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.     

Saccharomyces cerevisiae 2-mum DNA. An analysis of the monomer and its multimers by electron microscopy.

Summary The non-tandem inverted duplication in the 2-m DNA of Saccharomyces cerevisiae has a length of 0.19 m and is located asymmetrically along the molecule. The majority of the dumb-bell structures that are formed upon denaturation and selfannealing of the 2-m monomer consists of the renatured inverted duplication sequences as double stranded stem and two single stranded loops of 0.67 m±0.06 m (S-loop) and 0.86 m±0.05 m (L-loop) length. Two additional size classes which comprised 5–10% of the measured molecules had contour lengths of around 1.7 m and 2.1 m. The smaller dumb-bells contained two S-loops and the larger dumb-bells contained two L-loops as was shown by heteroduplex mapping with an HindIII fragment from the L-loop. Two models which assume illegitimate or site specific recombination, are presented to explain the generation of double S-loop and double L-loop molecules. At least part of the 4-m and 6- circular molecules present in the yeast supercoiled DNA fraction are shown to be dimers and trimers of 2-m monomers, but often with inverted loop segments most probably due to intramolecular recombination between sequences of the inverted duplication.2-m DNA is used to indicate the supercoiled DNA fraction although in our measurements the average monomeric length is 1.9 mPart of this work has been presented at the Conference: The Genetics and Biogenesis of Chloroplasts and Mitochondria, Munich, August, 1976     

The spermatozoon of Oikopleura dioica Fol (Larvacea,Tunicata)

Summary The spermatozoon of Oikopleura dioica is about 30 m long, with a spherical head, about 1 m wide, a 3 m long and 1 m wide midpiece, and a 25 m long tail with a tapered end piece. The head contains a nucleus with the chromatin volume limited to about 0.1 m³. A small acrosome is found in an anterior inpocketing, and a flagellar basal body in a posterior inpocketing of the nucleus. The midpiece contains a single mitochondrion with the flagellar axoneme embedded in a groove along its medial surface. The flagellar axoneme has the typical 9 + 2 substructure, and the basal body the typical 9+0 substructure. A second centriole and special anchoring fibres are absent.     

Phosphorylation-dephosphorylation is involved in Ca2+-controlled cytoplasmic streaming of characean cells

Summary The mechanism of Ca²⁺ regulation of the cytoplasmic streaming in characean cells was studied in relation to protein phosphorylation and dephosphorylation. A tonoplast-free cell model was developed which was sensitive to Ca²⁺. Protein phosphatase-1 and its inhibitor-1 were applied into the tonoplast-free cells. A synthetic inhibitor of protein phosphatase, -naphthylphosphate, was applied either to tonoplast-free cells from inside or to the outside of plasmalemma-permeabilized cells which are known to be very sensitive to Ca²⁺. ATP--S applied to permeabilized cells strongly inhibited the recovery of the streaming which had been stopped by 10 M Ca²⁺. Both inhibitor-l and -naphthylphosphate inhibited the streaming even in the absence of Ca²⁺. On the other hand, protein phosphatase-l recovered the streaming even in the presence of Ca²⁺.The results indicate that characean streaming is regulated by the phosphorylation state of a regulatory and/or motile protein component. Streaming is activated when the component is dephosphorylated and inactivated when the component is phosphorylated. Ca²⁺ is assumed to stimulate both phosphorylation and dephosphorylation of the component. Involvement of Ca²⁺/calmodulin in the streaming recovery was discussed in terms of the stimulation of dephosphorylation.Abbreviations ATP -S, Adenosine-5-O-(3-thiotriphosphate) - -NP -naphthylphosphate - EGTA ethylenglycol-bis-(-aminoethylether)N,N-tetraacetic acid - PIPES piperazine-N,N-bis(2-ethanesulfonic acid)     

Trois nouveaux Nadejdolepis Spasskii & Sasskaya, 1954 (Cestoda: Hymenolepididae) parasites de Charadrii (Aves) de Tasmanie

Three species of Nadejdolepis from Tasmania, Australia, are described and illustrated. N. burgessi n. sp., a parasite of Charadrius ruficapillus, is 4-6 mm long, with rostellar nitiduloid hooks 63-66 m long, a short evaginated cirrus 13-16 m long with a short collar of thin spines 1 m long, a narrow and tubular sclerotinoid vagina 40-50 long and 3-4 m in diameter with a little ampulla 3-5 m in diameter at the proximal end, and a membranous atrial segment with smooth, short (1 m) and compact spines which are sometimes difficult to observe. N. smithi n. sp., a parasite of 40-50 long and 3-4 m in diameter with a little ampulla 3-5 m in diameter at the proximal end, and a membranous atrial segment with smooth, short (1 m) and compact spines which are     

The Biometric Analysis of Bacterial Cells in Soil

The biometric analysis of bacterial cells in soil by light, fluorescence, and scanning electron microscopy showed that their average size is 0.8 m in diameter, 1.4 m in length, and 0.7 m³ in volume. In soil loci with enhanced microbiological activity (the rhizoplane of plants and the intestinal tract of soil invertebrates), the average size of bacterial cells was found to be 40% smaller than that of cells occurring in other parts of soil. This is the first experimental evidence showing that the metabolic activity of soil bacteria, their concentration, and allometric parameters are related.     

