Characteristics of the ATP-ase activity of isolated neurons of rabbit

Summary Isolated and homogenised Deiters' neurons from the lateral vestibular nucleus of rabbit in a Krebs-Ringer solution containing no added Mg⁺⁺, 1.3 moles/ ml and 5 moles/ml Mg⁺⁺, broke down ATP at the maximal rate of 0.29+-0.20, 2.40+–0.20, and 5.95+–0.63 moles/cell/hr. In 1.3 mM Mg⁺⁺ solution the single cell homogenates took up phosphate at the mean rate of 2.6+–0.2 moles/cell/hr. If the rabbits were injected 1 hour before with 20 mg/kg body weight of 2-amino-1-propene-1,1,3, tricarbonitrile (triap), the breakdown of ATP in these latter media was 0.82+–0.44, 2,5+–0.60, and 6.7+– 1.1 moles/cell/hr, respectively, and the quantity of inorganic liberated did not decrease.     

Organogenesis and somatic embryogenesis in callus cultures ofRosa hybrida andRosa chinensis minima

Pre-incubation of somatic tissues of the cut rose Carefree Beauty and miniature roses Red Sunblaze and Baby Katie in 10, 100, or 200 M 2,4-D induced the development of highly rhizogenic callus. Upon transfer of rhizogenic callus to a regeneration medium containing 23 M TDZ and 3 M GA₃, a low frequency of shoot organogenesis (3.3%) and somatic embryogenesis (6.6%) was observed. Incubation of leaf and stem internodes in 11, 27, 54, 81, or 108 M NAA followed by transfer of explants to the regeneration medium resulted in up to a 25% increase in shoot organogenesis from callus-derived internodal explants of Red Sunblaze, but no somatic embryogenesis was observed. The influence of glucose versus sucrose in the regeneration medium on leaf explants of Carefree Beauty and Baby Katie pre-incubated in 100 M 2,4-D revealed a difference in genotypic response to shoot organogenesis and somatic embryogenesis. For Carefree Beauty, a concentration of 111 mM glucose induced higher frequencies of both organogenic (33%) and embryogenic calluses (25%) than either 59 mM or 117 mM sucrose. For Baby Katie, no significant difference was found for number of organogenic calluses induced on 59 mM sucrose and 111 mM glucose.Abbreviations ABA Abscisic acid - BA Benzyladenine - 2,4-D 2,4-Dichlorophenoxyacetic acid - GA₃ Gibberellic acid - IAA Indoleacetic acid - MS Murashige & Skoog (1962) - NAA -Naphthaleneacetic acid - TDZ Thidiazuron     

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.     

Plantlet regeneration from callus initiated from flower buds in the wild species Allium senescens var. minor

Callus regeneration was observed from flower buds of Allium senescens var. minor inoculated in BDS, MS or B5 medium supplemented with 4.4 M benzyladenine alone or in combination with 4.5 M 2,4-dichlorophenoxy-acetic acid (2,4-d), with 2,4-d and kinetin (4.5 M/4.6 M) or with 5.3 M naphthaleneacetic acid. Ovules enlarged initially but the embryogenic tissue degenerated as callus development progressed from the nectar regions of the petals. Shoot buds and leaf primordia developed from the meristematic protuberances that originated from the surface of the callus. BDS medium with 4.5 M 2,4-d and 13.3 M BA was most suitable for shoot multiplication. The regenerated shoots were rooted in respective liquid medium without any growth regulators and successfully transferred to soil with 90% survival rate.Abbreviations BA N⁶-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid     

Modulation of brain mitochondrial membrane permeability and synaptosomal Ca2+ transport by dopamine oxidation

Effects of dopamine on the membrane permeability transition, thioredoxin reductase activity, production of free radicals and oxidation of sulfhydryl groups in brain mitochondria and the Ca²⁺ uptake by Na⁺-Ca²⁺ exchange and sulfhydryl oxidation in brain synaptosomes were examined. The brain mitochondrial swelling and the fall of transmembrane potential were altered by pretreatment of dopamine in a dose dependent manner. Depressive effect of dopamine on mitochondrial swelling was reversed by 10 g/ml catalase, and 10 mM DMSO. The activities of thioredoxin reductase in intact or disrupted mitochondria were decreased by dopamine (1-100 M), 25 M Zn²⁺ and 50 M Mn²⁺. Dopamine-inhibited enzyme activity was reversed by 10 g/ml SOD and 10 g/ml catalase. Pretreatment of dopamine decreased Ca²⁺ transport in synaptosomes, which was restored by 10 g/ml SOD and 10 mM DMSO. Dopamine (1-100 M) in the medium containing mitochondria produced superoxide anion and hydrogen peroxide, while its effect on nitrite production was very weak. The oxidation of sulfhydryl groups in mitochondria and synaptosomes were enhanced by dopamine with increasing incubation times. Results suggest that dopamine could modulate membrane permeability in mitochondria and calcium transport at nerve terminals, which may be ascribed to the action of free radicals and the loss of reduced sulfhydryl groups.     

Diurnal changes in proline content of desert plants

Summary The diurnal changes in proline and water contents of Zygophyllum quatarense and Francoeuria crispa were investigated under desert conditions. In both plants, the proline content was low before sunrise amounting to 10.42 moles/g fresh weight in Zygophyllum and 6.3 moles/g fresh weight in Francoeuria. By the progress of the day and the intensification of the evaporative power of the atmosphere, the proline content increased considerably in the two plants. The proline content reached maximal values at 5 p.m., reaching 23.36 moles/g in Zygophyllum and 11.16 moles/g in Francoeuria. The proline content of the artificially watered plants was kept at a low level throughout the day. The conditions and role of proline accumulation were discussed.     

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.     

Zur Beziehung zwischen Hautleistenverlauf und Morbus Wilson

Summary The content of free cystine was measured in cultures of fibroblasts from heterozygotes and children homozygous for the infantile type of cystinosis and from normal controls by a modified procedure. The values in 4 homozygotes ranged between 4.2–8.0 moles 1/2 cystine/g protein; those in 7 heterozygotes between 0.2–0.7 moles 1/2 cystine/g protein; and those in the 5 normal controls between less than 0.1–0.25 moles 1/2 cystine/g protein. This means that in the individual case normals and heterozygotes cannot be differentiated excactly in the lower heterozygote range. This fact should be taken into account in genetic counseling.

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Zusammenfassung In kultivierten Fibroblasten von Heterozygoten und Homozygoten für Cystinose vom frühkindlichen Typ und von Normalpersonen wurde der Gehalt an freiem Cystin mit einer methodisch modifizierten Aufarbeitung gemessen. Die Werte für 4 untersuchte Homozygote liegen im Bereich von 4,2–8,0 Mol 1/2 Cystin/g Protein, die von 7 Heterozygoten zwischen 0,2–0,7 Mol und die von 5 Normalkontrollen zwischen weniger als 0,1–0,25 Mol 1/2 Cystin/g Protein. Während die Gesamtgruppe der Heterozygoten signifikant über der der Normalpersonen liegt, ist im Einzelfall in einem Grenzbereich eine klare Unterscheidung zwischen Normalen und Heterozygoten nicht möglich. Bei der Familienberatung muß dies berücksichtigt werden.

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Supported by the Deutsche Forschungsgemeinschaft (SFB 35).     

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     

Use of mitochondrial inhibitors to differentiate kinetic properties of the ATP-dependent Ca2+ uptake system in synaptic membranes

Synaptosomal membranes accumulate 3–6 times more Ca²⁺ in the presence of ATP (50–1000 M) than basal Ca²⁺ accumulation (-ATP). The location of this Ca²⁺ accumulation appears to reside on the cytosolic face of the synaptosome since lysed synaptosomes accumulate 4-times more Ca²⁺ than intact synaptosomes. The inclusion of mitochondrial inhibitors, oligomycin (0.7 g/ml), sodium azide (100 M) and dinitrophenol (100 M) differentiate mitochondrial from nonmitochondrial Ca²⁺ accumulation under conditions that are [Ca²⁺]- and ATP-dependent. In the presence of low concentrations of ATP (<150 M) and Ca _free ²⁺ (2.5 or 6.8 M), Ca²⁺ accumulation occurs as one process in both lysed synaptosomal membranes and purified synaptic plasma membranes in the presence and/or absence of MI. When ATP levels are increased (>200 M), the Ca²⁺ accumulation process remains independent of the presence of mitochondrial inhibitors when Ca _free ²⁺ =2.5 M. When Ca _free ²⁺ is increased to 6.8 M, mitochondrial inhibitors differentiate mitochondrial from nonmitochondrial accumulation. These studies suggest that optimal conditions for the measurement of Ca²⁺ accumulating mechanisms in synaptosomal membranes depend on both [Ca²⁺] and ATP. Use of these assay conditions provide evidence that ATP-dependent Ca²⁺ uptake may be a viable mechanism for the regulation of synaptosomal Ca²⁺ levels.     

Effects of Anion Channel Blockers on Hyposmotically Induced Amino Acid Release From the In Vivo Rat Cerebral Cortex

A cortical cup model with continuous perfusion of artificial cerebrospinal fluid (containing 134 mM NaCl) was used to investigate the effects of anion channel blockers on the hyposmotically-induced release of amino acids from the in vivo rat cerebral cortex. The hyposmotic stimulus (25 mM NaCl) evoked a release of taurine, glutamate, aspartate, glycine, phosphoethanolamine and GABA. Topically applied anion channel blockers 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (1 mM); 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (2 mM); 5-nitro-2-(3-phenylpropylamino) benzoic acid (350 M); niflumic acid (500 M); tamoxifen (20 M) and arachidonic acid (0.5 M) all significantly reduced the hyposmotically-induced release of taurine. The releases of glutamate, aspartate, glycine, phosphoethanolamine and GABA were variably susceptible to inhibition by these compounds. These results demonstrate that osmoregulatory processes in cortical cells, in vivo, involve amino acids, with taurine playing a dominant role. The efflux of taurine and, to a lesser extent, the other amino acids may be mediated by anion channels.     

Micropropagation of Hemidesmus indicus (L.) R. Br. through axillary bud culture

A procedure for rapid in vitro propagation of the aromatic and medicinal plant Hemidesmus indicus (L.) R.Br. (Family Asclepiadaceae) from nodal explants is described. The highest shoot multiplication rate of 8.2 ± 0.4 shoots/explant with a 95% frequency was achieved in S weeks culture period on Murashige and Skoog medium supplemented with 1.15 M kinetin and 0.054 M -naphthaleneacetic acid. Excised shoots were rooted on the same basal medium supplemented with 1.15 M kinetin and 7.35 M indole-3-butyric acid. Shoots derived from subcultures exhibited better rooting response than those from primary cultures. After a hardening phase of 2 weeks, there was a 70% transplantation success in the field.Abbreviations MS Murashige and Skoog (1962) medium - BA N⁶ benzyladenine; KN kinetin - NAA a-naphthaleneacetic acid - IBA indole-3-butyric acid - IAA indole-3-acetic acid     

Gibberellin-like activity in suspensors of Tropaeolum majus L. and Cytisus laburnum L.

Gibberellins in the embryo-suspensor system have been considered so far only in Phaseolus coccineus. We present in this report the localization of gibberellin-like substances in the suspensors of Tropaeolum majus L. and Cytisus laburnum L. The total gibberellin activity (expressed as gibberellic-acid equivalent in the -amylase bioassay) in 2000 suspensors (106 mg fresh weight; FW) of C. laburnum and in 600 suspensors (236 mg FW) of T. majus were 50.9 g g^-1 FW and 8.9 g g^-1 FW respectively.Abbreviation GA gibberellin     

Effects of feeding of β-carotene-supplemented rotifers on survival and lymphocyte proliferation reaction of fish larvae (Japanese parrotfish ( Oplegnathus fasciatus) and Spotted parrotfish(Oplegnathus punctatus)): preliminary trials

Japanese parrotfish (Oplegnathus fasciatus) and Spotted parrotfish(Oplegnathus punctatus) larvae were fed with -carotene supplementedrotifers or unsupplemented control rotifers for 24 days after hatchout.Results show that survival rates of -carotene supplemented groups ofboth Japanese parrotfish and Spotted parrotfish larvae were higher than thatof control groups. -carotene supplemented and unsupplemented controlgroups exhibited similar growth in both species during this experiment.Proliferation of spleen lymphocytes with 100g ml^–1 ofConA, 10 or 50g ml^–1 of Poke weed mitogen from-carotene supplemented group was higher than that of a control group ofJapanese parrotfish. In Spotted parrotfish treated with 100gml^–1 of ConA from the -carotene supplemented groupslymphocytes proliferated to a higher degree than in the control. Resultssuggest that the supplementation of -carotene to rotifers, might be ofbenefit in production of healthy, resistive larvae against infectiousdisease.     

Callus induction and plant regeneration in Vetiveria zizanioides

Callus induction was obtained from basal parts of Vetiveria zizanioides Stapf. leaves cultured on Murashige and Skoog (MS) medium supplemented with 9.0 M 2,4-dichlorophenoxyacetic acid (2,4-d), 5.7 M indoleacetic acid (IAA) and 4.6 M kinetin. Calli were maintained on MS medium with the addition of 0.9 M 2,4-d and 2.3 M kinetin. Shoot formation was obtained from fast growing 14-day-old callus on the same basal medium supplemented with 0.9 M 2,4-d and 9.3 M kinetin. Embryo-like structures were observed. When transferred to basal medium, shoots readily developed roots. Fully developed regenerated plants were then successfully established in soil.     

Anti-Auxin Enhance Rosa Hybrida L. Micropropagation

Shoots of rose cultivars Super Star and Sonia were multiplied for ten subcultures at 4-week intervals on solidified Murashige and Skoog's medium supplemented with 22.19 M benzylaminopurine + 1.07 M napthalene acetic acid + 0.05 M gibberellic acid. Addition of anti-auxins 2,3,5-triiodobenzoic acid (TIBA; 2.0 M) and 2,4,6-trichlorophenoxy-acetic acid (2,4,6-T; 0.39 M) into proliferation medium increased number of shoots per explant and length of shoots in both cultivars. Treatment with TIBA increased also number of leaves per shoot and leaf chlorophyll content.     

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%.     

Nicotinic receptor-elicited sodium flux in rat pheochromocytoma PC12 cells: Effects of agonists,antagonists, and noncompetitive blockers

Nicotinic agonists stimulate²²Na flux in rat pheochromocytoma PC12 cells. The stimulatory effect of carbamylcholine is maximal at 1 mM, while the stimulatory effect of nicotine and anatoxin maximize at the same level at 100 M and 10 M, respectively. The tertiary amines arecolone and isoarecolone have no effect on flux at 100 M, while the methiodides at 100 M stimulate flux to an extent similar to 1 mM carbamylcholine. Dihydro and alcohol analogues of isoarecolone methiodide have markedly smaller effects on flux. A preincubation for 2 to 20 min with carbamylcholine (2 mM), nicotine (300 M), anatoxin (30 M) or isoarecolone methiodide (100 M) causes marked desensitization to a subsequent carbamylcholine-elicited stimulation of flux. d-Tubocurarine, mecamylamine, hexamethonium, and chlorisondamine inhibit carbamylcholine-elicited flux with IC₅₀ values of 1.0, 0.8, 43, and 0.020 M, respectively. Atropine has no effect at 1 M, but reduces the response to carbamylcholine by 50% at 8.6 M, presumably as a noncompetitive blocker. Other noncompetitive blockers of nicotinic acetylcholine-receptors, such as histrionicotoxins, gephyrotoxin, pumiliotoxin C, phencyclidine, bupivacaine and piperocaine, inhibit carbamylcholine-elicited stimulation of²²Na flux with IC₅₀ values from 0.3 to 1.8 M. In contrast to d-tubocurarine, which inhibits carbamylcholine-elicited desensitization, and mecamylamine, which has no apparent effect on desensitization, chlorisondamine and certain noncompetitive blockers appear to enhance desensitization. The effects of agonists, antagonists and noncompetitive blockers at the neuronal nicotinic acetylcholine receptor-channel of PC12 cells are compared to their effects on binding of [¹²⁵I]-bungarotoxin to agonist-recognition sites and of [³H]perhydrohistrionicotoxin to noncompetitive blocker sites of the nicotinic acetylcholine receptor-channel of electric ray (Torpedo) electroplax membranes. There are marked differences in relative potencies for the two types of nicotinic acetylcholine receptor-channel.     

The effects of triethyl lead on the development of hippocampal neurons in culture

Triethyl lead is the major metabolite of tetraethyl lead, which is used in industrial processes and as an antiknock additive to gasoline. We tested the hypothesis that low levels of triethyl lead (0.1 nmol/L to 5mol/L) interfere with the normal development of cultured E18 rat hippocampal neurons, possibly through increases in intracellular free calcium ion concentration, [Ca²⁺]_in. The study assessed survival and differentiation using morphometric analysis of individual neurons. We also looked at short-term (up to 3.75-h) changes in intracellular calcium using the calcium-sensitive dye fura-2. Survival of neurons was significantly reduced at 5 mol/L, and overall production of neurites was reduced at 2 mol/L. The length of axons and the number of axons and dendrites were reduced at 1 mol/L. Neurite branching was inhibited at 10 nmol/L for dendrites and 100 nmol/L for axons. Increases in intracellular calcium were observed during a 3.75-h exposure of newly plated neurons to 5 mol/L triethyl lead. These increases were prevented by BAPTA-AM; which clamps [Ca²⁺]_in at about 100 nmol/L. Culturing neurons with BAPTA-AM and 5 mol/L triethyl lead did not reverse the effects of triethyl lead, suggesting that elevation of [Ca²⁺]_in is not responsible for decreases in survival and neurite production. Triethyl lead has been shown to disrupt cytoskeletal elements, particularly neurofilaments, at very low levels, suggesting a possible mechanism for its inhibition of neurite branching at nanomolar concentrations.Abbreviations BAPTA-AM 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid acetoxymethyl ester - [Ca²⁺]_in intracellular free calcium ion concentration - DMSO dimethyl sulfoxide - E18 embryonic day 18 - FBS fetal bovine serum - fura-2AM fura-2 acetoxymethyl ester - HBSS Hanks' Balanced Salt Solution - MEM Eagle's Minimum Essential Medium     

Effect of the insecticides methylpyrimifos and chlorpyrifos on soil microflora in an agricultural loam

A study of the effects of two selected organophosphorus insecticides methylpyrimifos and chlorpyrifos on soil microflora in an agricultural loam was made. The insecticides had concentrations of 10 to 300 g g^–1. The presence of methylpyrimifos at concentrations of 100 to 300 g g^–1 or chlorpyrifos at concentrations from 10 to 300 g g^–1 significantly decreased aerobic dinitrogen fixing bacteria and dinitrogen fixation. Nitrifying bacteria decreased at concentrations of 200 and 300 g g^–1 of methylpyrimifos. The presence of 10 to 300 g g^–1 of chlorpyrifos decreased the total number of bacteria. However, fungal populations and denitrifying bacteria were not affected as a consequence of the addition of the organophosphorus insecticides to the agricultural soil, showing that these microorganisms can tolerate high amounts of those insecticides.