparison with citric acid in three soils differing in chemical properties. The soils were incubated with organic compounds at concentrations of 0 to 100 mol g^–1 soil for 30 min. While the addition of phenolic acid anions and phenol decreased soil pH, citrate either increased or did not affect soil pH depending on the soil type. All the organic compounds increased the amounts of easily-mobilised P fractions in the order of catechol citric acid proto-catechuic acid caffeic acid for the acid soils and citric acid > catechol = proto-catechuic acid > caffeic acid for the calcareous soil. Phosphorus mobilisation did not correspond to the amounts of Ca, Fe or Al ions released from the soils. These results suggest that organic acids and phenols altered the P fractions from stable, sparingly-soluble forms to easily dissolvable forms, and that chelation or precipitation of cations with organic ligands occur before biodegradation of the ligands.     

Restoration of regeneration potentiality in prolonged culture of Digitalis purpurea

Regeneration potential was restored in 2-year-old cultures of proliferating shoots of Digitalis purpurea L. under the influence of 14.43 M gibberellic acid, while cytokinins were ineffective. The retrieved shoots grew normally in a modified Murashige and Skoog's medium supplemented with 0.98 M indole-3-butyric acid (IBA) and 27.1 M adenine sulphate and rooted 100% in the presence of 0.49 M IBA alone.NBRI Research Publication No. 415 (N.S.)     

Rapid in vitro Regeneration of Sesbania drummondii

This paper describes rapid propagation of Sesbania drummondii using nodal explants isolated from seedlings and young plants. The nodal segments proliferated into multiple shoots on Murashige and Skoog's (MS) medium supplemented with 22.2 M benzyladenine. MS medium containing 2.2 and 4.5 M thidiazuron induced 5 – 6 shoots per stem node from 3-month-old plants. Nodal explants when cultured on MS medium containing combinations of benzyladenine (8.8 and 11.1 M) and indole-3-butyric acid (0.24 – 2.46 M) or indole-3-acetic acid (0.28 – 2.85 M) gave lesser number of shoots. Callus induced on cotyledonary explants when subcultured on 2.2 M thidiazuron containing medium resulted in its mass proliferation having numerous embryoid-like structures. Indole-3-butyric acid (0.24 – 2.46 M) was found suitable for root induction. In vitro regenerated plants were acclimatized in greenhouse conditions.     

Regeneration from long-term cell suspension cultures of tepary bean (Phaseolus acutifolius)

Plant regeneration has been achieved from long-term cell suspension cultures established from leaf derived callus of tepary bean (Phaseolus acutifolius). The proportion of densely cytoplasmic cells in suspension culture increased when cultured in the L-6 medium with 54 M NAA and 2 M KN. Filtration of the cells at each of five consecutive subcultures resulted in the isolation of a plant regenerating cell line (TB 686), which is being maintained in L-6 medium with 4.5 M 2,4-D and 2.3 M zeatin. Differentiated green cell aggregates were obtained when cells from maintenance medium were transferred to the same medium with 10 M BA. Embryo-like structures developed from these aggregates on L-6 medium with 2.3 M zeatin, 0.69 M GA₃ and 1.5 M NAA. Plantlets regenerated from these structures when they were cultured on L-6 medium with 7.0 M NAA and 1.0 M KN. Plant regeneration from the cell line remained relatively constant for 270 days. Regenerated plants were grown to maturity in the greenhouse.Abbreviations BA Benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ Gibberellic acid - IPA Isopentenyladenine - KN Kinetin - NAA Naphthaleneacetic acid - AA Amino acid medium (Toriyama and Hinita, 1985) The research was sponsored by United States Agency for International Development, Washington D.C., Cooperative Agreement DAN-4137-A-00-4053-00     

Heterogeneous Long Chain Acyl-CoA Synthetases Control Distribution of Individual Fatty Acids in Newly-Formed Glycerolipids of Neuronal Cells Undergoing Neurite Outgrowth

