Somatic embryogenesis in callus cultures of Rosa hybrida L. cv. Landora

Callus was initiated from immature leaf and stem segments of rose (Rosa hybrida cv. Landora) and subcultured every four weeks on a basal medium of half-strength Murashige & Skoog (1962) salts plus 30 g l^-1 sucrose (1/2 MS) and supplemented with 2.2 M BA, 5.4 M NAA and 2.2–9.0 M 2,4-D. Embryogenic callus and subsequently somatic embryos were obtained from 8-week-old callus culture on 1/2 MS+2.2 M BA+0.05 M NAA+0.3 M GA₃+200–800 mg l^-1 L-proline. Long-term cultures were established and maintained for up to 16 months by repeated subculture of embryogenic callus on L-proline deficient medium. About 12% of cotyledonary stage embryos taken from cultures cold-stored at 8±1°C for 4 days germinated on 1/2 MS+2.2 M BA+0.3 M GA₃+24.7 M adenine sulphate.Abbreviations BA benzyladenine - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid     

Asymmetric diffusion into the postsynaptic neuron: An extremely efficient mechanism for removing excess GABA from synaptic clefts on the Deiters' neurone plasma membrane

Microdissected Deiters' neuron plasma membranes have been used for studying the passage of GABA through the membrane both in the inward and outward direction. Working with 0.2 mM GABA in the compartment simulating the outside of the neurone and with 2.0 mM GABA in the one simulating the inside we found a net transport of GABA towards the inside. This mechanism does not require a Na⁺ ion gradient across the membrane. The nature of the transport process involved was studied by determining the rate of [³H]-GABA inward passage as a function of GABA concentration (1 nM–800 M) on the outward side of the membrane. The results have shown that until 50 M a diffusion process (v=D₁×C, where D₁=3.1×10^–11 1/m²×sec) is the sole mechanism involved. Above 50 M a second diffusion process is activated v=D₂×(C–50×10^–6), where D₂=2.8×10^–11 1/m²×sec. Taking in account both inward and outward directed diffusion, one can calculate 16 M as the equilibrium concentration of GABA on the outward side of the membrane. From a kinetic point of view, these diffusion processes are able to reduce GABA concentration in a synaptic cleft from 3 mM to 20 M within 3 sec. These diffusion systems are discussed as extremely efficient in removing the excess of released GABA in the synaptic cleft.     

Stereospecificity and electrogenicity of amino acid transport in Riccia fluitans

In the aquatic liverwort Riccia fluitans, membrane depolarization (_m), change in membrane conductance (g_m), and current-voltage (I-V) characteristics in the presence of different amino acids as well as the uptake of ¹⁴C-labeled amino acids were measured. L-isomers of the tested amino acids generate larger electrical effects (_m, g_m) than D-isomers, and the I-V characteristics show that the positive electrical inward-current of 20 mA m^-2 generated by 0.5 mM D-serine is only about 50% of the current generated by adding 0.5 mM L-serine. Whereas - and -amino acids rapidly depolarize the membrane to the same extend, with -aminobutyric acid (-AB) and dipeptides no significant electrical effects have been measured. The uptake kinetics of ¹⁴C-labeled amino acids display three components: (I) A saturable high-affinity component with K_s-values of 48 M D-alanine, 12 M -aminoisobutyric acid (AIB), 9 M L-alanine, 8 M L-proline, and 6 M L-serine, respectively; (2) an apparently linear low-affinity component, and (3) an also linear but unspecific component at concentrations >20 times the given K_s-value. Uptake of ¹⁴C-labeled AIB can be inhibited competitively by all tested neutral amino acids, the L-isomers being more effective than the D-isomers, as well as by ammonium or methylamine. Vice versa, AIB competitively inhibits uptake of L-serine and L-alanine. It is concluded that an uncharged stereospecific carrier for the investigated amino acids exists in the plasmalemma of Riccia fluitans. Accumulation ratios of about 50 suggest secondary active transport driven by a transmembrane electro-chemical gradient (mainly _m) which is generated by the electrogenic proton pump. It is suggested that this carrier binds to the amino group forming either a charged binary complex with positively charged amines (Felle 1980), or an uncharged complex with -AB or dipeptides, whereas electrogenic transport of - and -amino acids is mediated by a ternary carrier complex, probably charged by a proton.Symbols and Abbreviations _m membrane potential (mV) - E_co equilibrium potential (mV) of the transport system - g_m membrane (slope) conductance (Sm^-2) - g_m change in g_m - I-V curve current-voltage curve - AIB -aminoisobutytric acid - -AB -aminobutyric acid     