Two sites for adenine-nucleotide regulation of ATP-sensitive potassium channels in mouse pancreatic β-cells and HIT cells

Summary ATP-inhibited potassium channels (K(ATP)) were studied in excised, inside-out patches from cultured adult mouse pancreatic -cells and HIT cells. In the absence of ATP, ADP opened K(ATP) channels at concentrations as low as 10 m and as high as 500 m, with maximal activation between 10 and 100 m ADP in mouse -cell membrane patches. At concentrations greater than 500 m, ADP inhibited K(ATP) channels while 10 mm virtually abolished channel activity. HIT cell channels had a similar biphasic response to ADP except that more than 1 mm ADP was required for inhibition. The channel opening effect of ADP required magnesium while channel inhibition did not. Using creatine/creatine phosphate solutions with creatine phosphokinase to fix ATP and ADP concentrations, we found substantially different K(ATP)-channel activity with solutions having the same ATP/ADP ratio but different absolute total nucleotide levels. To account for ATP-ADP competition, we propose a new model of channel-nucleotide interactions with two kinds of ADP binding sites regulating the channel. One site specifically binds MgADP and increases channel opening. The other, the previously described ATP site, binds either ATP or ADP and decreases channel opening. This model very closely fits the ADP concentration-response curve and, when incorporated into a model of -cell membrane potential, increasing ADP in the 10 and 100 m range is predicted to compete very effectively with millimolar levels of ATP to hyperpolarize -cells.The results suggest that (i) K(ATP)-channel activity is not well predicted by the ATP/ADP ratio, and (ii) ADP is a plausible regulator of K(ATP) channels even if its free cytoplasmic concentration is in the 10–100 m range as suggested by biochemical studies.We would like to thank Mr. Louis Stamps for expert technical assistance and Dr. Wil Fujimoto and Ms. Jeanette Teague for generously providing HIT cells obtained from Dr. Robert Santerre at Eli Lilly. We would also like to thank Dr. Michel Vivaudou for providing the program ALEX. Support was provided by the NIH and the Department of Veterans Affairs.     

Plant regeneration via somatic embryogenesis in Styrian pumpkin: cytological and biochemical investigations

Somatic embryo formation was induced from cotyledon explants of Styrian pumpkin (Cucurbita pepo L. subsp. pepo var. styriaca Greb.) by using a solid MS medium supplemented with 16.11M NAA and 4.44M BA or 26.85M NAA and 13.32M BA. The callus proliferation was more efficient on medium supplemented with 26.85M NAA and 13.32M BA. In contrast, the embryogenic response was higher on medium with lower concentrations of growth regulators (16.11M NAA and 4.44M BA). The time needed for embryo induction did not depend on medium composition. Embryos in globular stage were transferred to three different maturation media, containing 2.89M GA₃ in combination with 0.54M NAA, 11.42M IAA and growth regulator-free medium. The germination rate was the highest when embryos were cultured on medium with 11.42M IAA. Plantlets grown on this medium achieved maturity suitable for transplantation into soil within 9 to 10weeks. The regenerated plants were successfully transferred into field and developed fertile flowers and set fruits. Biochemical analysis showed significant lower total glutathione levels among in vitro grown plantlets compared to seedlings grown in soil. When the plantlets were transferred into soil, they reached a normal size within a month and the glutathione concentration was comparable to seed-derived plants at the same developmental stage. Transmission electron microscopy was used to investigate possible differences in the ultrastructure of cells from callus cultures, and leaf cells of regenerated and seed-derived plants. Differences in the ultrastructure were found within chloroplasts which contained only single thylakoids, large starch grains and small plastoglobuli in callus cells in comparison to leaf cells, which possessed a well developed thylakoid system, small starch grains and large plastoglobuli.     