Using PC12 cells undergoing neurite outgrowth, we studied the activation of various fatty acids, of different chain lengths and degrees of saturation, by long chain acyl-CoA synthetases (LCASs). Cells treated with nerve growth factor (NGF) were labeled with [³H]glycerol, [³H]oleic acid (OA) or [³H]arachidonic acid (AA) in the presence of other unlabeled fatty acids of endogenous or exogenous origin. Triacsin C (4.8 M), an inhibitor of acyl-CoA synthetase, decreased the incorporation of exogenous [³H]OA into glycerolipids by 30–90%, and increased by about 60% the accumulation of free [³H]OA in the cells. However it did not affect the incorporation of endogenous fatty acids nor of exogenous [³H]AA into phospholipids, suggesting that LCASs which activate exogenous AA and at least some endogenous fatty acids are relatively insensitive to this drug. Activities of the LCAS that is specific for AA (ACS), or of the non-specific LCAS which activates OA and other fatty acids (OCS), were much higher in microsomal and cytoplasmic fractions than in mitochondria or nuclei. The Vmax and Km values of ACS and OCS in microsomes were 12 and 0.7 nmol/min/mg protein and 70 and 37 M, respectively; and in cytoplasm, 6 and 0.6 nmol/ min/mg protein and 38 and 60 M, respectively. Triacsin C (2–33 M) did not affect ACS activity in microsomal or cytoplasmal fractions, but inhibited OCS activities dose-dependently and competitively: IC₅₀ and apparent Ki values were 13.5 M and 14 M in microsomes, and 3.8 M and 4 M in cytoplasm. NGF stimulated the activities of the LCASs, and, consistently, the incorporation of the various fatty acids into glycerolipids. These data indicate that LCASs are heterogeneous with respect to their intracellular locations, substrate specificities, kinetic characteristics and sensitivities to triacsin C; and that this heterogeneity affects the extents to which individual fatty acids are utilized to form glycerolipids.     

Effect of GABAmimetics on electrocorticographic spike discharges induced by guanidinoethanesulfonic acid (amidino-taurine) in the rat

The effect of guanidinoethanesulfonic acid (GES) on rat electrocorticograms (ECoG) and the effects of -aminobutyric acid (GABA) and GABA-agonists on the ECoG changes induced by GES were studied. Sporadic spike discharges began 2–5 min after 1 mol GES/10 l on filter paper was applied to the pia mater of the left sensorimotor cortex; spike discharges extended to the opposite cerebral hemisphere 60 min after the onset of the ipsilateral spike discharges. The spike discharges with a frequency of 5–10 spikes/min lasted until the end of the 4 hour recording. The induced spike discharges were suppressed when the original GES soaked filter paper was replaced by one containing GES (1 mol) supplement combined with taurine (1 mol/10 l). GABA (1 mol) and its receptor agonist, muscimol (10nmol) and (3R)-(–)-4-amino-3-hydroxybutyric acid (1 mol) also suppressed the GES-induced spike discharges when applied topically. Diazepam (DZP) (10 mg/kg) suppressed the GES-induced spike discharges 10 min after i.p. injection, but phenobarbital (20 mg/kg) increased the frequency and voltage of spike discharges 100 min following subcutaneous administration. Intraperitoneal injection of either valproate (200 mg/kg) or phenytoin (25 mg/kg), after the completion of the spike discharges, showed no effect. These findings suggest that neurotransmission or neuromodulatory effects of taurine participate in GES-induced seizure activity, and that GABA_A and DZP receptors may play a role in the mechanism that suppresses GES-induced seizures.     

In vitro meristem cloning of Eucalyptus tereticornis Sm.

Rapid multiplication of axillary meristems and direct shoot development occurred from nodal explants of mature Eucalyptus tereticornis Sm. with 5.3 M NAA, 1.1 M IAA and 4.4 M BA in Murashige-Skoog medium. Repeated subcultures of the second generation shoot cultures into low cytokinin-auxin containing media (0.44–0.88 M BA+0.1 M NAA) yielded axillary microshoots in large numbers. Half-strength MS liquid medium with 4.9 M IBA, 5.5 M IAA and 5.3 M NAA for four days, half-strength semi-solid hormonefree MS medium with charcoal, and MS liquid medium without charcoal and hormones, in sequence, induced rooting of shoots in the dark. This system is suitable for the mass propagation of this difficult-to-root eucalypt.Abbreviations BA 6-benzyladenine - IAA indole-3-acetic acid - IBA -indole-3-butyric acid - 2-iP isopentyl adenine - Kn kinetin - MS Murashige-Skoog - NAA -naphthalene acetic acid - PVP polyvinylpyrrolidone     

Organic acids in the rhizosphere of Banksia integrifolia L.f.

Water leachates of the proteoid root layer of a mature stand of Banksia integrifolia were analysed for low molecular weight (LMW) organic acids by GC, HPLC and colorimetric techniques. Large amounts of organic acids (2500 g in 100 mL of leachate) were found in the proteoid root layer compared to the surrounding soil and leaf litter (230 g in 100 mL of leachate). Citric acid represented approximately 50% of the total organic acids leached, malic acid approximately 18%, and aconitic acid constituted approximately 17%. Concentration of citric acid in the proteoid root layer may enhance the availability of phosphorus for plant uptake.     