Influence of 2,3,5-triiodobenzoic acid on the transport and metabolism of IAA in lupin hypocotyls

The influence of 2,3,5-triiodobenzoic acid (TIBA) on the transport and metabolism of indolyl-3-acetic acid (IAA) was studied in etiolated lupin (Lupinus albus L) hypocotyls. Double isotope-labeled IAA [(5-³H)-IAA plus (1-¹⁴C)-IAA] was applied to the cut surface of decapitated seedlings. This confirmed that the species mobilized was unaltered IAA and permitted us to measure the in vivo decarboxylation of applied IAA. A pretreatment with TIBA applied to the cut surface produced a partial or drastic inhibition in the basipetal IAA movement at 0.5 or 100 M, respectively. Since TIBA inhibits auxin polar transport by interfering with the efflux carrier, the above results suggest that 100 M TIBA is sufficient to saturate the binding sites in the transporting cells. Compared to the control plants, in vivo decarboxylation of IAA was enhanced in 0.5 M TIBA-treated plants, while no decarboxylation was detected after treatment with 100 M TIBA. The in vitro decarboxylation of (1-¹⁴C)-IAA catalyzed by purified peroxidase was moderately activated by 100 M and unaffected by 0.5 M TIBA. The paradoxical effect of TIBA in vivo vs in vitro assays suggests that the in vivo effect of TIBA on IAA oxidation might be the consequence of the action of TIBA on the auxin transport system. Thus, transport reduction by 0.5 M TIBA caused a temporary accumulation of IAA in that apical region of the hypocotyl which has the highest capacity to decarboxylate IAA. In the presence of 100 M TIBA, a concentration which presumably saturates the efflux carriers, most of the added IAA can be expected to be located in the transporting cells where, according to the present data, IAA decarboxylation cannot take place.     

In vitro organogenesis of elephant apple (Feronia limonia)

Elephant apple (Feronia limonia L.). was micropropagated on MS medium containing 4.4 M benzyladenine and 4.6 M kinetin using cotyledon explants taken from in vitro-grown seedlings. Adventitious buds formed on the cotyledon developed into shoots that were rooted in half-strength MS medium containing 0.57 M indoleacetic acid and 0.49 M indolebutyric acid. Plants were successfully established in soil.Abbreviations BA 6-benzyladenine - IAA 3-indoleacetic acid - IBA 3-indolebutyric acid - MS Murashige & Skoog     

Sisal plant regeneration via organogenesis

Callus was initiated from in vitro grown immature leaf and ex vitro grown mature leaf and rhizome explants of Agave sisalana Perr. ex. Engelm, on MS medium containing 2,4-D (9.05 M) and kinetin (4.6 M) or 2,4-D (9.05 M), kinetin (4.6 M) and CH (1000 mg l^–1) or mod. MS (NH₄NO₃, 1500 mg l^–1) containing 2,4-D (9.05 M) and kinetin (4.6 M). Light was essential for callus formation which, however, was different in three types of explants on three different media compositions. Increasing NH₄ ⁺had a negative impact while addition of CH had a positive impact on callus formation. Shoot regeneration from callus from CH-supplemented medium only was achieved for rhizome and immature leaf tissues. The highest rate of regeneration was obtained with BA (26.6 M) as the sole hormone. Shoot buds g^–1 callus varied according to BA concentrations. Shoot proliferation rate increased on half-strength MS medium containing BA (8.9 M). Microshoots developed on MS medium containing BA (2.22 M) and GA₃ (1.44 M) and finally rooted on MS medium containing IAA (11.42 M). Acclimatized rooted plantlets are growing satisfactorily in ex vitro. This is the first report on plant regeneration via organogenesis of A. sisalana.     

Inhibitory actions of muscarinic cholinergic receptor agonists on serotonin N-acetyltransferase in bovine pineal explants in culture

We have previously identified muscarinic cholinergic receptors in the bovine pineal gland with a K_D value of 0.423±0.01 nM and a B_max value of 69.75±20.91 fmol/mg protein. Similarly, we have shown that the bovine pineal gland possesses a specific choline acetyltransferase with an activity of 0.034±0.004 nmol/mg protein/min. In order to delineate the function of these cholinergic receptor sites, we have studied the effects of muscarinic cholinergic receptor agonists on the activity of serotonin N-acetyltransferase, the melatonin synthesizing enzyme. Cholinergic receptor agonists such as methacholine (10 M), carbachol (10 M), and oxotremorine (10 M) inhibited the activity of serotonin N-acetyltransferase in the bovine pineal explants in culture, from a control value of 5.02±0.45 to 1.25±0.25, 1.30±0.15, and 1.22±0.20 pmol/mg protein/min, respectively. These inhibitory effects were blocked by muscarinic cholinergic receptor antagonists such as atropine (20 M) or QNB (20 M). The presence of high affinity muscarinic cholinergic binding sites, of a specific choline acetyltransferase, along with an inhibitory action of cholinomimetic agents on the activity of serotonin N-acetyltransferase, are interpreted to suggest that muscarinic cholinergic fibers may modulate the synthesis and actions of pineal melatonin.     

Freeze-fracture studies on the cockroach hemocyte membrane complex: Symmetric and asymmetric membrane particle distributions

Summary Freeze-fracture studies were conducted on the membranes of normal cockroach hemocytes. The plasmalemma is asymmetric with the A fracture face containing 80–100 Å membrane intercalated particles at a concentration of 2500/². The B fracture face contains 120–150 Å particles with a relatively low density (800/²). The nuclear envelope displays an asymmetry with the A fracture face containing 1500 particles/² and the B face containing 300/ ². No significant particle size differences were observed in nuclear envelope fracture faces. Two types of symmetric membranes were also found in these cells. Both A and B fracture faces of the membrane surrounding the numerous cytoplasmic inclusion bodies contain particle sizes and concentrations similar to the B face of the plasmalemma. A second type of symmetry was observed in cells apparently engaged in exocytosis. Vesicles (0.1 D) from this process were completely particle free on both fracture faces. Such particle free vesicles could be found in the cytoplasm, attached to the plasmalemma, or completely separated from the cell.Supported by a Pharmaceutical Manufacturers Association Foundation Fellowship.The author wishes to thank Ms. Annalena K. Charla for assistance in plate preparation, Dr. Julius Schultz and the Papanicolaou Cancer Research Institute for use of the freeze-etch device, and Dr. David Smith for the electron microscope facilities.     

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.     

High-affinity taurine uptake in developing retina

A specific system for taurine transport is present at the early stages of development in both chick and rat retinas. The results obtained with taurine analogs indicate a high degree of specificity of taurine uptake. Two transport systems were detected for the adult rat retina: a high-affinity (K _m 21 M) and a low-affinity transport system (K _m 312 M). On the other hand, in the adult chick retina, only a low-affinity transport system (K _m 580 M) could be detected. Nevertheless, embryo chick retina accumulated [³H]taurine by two different kinetic mechanisms withK _m s of 242 M and 21 M for the low- and high-affinity processes, respectively. Taurine uptake systems were absolutely Na⁺ dependent. The sodium-dependence curve for taurine uptake was sigmoid. These mechanisms appear not to be mediated by a Na⁺ cotransport system. In spite of the differences observed in taurine uptake in both species, in each of them it closely parallels the changes brought about by the morphological and functional maturation of the retina.     

Micropropagation of Tamarix gallica from nodal explants of mature trees

Tamarix gallica L. was micropropagated from four-to six-node explants taken from mature trees. Shoot proliferation was induced on Linsmaier and Skoog medium containing 30 g l^-1 sucrose, 7 g l^-1 agar, 200 mg l^-1 reduced glutathione (basal medium) and supplemented with 3.3 M benzyladenine. Adding 0.5 or 1.0 M indole-3-butyric acid (IBA) to the basal medium increased lateral shoot formation and ease of rooting. Microcuttings repeatedly subcultured on 1.0 M IBA produced well-developed roots, a high number of axillary shoots and could be acclimatized in the greenhouse.     

Lead‐related effects on rat fibroblasts

Lead (Pb) is an environmental toxicant that can induce structural and functional abnormalities of multiple organ systems, including the central nervous and the immune systems. The aim of this study was to evaluate the effects of extracellular Pb supplementation on the cellular content of the metal and on the proliferation and the survival of normal rat fibroblasts.We found that the concentration of Pb in the culture medium was 0.060 M and the normal Pb concentration in rat fibroblasts was 3.1 ± 0.1 ng/10⁷ cells. Then we exposed the cells to increasing concentration of Pb (as Pb acetate) from 0.078–320 M. We observed a dosedependent inhibition of cell proliferation after 48 h, which was already apparent at a concentration of 0.312 M (p = 0.122) and became statistically significant for concentration higher than 0.625 M (p = 0.0003 at 5 M). Cell proliferation was completely compromised at 320 M Pb total inhibition of cell proliferation.To investigate the mechanisms of Pbmediated inhibition of cell proliferation, we evaluated the occurrence of apoptosis in the same cells and found that cytosolic DNA fragments, hallmark of apoptotic cell death, increased significantly at Pb concentrations from 2.5–10.0 M. The occurrence of apoptosis was also confirmed by FACS analysis which showed the appearance of a subdiploid peak at Pb concentrations from 5–20 M. The distribution of cells in the cell cycle showed a dosedependent accumulation of cells in the G₀/G₁ phase mainly compensated by a decrease in the percentage of cells in the S phase. In conclusion, our results demonstrate that induction of apoptosis contributes to the Pbinduced inhibition of cell proliferation in rat fibroblasts.     

Cytoskeletal-associated protein kinase and phosphatase activities from cerebral cortex of young rats

We describe the phosphorylation system associated with the Triton-insoluble cytoskeletal fraction that phosphorylates in vitro the 150 kDa neurofilament subunit (NF-M) and alpha and beta tubulin from cerebral cortex of rats. The protein kinase activities were determined in the presence of 20 M cyclic AMP (cAMP), 1 mM calcium and 1 M calmodulin (Ca²⁺/calmodulin) or 1 mM calcium, 0.2 mM phosphatidylserine and 0.5 M phorbol 12,13-dibutyrate (Ca²⁺/PS/PDBu). Phosphorylation of these cytoskeletal proteins increased approximately 35% and 65% in the presence of cAMP and Ca²⁺/calmodulin, respectively, but was unaffected in the presence of Ca²⁺/PS/PDBu. Basal phosphorylation of these proteins studied increased approximately 35% and 72% in the presence of 0.5 M okadaic acid and 0.01 M microcystin-LR, respectively, suggesting the presence of phosphatase type 1. Results suggest that at least two protein kinases and one protein phosphatase are associated with the Triton-insoluble cytoskeletal fraction from cerebral cortex of rats.     

Rapid plant regeneration from callus cultures of Plumbago zeylanica

Rapid plant regeneration was achieved in callus cultures derived from leaf and stem explants of Plumbago zeylanica Linn. on MS basal medium supplemented with 4.44 M 6-BA, 1.42 M IAA and 3% (w/v) sucrose. The rate of shoot bud regeneration was positively correlated with the concentration of growth regulators in the nutrient media. The leaf explants were more responsive (82.3%) than the stem explants on medium containing 1.42M IAA in combination with 4.44 M BA. The rate of regeneration was found to maintain the same level for 12 months without loss of vigour. Rooting of the differentiated shoots was achieved in media having 0.57 M IAA with 2% (w/v) sucrose within 10 days of culture. Regenerated plantlets were transferred to soil which grew normally with a survival rate of 90%. This protocol may help in the conservation of the species and selection of variants that may be induced to widen the genetic base of the genus.     

Sodium dependency of GABA uptake into glial cells in bullfrog sympathetic ganglia

The kinetics of sodium dependency of GABA uptake by satellite glial cells was studied in bullfrog sympathetic ganglia. GABA uptake followed simple Michaelis-Menten kinetics at all sodium concentrations tested. Increasing external sodium concentration increased bothK _m andV _max for GABA uptake, with an increase in theV _max/K _m ratio. The initial rate of uptake as a function of the sodium concentration exhibited sigmoid shape at 100 M GABA. Hill number was estimated to be 2.0. Removal of external potassium ion or 10 M ouabain reduced GABA uptake time-dependently. The effect of ouabain was potentiated by 100 M veratrine. These results suggest that at least two sodium ions are involved with the transport of one GABA molecule and that sodium concentration gradient across the plasma membrane is the main driving force for the transport of GABA. The essential sodium gradient may be maintained by Na⁺, K⁺-ATPase acting as an ion pump.     

Regeneration of simon poplar (Populus simonii) from protoplast culture

Simon poplar (Populus simonii) protoplasts were isolated from suspension cells, with protoplast yield of 3.8×10⁷ g^–1 F. W. They were cultured in a K8P liquid medium containing 13.57M 2,4-D, 1.07M NAA and 0.93 M KT. Protoplast culture was influenced by the plating density, osmotic pressure, and the sources and amounts of nitrogen and carbon in the culture medium. Multiple shoots were produced from protoplast-derived callus after culture on MS medium containing 4.44 M BA, 2.32M KT, 2.28 M ZT, and 0.54M NAA. Shoots 2–3 cm in height were isolated from the calli and rooted on 1/2 MS medium. After transplantation into pots, the regenerated plants grew vigorously in greenhouse.Abbreviations BA N⁶-benzyladenine - NAA 1-naphthalene acetic acid - 2,4-D 2,4-dichlorophenoxy acetic acid - KT Kinetin - ZT Zeatin - 2ip 2-isopentenyl-adenine - FDA fluorescein diacetate - MES 2-(N-morpholino) ethane sulfonic acid - MS Murashige and Skoog basal medium (1962) - K8P Kao basal medium (1977) - CPW Cell and Protoplast Wash solution (Power and Davey 1980)     