A novel technique for the study of bacterial cell mechanical properties

A micromanipulation method is described for measuring the bursting forces of bacteria and relating them to cell size. At a compression speed of 6.2 m s^–1, bursting forces of three samples of rapidly growing Staphylococcus epidermis from a batch culture varied from 3 to 34 N with an average value of 13.8 N (standard error 0.8 N). Escherichia coli grown in continuous culture at a specific growth rate of 0.5 h^–1 had bursting forces varying from 1 to 9 N with an average value of 3.6 N (standard error 0.4 N). In squeeze-hold experiments, force relaxation was observed, which was attributed to water loss from the cells, or viscoelasticity, or both. At high compression speed, such as 6.2 m s^–1, this relaxation could be neglected. Micromanipulation strength measurements might be used in studies of cell mechanical disruption and of the dependence of cell strength on cell physiology.     

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     

The pattern of innervation of a polyneural muscle: Axolotl iliotibialis

Summary The pattern of innervation on individual iliotibialis muscle fibres from axolotl (Ambystoma mexicanum) has been investigated histologically and elctrophysiologically. These polyneural fibres were found to be innervated on average at five end plate sites. The sites were distributed irregularly along each fibre. Average end plate length was found to be approxiamtely 70 m. Most end plates were separated by less than 1000 m; 26% by less than 150 m; the average separation was 516 m. Advantage was taken of the dual innervation of the muscle to investigate the separation between synaptic terminals from different axons. Some individual fibres were found to be innervated by axons from two different spinal nerves. End plate sites on dually innervated fibres were located by ACh iontophoresis. 30% of such sites were found to be innervated by more than one axon terminal. The average separation of such sites was found to be 950 m. Four different axons were found to innervate some individual muscle fibres. It is suggested that the unusual ability of axolotl muscle fibres to accept synaptic terminals from different axons at closely adjacent sites may be a major factor underlying selective reinnervation in this animal.Vacation student assisted by The Dale Fund of the Physiological Society     

Serum release of hepatic calcium-binding protein regucalcin by liver injury with galactosamine administration in rats

The incorporation and 9 desaturation of exogenous [¹⁴C]stearic acid were studied in HTC 7288c cells in suspension. We examined the uptake of the acid over a wide range of concentrations (0–160 M) after incubating the cells for 6 h in a chemically-defined medium. Under this experimental condition, the uptake of the labeled acid was more extensive than that obtained from static cultures or from monolayer of isolated hepatocytes of rats. At an external concentration of 160 Mca. 52 nmoles of acid per mg of cellular protein was taken up. The production of oleic acid from [¹⁴C]stearate (9 desaturation) correlated well with the uptake curve between 0–80 M concentration. For higher stearate concentrations, the biosynthesis of oleic acid declined substantially and a plateau of 22 nmoles/mg cellular protein was reached. The incorporation and desaturation of an initial exogeneous concentration of [¹⁴C]stearic acid (80 M) was also studied from 0–6 h. The results obtained demonstrated that the uptake of the substrate into cellular lipids was fast and non saturable. Quantitative gas-liquid chromatography of total cellular lipids under the different experimental conditions demonstrated a negative correlation between the decrease in the palmitic and palmitoleic acids and the increase in the intracellular levels of stearic and oleic acids. These analytical modifications took place with no changes in the saturated/monoenoic fatty acid ratio. This work also demonstrated a significant contribution of the stearoyl-CoA desaturase system to the high levels of oleic acid present in this kind of hepatoma cells.Abbreviations FAME Fatty Acid Methyl esters - GLC gas-liquid chromatography - HEPES N-2-hydroxyethyl-piperazine-N-2-ethanosulfonic acid - HTC Hepatoma Tissue Culture - IMEM-Zo Improved Minimal Essential Medium-zinc optional     

Micropropagation and ecorestoration of Vanda spathulata,an exquisite orchid

Node explants collected from flowering plants of Vanda spathulata, an endemic and exquisite orchid of Peninsular India and Sri Lanka, were cultured in Mitra medium with combinations of 4.4–88.8 m 6-benzyl adenine (BA) and 0.0–114.2 m indole-3-acetic acid (IAA). Combinations of 44.4 m BA with 17.1 or 28.5 m IAA and 66.6 mM BA with 28.5 or 40.0 m IAA induced maximum formation of 12.6 and 12.1 shoots / node, respectively, in a 6-month period. Subcultured nodal explants produced maximum of 6.1 shoots at combinations of 22.2–44.4 m 21 BA and 5.7–28.5 m IAA. Rooting of shoots occurred in medium containing 75 g l^–1 banana pulp and 5.7 m IAA within 3–9 weeks. Plantlets of 2–5 cm length possessing two to five roots established easily in community pots at 80–90% rates without hardening. Community potted plants introduced into forest segments at Ponmudi and Palode in Southern Western Ghats of India established at a rate of 50–70%.     

The effect of monoterpenes on astaxanthin production by a mutant ofPhaffia rhodozyma

Summary The total pigment and astaxanthin content ofPhaffia rhodozyma increased with increasing concentrations -pinene up to 500 l -pinene/l. Above this concentration the total pigment and astaxanthin content as well as the biomass production decreased. The addition of 500 l -pinene/l increased the total pigment content from 1652 g/g to 2201 g/g and the astaxanthin content from 1554 g/g to 1883 g/g. A sharp decrease in maximum specific growth rate occurred above 150 l -pinene/l.     

Growth and morphogenesis of immature embryos of sweet potato (Ipomoea batatas L.) in vitro

Sweet potato (Ipomoea batatas L.) embryos excised from the fertilized ovules of 6- to 12-day-old capsules were cultured on MS medium supplemented with NAA, BA, GA separately and in combinations. GA was found essential for initial morphogenesis of globular and heart stages. Seedlings were recovered from late globular stage onwards but recovery was best from advanced embryo stages. Differentiated embryos produced multiple shoots on MS medium +1M NAA÷2M BA +0.5M GA.     

Morphology and structural organization of spiracles in femaleArgas (Persicargas) walkerae (Acari: Argasidae)

Scanning electron microscopical investigations of fractures and corrosion casts of the spiracles from femaleA. walkerae ticks revealed a four-part structure, consisting of spiracular plate, ostium and macula forming the external closure, followed by the subostial space and the vestibulum of regulable volume, as well as the atrial chamber as the innermost part from which the main tracheal trunks originate. On the average, the spiracular plate was 158 m long and 188 m at the broadest width. It consisted of a thin, highly perforated external and a thick internal layer, which enclosed the interpedicellar space with numerous stout pedicels. In its posterior region, the spiracular plate was covered by the macula, which was up to 80 m in length and 110 m in width. The interpedicellar cavity opened into the subostial space measuring 95.5 m in length and 159.6m in width, which proceeded into the 112-m long vestibulum. The roof of the vestibulum was flexible and could be everted and inverted. Inverted, the roof formed a quadratic bulge with numerous deep cuticular folds, which confined the lumen of the vestibulum either partially or completely. In corrosion casts, the roof was everted to a length of up to 89.3 m. In the posterior part of the vestibulum, as well as in the initial fourth of the artrial chamber, numerous anvil-, cone-or drop-like cuticular projections were arranged in wedge-like fashion. The atrial chamber was almost spherical with a diameter of 138.4 m. Five main tracheal trunks of different luminal diameter as well as numerous channels opened into the atrial chamber.     

Oocysts of Isospora ernsti n. sp. and Isospora blagburni n. sp. (Apicomplexa: Eimeriidae) from the black-capped bulbul,Pycnonotus xanthopygos

Oocysts of Isospora ernsti n. sp. and Isospora blagburni n. sp. are described from the black-capped bulbul Pycnonotus xanthopygos from Lincoln Park Zoo, Chicago, Illinois. The bird came from southwestern Africa seven years earlier. I. ernsti oocysts are ellipsoidal to bluntly ovoid, 28–38 × 23–31m (mean 34 × 28 m) and have a single-layered oocyst wall. Micropyle, oocyst residuum and polar granules are absent. Sporocysts are elongate ovoid, 24–30 × 11–16 m (mean 27×13 m). Stieda and substiedal bodies and sporocyst residuum are present. I. blagburni oocysts are spherical to subspherical. 21–28 × 19–26 m (mean 25 × 23 m) and have a single oocyst wall. Sporocysts are ovoid and 17–23 × 10–13 m (mean 20 × 12 m). Stieda and substiedal bodies and sporocyst residuum are present.     

Flufenamic acid as an inducer of mitochondrial permeability transition

To assess the mechanism by which mitochondrial permeability transition (MPT) is induced by the nonpolar carboxylic acids, we investigated the effects of flufenamic acid (3-trifluoromethyl diphenylamine-2-carboxylic acid, FA) on mitochondrial respiration, electrical transmembrane potential difference (), osmotic swelling, Ca²⁺ efflux, NAD(P)H oxidation and reactive oxygen species (ROS) generation. Succinate-energized isolated rat liver mitochondria incubated in the absence or presence of 10 M Ca²⁺, 5 M ruthenium red (RR) or 1 M cyclosporin A (CsA) were used. The dose response-curves for both respiration release and dissipation were nearly linear, presenting an IC₅₀ of approximately 10 M and reaching saturation within 25-50 M, indicating that FA causes mitochondrial uncoupling by a protonophoric mechanism. Within this same concentration range FA showed the ability to induce MPT in energized mitochondria incubated with 10 M Ca²⁺, followed by dissipation and Ca²⁺ efflux, and even in deenergized mitochondria incubated with 0.5 mM Ca²⁺. ADP, Mg²⁺, trifluoperazine (TFP) and N-ethylmaleimide (NEM) reduced the extent of FA-promoted swelling in energized mitochondria by approximately one half, whereas dithiothreitol (DTT) slightly enhanced it. NAD(P)H oxidation and ROS generation (H₂O₂ production) by mitochondria were markedly stimulated by FA; these responses were partly prevented by CsA, suggesting that they may be implicated as both a cause and effect of FA-induced MPT. FA incubated with mitochondria under swelling assay conditions caused a decrease of approximately 40% in the content of protein thiol groups reacting with 5,5-dithiobis(2-nitrobenzoic acid) (DTNB). The present results are consistent with a ROS-intermediated sensitization of MPT by a direct or indirect FA interaction with inner mitochondrial membrane at a site which is in equilibrium with the NAD(P)H pool, namely thiol groups of integral membrane proteins.     

Ultrastructure of Sporothrix schenckii treated with iodine-potassium iodide solution

The ultrastructural changes produced by iodine-potassium iodide solution on yeast cells of Sporothrix schenckii were investigated by transmission electron microscopy in order to clarify the mechanism of oral potassium iodide therapy for sporotrichosis. Yeast cells were dipped with solutions containing various concentrations of iodine. The rate of germination decreased markedly between the range of iodine concentrations from 0.63 g/ml to 5.0 g/ml. No significant ultrastructural changes were seen at the concentration of the iodine of 1.25 g/ml (80% germination) or less. In the concentration of 2.5 g/ml (50% germination), normal cells and degenerated cells coexisted. When the cells were treated with 5.0 g of iodine per ml (0% germination) or more, their interior structures were completely destroyed. It is assumed that iodine treatment of the organism causes rapid destruction in the whole cell.     

In vitro analysis of ion channels in periaxolemmal-myelin and white matter clathrin coated vesicles: Modulation by calcium and GTPγS

This study reports the analysis of K⁺ channel activity in bovine periaxolemmal-myelin and white matter-derived clathrin-coated vesicles. Channel activity was evaluated by the fusion of membrane vesicles with phospholipid bilayers formed across a patch-clamp pipette. In periaxolemmal myelin spontaneous K⁺ channels were observed with amplitudes of 25–30, 45–55, and 80–100 pS, all of which exhibited mean open-times of 1–2 msec. The open state probability of the 50 pS channel in periaxolemmal-myelin was increased by 6-methyldihydro-pyran-2-one. Periaxolemmal-myelin K⁺ channel activity was regulated by Ca²⁺. Little or no change in activity was observed when Ca²⁺ was added to thecis side of the bilayer. Addition of 10 M total Ca²⁺ also resulted in little change in K⁺ channel activity. However, at 80 M total Ca²⁺ all K⁺ channel activity was suppressed along with the activation of a 100 pS Cl^– channel. The K⁺ channel activity in periaxolemmal myelin was also regulated through a G-protein. Addition of GTPS to thetrans side of the bilayer resulted in a restriction of activity to the 45–50 pS channel which was present at all holding potentials. Endocytic coated vesicles, form in part through G-protein mediated events; white matter coated vesicles were analyzed for G proteins and for K⁺ channel activity. These vesicles, which previous studies had shown are derived from periaxolemmal domains, were found to be enriched in the subunits of G₀, G_s, and G_i and the low molecular weight G protein,ras. As with periaxolemmal-myelin treated with GTPS, the vesicle membrane exhibited only the 50 pS channel. The channel was active at all holding potentials and had open times of 1–6 msec. Addition of GTPS to the bilayer fused with vesicle membrane appeared to suppress this channel activity at low voltages yet induced a hyperactive state at holding potentials of 45 mV or greater. The vesicle 50 pS K⁺ channel was also activated by the 6-methyl-dihydropyron-2-one (20 M).Abbreviations CNPase 2–3 cyclic nucleotide phosphohydrolase - EDTA ethylenediamine N,N,N,N-tetraacetic acid - G-protein GTP(guanosine triphosphate) binding protein - GTPS guanosine 5-O-(3-thiotriphosphate) - MAG myelin associated glycoprotein - Na⁺ K⁺ ATPase, Na⁺ and K⁺ stimulated adenosine triphosphatase - PLP myelin proteolipid protein Special issue dedicated to Dr. Majorie B. Lees.