Production of carbon monoxide from aromatic amino acids by Morganella morganii

Morganella morganii produced CO when cultured in a medium containing casamino acids or peptone as the sole carbon source. Although the production of CO was distinctly enhanced by the addition of hemin to the medium, the amounts of CO produced in the absence of hemin were nearly proportional to the amounts of peptone added to the culture media. Examination of 20 amino acids for their ability to produce CO by resting cells revealed that phenylalanine, tyrosine, histidine and tryptophan were the sources of CO. Oxygen and hemin were necessary for CO production from the amino acids except tryptophan which produced CO in the absence of hemin. When cells were incubated for 4 h at 30° C in the mixture containing 40 mol tyrosine and 1 mol hemin, about 15 mol CO was produced; the activity of CO production was about 1.2 mol CO/h · mg cell nitrogen. Phenylpyruvic acid, p-hydroxyphenylpyruvic acid and imidazolepyruvic acid also produced CO in the presence of hemin, while indolepyruvic acid produced CO regardless of the presence or absence of hemin. The production of CO by the 2-oxo acids proceeded spontaneously and did not require the presence of M. morganii cells.     

Development of biofilms in anaerobic reactors

The development of biofilms in polyethylene sheets and particles was studied using downflow reactors with synthetic nutrient media made up of a mixture of volatile fatty acids. Results suggest a preferential immobilization of acetoclastic organisms in the inner space of the surfaces and the colonization by the butyrotrophic bacteria in the outer layers. After 101 days the bioparticles reached a specific acetociastic activity of 72.45mol acetic acid/g protein ·h while the biofilms had 58.80 mol acetic acid/g protein attached ·h. Due to the low density of the polyethylene particles low fluidization velocities would be needed (2m/h) in a downflow fluidized bed reactor.     

The in vitro culture of Gerbera aurantiaca

Multiple shoot cultures were obtained from elongating axillary buds of Gerbera aurantiaca Sch. on the medium of Murashige & Skoog (MS) supplemented with 5 M benzyladenine. Shoots rooted in vitro on MS medium supplemented with 5 to 10 M indolebutyric acid or 5 to 10 M -naphthaleneacetic acid. Plantlets were hardened in a mist bed and transferred to soil.     

Minimal growth in vitro conservation of coffee (Coffea spp.)

We have studied the influence of low concentrations of 6-benzyladenine on growth limitation, in order to preserve coffee germplasm through a microcutting collection. Concentrations of 0 M, 1.3 M and 4.4 M were compared in four species: Coffea congensis, C. canephora, C. liberica and C. racemosa. After six months, microcutting behaviour varied between the different treatments, and a species effect was observed. The slow growing species (C. liberica and C. congensis) needed 1.3 M; the others coffee species (C. canephora and C. racemosa) exhibited moderate caulogenesis on 6-benzyladenine-free medium. Zero and low concentrations did not affect survival rates. In conclusion 1.3 M seems most appropriate for conserving all four species.Abbreviation BA 6-benzyladenine     

Clonal propagation of Virginia Pine (Pinus virginiana Mill.) by organogenesis

Clonal propagation of Virginia Pine (Pinus virginiana Mill.) was achieved by organogenesis on cotyledon explants. The influence of several cytokinins and abscisic acid on adventitious shoot production from cotyledon explants was investigated. Benzyladenine was more effective in shoot induction than three other cytokinins tested. Benzyladenine (22.2 M) in combination with naphthaleneacetic acid(0.05 M) in a Gresshoff and Doy (1972) medium was found to increase shoot bud production. Abscisic acid (7.6 M) in combination with benzyladenine and naphthaleneacetic acid enhanced shoot formation by an additional 65%. Root initiation was achieved with 0.5 strength Gresshoff and Doy media amended with naphthaleneacetic acid (1.3 M), indole-3-butyric acid (1.2 M) and benzyladenine (0.4 M). Over 2400 plantlets from 2 families survived and were transferred to a greenhouse in preparation for field planting. After ten months, the maximum number of surviving plantlets/seed explant from these two sources was 57 for family ALPV-38 and 41 for family ALPV-78, respectively.Abbreviations ABA Abscisic acid - BA N⁶-Benzyladenine - GD Gresshoff and Doy (1972) nutrient media - IBA Indole-3-butyric acid - KN Kinetin - NAA -Naphthaleneacetic acid - 2iP 2-isopentenyl adenine - ZN Zeatin     