In vitro propagation of Glehnia littoralis from shoot-tips

Shoot cultures of Glehnia littoralis F. Schmidt ex Miq. (Umbelliferae) were established by placing shoot tip explants on Linsmaier and Skoog medium with 1 M NAA and 10 M BAP. Shoots were multiplied on the basal medium supplemented with 0.3 M NAA and 3 M BAP and rooted on medium containing either 1 M IBA or 3–10 M IAA. Plantlets survived in pots without any covering. This unique characteristic of the plantlets was ascribed partly to a well-developed cuticle on the surface of the leaf and the small ratio of surface area to fresh weight of a leaf blade in comparison with those of other species whose plantlets needed coverings after potting. The regenerated plantlets were finally transferred to soil.Abbreviations IAA potassium indole-3-acetate - IBA indole-3-butyric acid - IPA indole-3-propionic acid - NAA potassium 1-naphthaleneacetate - 2,4-D sodium 2,4-dichlorophenoxyacetate - BAP 6-benzylaminopurine - 2-iP N⁶-(2-isopentenyl)adenine     

Factors influencing shoot regeneration from cotyledonary explants ofCucumis melo

Cotyledonary explants of 4-day-oldCucumis melo cv. Hale's Best Jumbo in vitro seedlings showed maximum initiation of shoot buds when cultured onto a revised Murashige & Skoog medium supplemented with 5 M indole-3-acetic acid and 5 M benzylaminopurine and cultured at 25–29°C under low light intensity (5–30 mol m^-2 s^-1). Subculture of the shoot buds onto the same medium without auxin and supplemented with 3 M benzylaminopurine caused the development of shoots from 30% of the buds. The presence of abscisic acid significantly increased the number of explants producing shoot buds. Bud initiation was affected by genotype, seedling age, light intensity, and temperature. Addition of gibberellic acid, thidiazuron or silver nitrate to regeneration medium did not improve either bud initiation or shoot regeneration.     

Tissue culture and micropropagation of Cuphea ericoides,a potential source of medium-chain fatty acids

Plant rgeneration occurred on leaf-and stem-derived callus of Cuphea ericoides Cham. & Schlechtd obtained in Murashige and Skoog medium supplemented with auxins [indole-3-acetic acid (IAA), -naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-d)] plus cytokinins [6-benzyladenine (BA) or kinetin]. These calluses were subcultured and showed vigorous growth. When subcultured on medium containing 2.22 or 4.44 M BA, the calluses showed profuse regeneration of shoots whereas those subcultured on medium supplemented with 2.69 M NAA or 0.226 M 2,4-d produced numerous roots. Isolated shoots rooted on Murashige and Skoog medium lacking growth regulators or containing 0.54 M NAA or 0.49 M indole-3-butyric acid (IBA). Plantlets were acclimatized to greenhouse conditions.Abbreviations BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige & Skoog medium - NAA 1--naphthaleneacetic acid     

Mitochondria in fine afferent nerve fibres of the knee joint in the cat: a quantitative electron-microscopical examination

The distribution of mitochondria, their content and concentration (expressed as the ratio of the mean volume of mitochondria and the surface of the sensory axon) were determined in group-III and-IV nerve fibres innervating the knee joint capsule in the cat. Mitochondria mainly accumulated in axonal swellings (beads) and end bulbs of the terminal branches. Between single nerve fibres, marked differences in the content and the concentration of mitochondria were obtained in proximal portions (inside of the perineurium) and in distal portions (unmyelinated sensory endings). In group-III nerve fibres, the mitochondrial concentration ranged from 0.005 to 0.030 m³/m² (proximal portion) and from 0.016 to 0.080 m³/m² (distal portion). In unmyelinated group-IV nerve fibres, the values also showed a broad variation ranging from 0.001 to 0.011 m³/m² (proximal portion) and from 0.003 to 0.019 m³/m² (distal portion). The wide range of mitochondrial concentrations may reflect different energy consumption during receptive processes: nerve fibres with a low mechanical threshold and a high probability of excitatory events may be rich in mitochondria, whereas fibres with a high mechanical threshold and a low probability of excitatory events may be poor in mitochondria.