Experimental studies upon a bundle of tonic fibres in the locust extensor tibialis muscle

The bundle of tonic fibres situated at the proximal end of the locust metathoracic extensor tibialis muscle is innervated by the dorsal unpaired median neurone (DUMETi) as well as by the slow excitatory (SETi)) and common inhibitor (CI) neurones. It is not innervated by the fast excitatory neurone (FETi).These fibres contract spontaneously and rhythmically. The myogenic rhythm can be modified by neural stimulation.Spontaneous slow depolarizing potentials resembling the pacemaker potentials of insect cardiac muscle were demonstrated in these fibres.The actions of glutamate on the tonic muscle fibres are not compatible with its being a specific excitatory transmitter. Glutamate can stimulate weak contractions of the muscle, but this action is inhibited when chloride ions are removed from the saline.10^?6 M Octapamine hyperpolarizes the tonic fibre membrane. Octopamine, GABA and glutamate all inhibit the myogenic contractions and reduce the force of the neurally evoked contractions.The tonic muscle is very responsive to proctolin. At 5 × 10^?11 M proctolin enhances the force and increases the frequency of myogenic contractions. At 10^?9 M it depolarizes the muscle membrane potential, and at that and higher concentrations it causes the muscle to contract. At 2 × 10^?7 M proctolin induces contractures which resemble those evoked by sustained high-frequency neural stimulation. Iontophoretic experiments show that proctolin receptors occur at localized sites on the tonic fibre membrane.     

A peptide action in a lobster neuromuscular preparation

The neuropeptide proctolin causes a sustained contraction of the opener muscle of the dactyl of the lobster walking leg. This substance acts directly on the muscle at concentrations as low as 10^?10M. The contraction is dependent on extracellular calcium. Neither a significant depolarization nor a detectable change in the input resistance accompanies the response. No presynaptic action of proctolin is indicated; excitatory and inhibitory junctional potential sizes and the frequency of spontaneous miniature excitatory junctional potentials are unaffected.     

Inhibiton and gamma-aminobutyric acid-induced conductance on locust (Schistocerca gregaria) extensor tibiae muscle fibres

• 1.1. Increases in membrane conductance (g_m) were induced by GABA in distal bundles 32, 33 and 34 of extensor tibiae muscles of the locust (Schistocerca gregaria).
• 2.2. Bath application of GABA (10⁻⁵−5 × 10⁻³ M) induced reductions in muscle fibre space constant (λ).
• 3.3. GABA (5 × 10⁻³ M) induced additional membrane conductance of 2.21 ± 0.03 × 10⁻⁶ S/mm, 0.38 ± 0.03 × 10⁻⁶ S/mm and 0.29 ± 0.06 × 10⁻⁶ S/mm on muscle bundles 34, 33 and 32 respectively. The greater sensitivity of muscle fibres in bundle 34 to GABA is due at least in part to a larger number of GABA receptors on bundle 34 muscle fibres.
• 4.4. The decrement of electrotonic potentials in the presence of GABA were measured over distances of both half fibre length and whole fibre length. Good agreement was obtained between changes in space constant produced by GABA using half fibre length and whole fibre length data.
• 5.5. By taking into account changes in space constant induced by GABA it was possible to demonstrate that presynaptic GABA receptors were involved in the inhibition of slow excitatory postsynaptic potentials by GABA.
• 6.6. “Slow” excitatory postsynaptic potentials recorded under current clamp were inhibited in a dose-dependent manner by GABA. This inhibition was not dependent on muscle-fibre GABA sensitivity and could not be completely accounted for by GABA-induced changes in the cable properties of the muscle fibres.

     

Alcohol-induced potentiation of the neurally evoked contractions of the retractor unguis muscle of the locust, Schistocerca gregaria

Each of a series of seven monohydroxyl alcohols caused an increase in the force of the neurally evoked contractions of the innervated retractor unguis muscle isolated from the metathoracic femurs of the locust, Schistocerca gregaria. The negative logarithm of equipotent concentrations of the alcohols was proportional to the logarithm of the partition coefficient (1-octanol/water) of the alcohols: $\text{log}\text{1}\text{C}\text{= 1·282}\text{log}\text{P+1·684}$, where C is concentration, and P is the partition coefficient.Ethylene glycol and glycerol did not potentiate the contractions, possibly due to their very low lipophilic character. Acetone (10^?2 M) and benzene (10^?5 M) also potentiated the neurally evoked contractions of the insect muscle.     

The mechanism of block of glutamate synapses by dipicolinic acid

The effect of dipicolinic acid (2,6-pyridine dicarboxylic acid) on the mealworm neuromuscular junction was studied using conventional microelectrode recording techniques. Dipicolinic acid (10^?5-10^?3 M) added to the bathing solution reversibly blocked neuromuscular transmission. The depolarization in response to iontophoretically applied L-glutamate (glutamate potential) was not affected by dipicolinic acid even when the neurally evoked excitatory postsynaptic potential (EPSP) was totally abolished. Focal extracellular recordings from single synaptic sites revealed that in the presence of 1 x 10^?4 M dipicolinic acid the presynaptic spike was unchanged, but the quantal content for evoked transmitter release was reduced. The calcium-dependent action potential elicited by direct stimulation of the muscle fiber was not impaired by dipicolinic acid. These results suggest that dipicolinic acid interferes with the transmitter-releasing mechanism from the presynaptic terminal.     

Proctolin: a peptide transmitter candidate in insects.

The slow, striated muscles of the proctodeum (hindgut) of the cockroach, $\text{Periplaneta americana}$ (L.), were examined pharmacologically with reference to the responses evoked by nerve stimulation, glutamate, 5-HT, and proctolin, a myotropic peptide from $\text{Periplaneta}$ recently isolated and identified. The graded contractions evoked by repetitive nerve stimulation were simulated by 5-HT and proctolin at threshold concentrations of about 10⁻⁷ and 10⁻⁹ M respectively; responses to glutamate (∼10⁻⁴ M) were not similarly graded. The 5-HT receptors are distinct from other receptors, including the post-synaptic receptors, since they were specifically blocked by bromolysergic acid diethylamide. Proctolin was fully active on TTX-treated or surgically denervated muscle indicating that the proctolin receptors are located on the muscle fibre membrane. Tyramine, at threshold levels 5×10⁻⁸ M, reversibly antagonized the responses evoked by proctolin and by nerve stimulation but was without effect on the 5-HT and glutamate responses. Neurally evoked responses were potentiated by subthreshold concentrations of proctolin but not by glutamate. Pharmacologically, the proctolin and post-synaptic receptors appear to be identical and distinct from the glutamate and 5-HT receptors. Since proctolin is known to be a constituent of an efferent pathway of the proctodeal nerves, the evidence suggests that it may function as an excitatory transmitter substance. Peptidergic transmission is discussed in relation to the ultrastructural organization of the proctodeal nerve terminals which contain neurosectory granules in addition to electron-lucent, synaptic vesicles.     

Modulation of transmitter release from the locust forewing stretch receptor neuron by GABAergic interneurons activated via muscarinic receptors

The role of muscarinic receptors in the down‐regulation of acetylcholine (ACh) release from the locust forewing stretch receptor neuron (fSR) terminals has been investigated. Electrical stimulation of the fSR evokes monosynaptic excitatory postsynaptic potentials (EPSPs) in the first basalar motoneuron (BA1), produced mainly by the activation of postsynaptic nicotinic cholinergic receptors. The general muscarinic antagonists scopolamine (10⁻⁶ M) and atropine (10⁻⁸ to 10⁻⁶ M) caused a reversible increase in the amplitude of electrically evoked EPSPs. However, scopolamine (10⁻⁶ M) caused a slight depression in the amplitude of responses to ACh pressure‐applied to the soma of BA1. These observations indicate that the EPSP amplitude enhancement is due to the blockade of muscarinic receptors on neurons presynaptic to BA1. The muscarinic receptors may be located on the fSR itself and act as autoreceptors, and/or they may be located on GABAergic interneurons which inhibit ACh release from the fSR. Electron microscopical immunocytochemistry has revealed that GABA‐immunoreactive neurons make presynaptic inputs to the fSR. The GABA antagonist picrotoxin (10⁻⁶ M) caused a reversible increase in the EPSP amplitude, which does not appear to be due to an increase in sensitivity of BA1 to ACh, as picrotoxin (10⁻⁶ M) slightly decreased ACh responses recorded from BA1. Application of scopolamine (10⁻⁶ M) to a preparation preincubated with picrotoxin did not cause the EPSP amplitude enhancement normally seen in control experiments; in fact, it caused a slight depression. This indicates that at least some of the presynaptic muscarinic receptors are located on GABAergic interneurons that modulate transmission at the fSR/BA1 synapse. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 420–431, 1999     

Protoplast isolation optimization and regeneration of cell wall in Gracilaria gracilis (Gracilariales, Rhodophyta)

This paper reports the first successful isolation and cell wall regeneration of Gracilaria gracilis (Stackhouse) Steentoft, Irvine et Farnham protoplasts. These results form an important foundation for the development of a successful tissue culture system for G. gracilis. Initially, an isolation protocol was optimized by investigation of the effects of the enzyme constituents and concentrations, the pre-treatment of thalli, the incubation period and temperature, and the pH of the enzymatic medium on protoplast yields. A pre-treatment of G. gracilis thalli with 1 % (w/v) papain for 30 min followed by a 3-h enzymatic digestion of thalli with an enzymatic mixture containing 2 % (w/v) cellulase Onozuka R-10, 1 % (w/v) macerozyme R-10, and 10 U mL^?1 agarase at pH 6.15 was found to produce the highest yield of protoplasts at 22 °C. Reliably high yields (20–30?×?10⁵ protoplasts g^?1 f.wt) of protoplasts could be obtained from G. gracilis thalli when this optimized protocol was used. Cell wall re-synthesis by G. gracilis protoplasts, which constitutes the first step towards whole plant regeneration, was followed using calcoflour staining and scanning electron microscopy. Protoplasts were shown to complete the initial stages of cell wall re-synthesis within the first 24 h of culturing.     

L-glutamate as an excitatory transmitter at the neuromuscular junction of a beetle larva

The effects of L-glutamate and acetylcholine on the ventral muscle fibres of the larval mealworm Tenebrio molitor were studied by means of microelectrodes. Bath application of L-glutamate at concentrations higher than 1 × 10 ⁴M suppressed excitatory postsynaptic potentials (EPSPs) and evoked both a depolarisation and a reduction in the input resistance of the muscle fibre. In contrast, acetylcholine chloride (up to 1 mM) had no effect at all. Circumscribed spots could be detected on the fibre surface where iontophoretic applications of L-glutamate caused transient depolarizations (glutamate potentials). Focal extracellular recordings revealed that the glutamate sensitive spots were identical with synaptic sites. The reversal potentials of the EPSP and the L-glutamate potential were identical. These results are compatible with the hypothesis that L-glutamate is an excitatory transmitter at the neuromuscular junction.     

Ionic mechanism of 5-hydroxytryptamine induced hyperpolarization and inhibitory junctional potential in body wall muscle cells of Hirudo medicinalis

Five-hydroxy tryptamine (5-HT) causes a hyperpolarization and increased conductance of the leech body wall muscle cell membrane. If 5-HT is applied in the absence of the Cl^?ion, the response appears as a depolarization, whereas if 5-HT is applied in the absence of the K⁺ion, the response is a hyperpolarization. In both cases, the conductance of the muscle cell membrane is increased. Stimulation of the peripheral nerve to the body wall muscle produces a complex junctional potential in muscle cells. Exposing the muscle to d-tubocurarine (d-TC) eliminates the excitatory component (EJP) of the complex potential. The inhibitory potential (IJP) that remains has an equilibrium potential at approximately 65 m V. Furthermore, this IJP appears as a depolarization when the nerve is stimulated in the presence of d-TC and low CL^?, whereas this is not the case if the nerve is stimulated in the presence of d-TC and low K⁺. The drugs BOL-148 and cyproheptadine block the IJP's in the body wall muscle. These data are interpreted as indicating that 5'HT acts on leech body wall muscle cells by increasing the conductance to the Cl^?ion and that the IJP's caused by nerve stimulation are probably the result of 5-HT release at nerve terminals. As a final point, it has been shown that the inhibition by 5-HT of the spontaneous EJP's that occur on the leech body wall muscle results from an inhibition of central neurons and not from any direct effect on the muscle cell or on peripheral synapses.     

On the effect of the ionophore X-537A on neuromuscular transmission in the rat

In the rat phrenic nerve-diaphragm muscle preparation, X-537A at 6×10^?6 to 3×10^?5 M (1) depolarized muscle fibre membranes, (2) caused an occasional transient increase in and ultimate block of spontaneous transmitter release, (3) did not increase the amplitude of the end-plate potential (epp) but abruptly blocked stimulus-evoked transmitter release, and (4) produced an increase in the occurrence of “giant” miniature epp's (mepp's). The possibility is discussed that the sporadically raised mepp frequency was due to an ionophore-induced depolarization of nerve terminals. The increased occurrence of “giant” mepp's apparently reflected a X-537A-induced spontaneous multiquantal release of acetylcholine. This was not dependent on extracellular calcium but appeared to be of presynaptic origin.     

Modulation of transmitter release from the terminals of the locust wing stretch receptor neuron by muscarinic antagonists

The forewing stretch receptor (SR) neuron makes monosynaptic connections with wing depressor motoneruons; in this article the pharmacology of its output onto the first baslar motoneuron (BA1) has been investigated. The SR, like other insect afferents that have been studied so far, appears to be cholinergic; transmission was suppressed reversibly by the nicotinic antagonist gallamine (10^?4M) and irreversibly by α-bungarotoxin (10^?6 M). The choline reuptake blocker hemicholinium-3 (10^?4 M) also caused a reversible reduction in the amplitude of SR excitatory postsynaptic potentials (EPSPs) recorded in BA1. The receptor subtype nonselective muscarinic antagonists atropine (10^?4 M), scopolamine (10^?4 M), and quinuclidinyl benzilate (10^?5 M), unlike nicotinic antagonists, caused an augmentation in EPSP amplitude. This effect does not appear to be caused by an increase in sensitivity of the motoneuron to acetylcholine (ACh), since atropine produced a marked reduction rather than an increase in the amplitude of responses to ACh pressure applied to the soma of BA1. Scopolamine only caused a modest reduction in the amplitude of ACh somatic responses. The simplest explanation for these observations is that muscarinic antagonists bring about an increase in EPSP amplitude by blockade of presynaptic autoreceptors that normally down-regulate the release of ACh from SR terminals. The effects of muscarinic receptor subtype-selective antagonists indicate that presynaptic receptors in this preparation may have a pharmacological profile more similar to that of vertebrate M₂ receptors than to that of M₁ or M₂ subtypes. The functional significance of autoreceptors in this preparation are discussed. © 1995 John Wiley & Sons, Inc.     

Electrical properties and transmitter output associated with growth and transformation in shrimp muscle fibers

Summary Comparisons were made of the passive electrical properties of closer muscle fibers in the dimorphic claws of snapping shrimp,Alpheus armillatus. During claw transformation the small fibers of pincer claws grow to become much larger snapper claw fibers. As muscle fibers grow, the relationship of fiber input resistance (R ₀) to fiber diameter (d) is predicted by the proportionality,R ₀d ^–3/2. Muscle fiber membrane resistance,R _m, is independent of fiber diameter, but membrane capacitance,C _m, grows with diameter. This results in a 40 to 50 fold reduction in fiber input impedance as fiber diameter enlarges during transformation. Reductions of muscle fiber impedance are partially compensated by 2–5 fold increases in quantal content at excitatory synapses on snapper muscle fibers. However, changes in quantal content during transformation apparently are independent of fiber diameter per se. Excitatory junction potentials in both pincer and snapper muscle fibers have equal amplitude. Because fiber input impedance decreases precipitously during transformation, and in view of the relatively small compensatory changes in quantal content at excitatory synapses, additional pre- or post-synaptic modifications must supplement increased quantal content to maintain synaptic efficacy in transformed muscle fibers.Abbreviations ejp excitatory junctional potential - epp endplate potential - mepp miniature endplate potential     

灭多威对美洲大蠊腹六神经节突触传递的阻断作用

用电生理学方法研究了灭多威对美洲大蠊Periplanetaamerwana腹六神经节(A6节)突触传递的影响。用灭多威溶液浸泡A6节,电刺激尾须神经粗支,用甘露醇间隙法记录兴奋性突触后电位(EPSP)和突触后动作电位。给予弱刺激只记录到EPSP时,灭多威作用初期EPSP幅度增加、时程延长,能诱发突触后动作电位,随后EPSP逐渐减小至消失,冲洗可恢复,突触前反应保持不变。增加电刺激强度记录到突触后动作电位时,灭多威可阻断A6节的突触传递,阻断时间是浓度依赖性的,阻断是可逆的,但冲洗30 min仍保留一定的后作用。对美洲大蠊雄性成虫腹腔注射灭多威测定致死中量(LD₅₀)为(3.56±0.01) μg/g体重。根据灭多威的作用机理对其阻断A6节突触传递的特点以及对虫体的毒杀机制进行了讨论。     

Callus and Cell Suspension Cultures of Carnation

Callus cultures of carnation, Dianthus caryophyllus L. ev. G. J. Sim, were grown on a synthetic medium of half strength Murashige and Skoog salts, 3 % sucrose, 100 mg/l of myo-inositol, 0.5 mg/l each of thiamin, HCl, pyridoxin, HCl and nicotinic acid and 10 g/l agar. Optimal concentrations of growth regulators were observed to be 3 × 10^?6M indoleacetic acid (JAA) combined with 3 × 10^?6M benzylaminopurin (BAP) or 10^?6M 2,4-dichlorophenoxy acetic acid (2,4-D) alone. IAA + BAP caused a 100 fold increase in fresh weight over 4 weeks at 25°C. Addition of casein hydrolysate increased growth further. Cell suspension cultures worked best in media containing 2,4-D in which they had a doubling time of about 2 days. Filtered suspensions were successfully plated on agar in petri dishes, but division was never observed in single cells. The cultures initiated roots at higher concentrations of IAA or NAA, but all attempts to induce formation of shoots or em-bryoids gave negative results.     

Feasibility of SRC Species for Growing in Mediterranean Conditions

The suitability of poplar (Populus × canadensis Moench.—genotype ‘Neva’), black locust (Robinia pseudoacacia L.), and eucalyptus (Eucalyptus bridgesiana R. Baker) growing in short rotation coppice (SRC) system in a Mediterranean area (southern Italy), and under two management regimes, was evaluated in terms of survival, biomass yield, biomass quality, and soil fertility. The high management regime (H treatment) consisted of high plant density (6667 trees ha^?1) and a 2-year harvesting cycle; the low management regime (L treatment) consisted of low plant density (1667 trees ha^?1) and a 4-year harvesting cycle. The dry biomass production was 36, 13, and 9 t dry matter (dm)?ha^?1 in the H treatment and 25, 14, and 7 t dm ha^?1 in the L treatment for eucalyptus, black locust, and poplar, respectively. The analysis of the biomass showed a superior quality for the black locust feedstock because of its low moisture and ash percentages, high heating value (HHV), and low alkali metal concentrations, although, from an environmental point of view, the high N (12.3 g kg^?1) and S (0.7 g kg^?1) biomass concentrations would increase the pollutant emissions generated by combustion. Eucalyptus showed a high HHV, especially for the H treatment (18.70 MJ kg^?1). Its high concentrations of K (4 g kg^?1) and Mg (0.8 g kg^?1) could provoke slagging and fouling in combustion equipment, and the high concentrations of S and N, if leaves are considered in the harvested biomass, indicate the low quality of its feedstock. No specific poplar feedstock stood out, although it had a good HHV (19.02 MJ kg^?1). The soil fertility was not affected negatively after the 4-year SRC cycle, while S content in soil showed a tendency to increase in the case of black locust cultivation.     

Myogenic contractions in locust muscle induced by proctolin and by wasp, Philanthus triangulum, venom

Apparently myogenic slow contractions of the extensor tibiae of Locusta migratoria can be induced by proctolin in a concentration of 10^?10 to 10^?9 mole per liter perfusion fluid. Proctolin in a concentration of 10^?8 mole/l causes a prolonged contraction interrupted by rhythmical relaxations. Higher concentrations of proctolin cause a powerful but irreversible contraction.In some preparations in which proctolin is ineffective in a concentration of 10^?9 mole/l, a short stimulation of nerve 3b can initiate a series of rhythmic contractions. If, however, nerve 3b is stimulated at the peak of such a contraction a rapid relaxation is induced.Administration of the venom of Philanthus triangulum in a concentration which blocks the excitatory and inhibitory neuromuscular transmission, induces similar myogenic contractions. Stimulation of nerve 3b at the peak of such a contraction again causes a relaxation. Similar myogenic contractions can also be induced by administration of a homogenate of a locust.     

Resonance light‐scattering enhancement effect of the protein–Y3+–TTA–SLS system and its analytical application

In this paper, a sensitive resonance light scattering (RLS) method for the determination of protein is reported. In the Tris–HCl (pH 7.50) buffer, protein enhanced the RLS intensity of the Y³⁺–2‐thenoyltrifluoroacetone (TTA)–sodium dodecyl sulphate (SLS) system. The enhanced RLS intensities were in proportion to the concentrations of proteins in the range 8.0 × 10^?9–1.0 × 10^?5 g/mL for BSA, 1.0 × 10^–8–1.0 × 10^?5 g/mL for HSA and 1.0 × 10^–8–1.0 × 10^?6 g/mL for EA, and their detection limits were 5.0, 5.4 and 6.7 ng/mL, respectively. Actual samples were satisfactorily determined. The interaction mechanism was also studied. Copyright © 2008 John Wiley & Sons, Ltd.     

Excitatory neuromuscular transmission in crayfish: Calcium dependence is unaffected by picrotoxin

Picrotoxin, 1 × 10^?5M to 1.6 × 10^?3M, had little or no effect on the amplitude of intracellularly recorded excitatory junctional potentials (EJPs) at extracellular calcium concentrations [Ca²⁺]₀ ranging from 0.5 to 15 mM. The slope of the log EJP vs. log[Ca²⁺]₀ relationship was approximately 1 with or without picrotoxin. The reduction of EJP amplitude resulting from the addition of 5 × 10^?5M GABA was largely reversed by 10^?5M picrotoxin.     

Tea stalks – a novel agro-residue for the production of tannase under solid state fermentation by Aspergillus niger JMU-TS528

A novel agro-residue, tea stalks, was tested for the production of tannase under solid-state fermentation (SSF) using Aspergillus niger JMU-TS528. Maximum yield of tannase was obtained when SSF was carried out at 28 °C, pH 6.0, liquid-to-solid ratio (v/w) 1.8, inoculum size 2 ml (1?×?10⁸ spores/ml), 5 % (w/v) ammonium chloride as nitrogen source and 5 % (w/v) lactose as additional carbon source. Under optimum conditions, tannase production reached 62 U/g dry substrate after 96 h of fermentation. Results from the study are promising for the economic utilization and value addition of tea stalks.