Stimulation of ethylene production by catecholamines and phenylethylamine in potato cell suspension cultures

The catecholamines (50 M dopamine, 50 M norepinephrine and 100 M epinephrine) and phenylethylamine (200 M) were found to stimulate ethylene production in potato suspension cultures. When 100 M amino-oxyacetic acid was added together with epinephrine, ethylene release returned to control levels. The endogenous 1-aminocyclopropane-1-carboxylic acid levels were increased in parallel with the release of ethylene, suggesting that the observed effect probably occurs via regulation of aCC synthase. Our results suggest that there is a link between these naturally occurring monoamines and ethylene in plants.Abbreviations AOA amino-oxyacetic acid - ACC 1-aminocyclopropane-1-carboxylic acid - DA dopamine - NE norepinephrine - E epinephrine - CA catecholamines - PEA phenylethylamine     

In vitro culture of Aloe Barbadensis Mill.: Micropropagation from vegetative meristems

A method for rapid and highly effective plant micropropagation from vegetative meristems was established for Aloe barbadensis Mill. Plant micropropagation was achieved culturing apices on medium containing 1.1 M 2,4-dichlorophenoxyacetic acid and 2.3 M kinetin for 15–30 days. High morphogenetic ability was maintained by transferring explants (after 60 days) on media containing 0.11 M 2,4-dichlorophenoxyacetic acid and 2.2 M 6-benzylaminopurine.     

Regulation of auxin transport in pea (Pisum sativum L.) by phenylacetic acid: effects on the components of transmembrane transport of indol-3yl-acetic acid

Phenylacetic acid (PAA), a naturally-occurring acidic plant growth substance, was readily taken up by pea (Pisum sativum L. cv. Alderman) stem segments from buffered external solutions by a pH-dependent, non-mediated diffusion. Net uptake from a 0.2 M solution at pH 4.5 proceeded at a constant rate for at least 60 min and, up to approx. 100 M, the rate of uptake was directly proportional to the external concentration of the compound. The net rate of uptake of PAA was not affected by the inclusion of indol-3yl-acetic acid (IAA) in the uptake medium (up to approx. 30 M) and, unlike the net uptake of IAA, was not stimulated by N-1-naphthylphthalamic acid (NPA) or 2,3,5-triiodobenzoic acid. At an external concentration of 0.2 M and pH 4.5, the net rate of uptake of PAA was about twice that of IAA. It was concluded that the uptake of PAA did not involve the participation of carriers and that PAA was not a transported substrate for the carriers involved in the uptake and polar transport of IAA. Nevertheless, the inclusion of 3–100 M unlabelled PAA in the external medium greatly stimulated the uptake by pea stem segments of [1-¹⁴C]IAA (external concentration 0.2 M). It was concluded that whilst PAA was not a transported substrate for the NPA-sensitive IAA efflux carrier, it interacted with this carrier to inhibit IAA efflux from cells. Over the concentration range 3–100 M, PAA progressively reduced the stimulatory effect of NPA on IAA uptake, indicating that PAA also inhibited carrier-mediated uptake of IAA. The consequences of these observations for the regulation of polar auxin transport are discussed.Abbreviations IAA indol-3yl-acetic acid - DMO 5,5-dimethyloxazolidine-2,4-dione - NPA N-1-naphthylphthalamic acid - PAA phenylacetic acid - TIBA 2,3,5-triiodobenzoic acid     

Organogenesis versus embryogenesis from long-term suspension cultures of cucumber (Cucumis sativus L.)

Suspension cultures were initiated from leaf explant-derived callus of cucumber,Cucumis sativus cv. Hokus, and maintained under two different conditions; (I) continuously in medium with 5 M 2,4-D + 5 M BA, and (II) alternately three cultures in medium containing 5 M NAA + 5 M BA and one culture in 5 M 2,4-D + 5 M BA. After plating on solid medium with 0.5 M KIN + 0.1 M IAA, suspension aggregates from long-term culture in medium with 2,4-D developed into callus, and subsequently formed somatic embryos. These embryos, however, hardly developed into plants. They showed growth arrest and several structural abnormalities. In contrast, organogenesis took place when suspension aggregates from NAA containing medium were plated on solid medium with 0.5 M KIN + 0.1 M IAA. Numerous adventitious buds were regenerated, which quite normally developed into plants. Sucrose at low concentration of 1% improved plant formation. On the average thirty complete plants were obtained from each ml of suspension. It is discussed why adventitious buds develop into plants so well, whereas somatic embryos are prone to growth arrest and abnormal development.Abbreviations BA 6-benzylaminopurine - KIN kinetin - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid