Nitric oxide control of large veins in the toad <Emphasis Type="Italic">Bufo marinus</Emphasis>

This study examined the nitric oxide (NO) control of the vascular smooth muscle of the ventral abdominal vein and vena cava of the toad, Bufo marinus, by using anatomical and physiological approaches. Nicotinamide adenine di-nucleotide phosphate-diaphorase histochemistry and immunohistochemistry using endothelial nitric oxide synthase (NOS) and neural NOS antibodies produced no evidence for endothelial NOS in the veins, but, neural NOS-immunoreactive perivascular nerves were present. Acetylcholine (10^–5 M) caused a vasodilation in both veins that was endothelium-independent, and which was blocked by the soluble guanylyl cyclase inhibitor, ODQ (10^–5 M). The NOS inhibitors, L-NNA (10^–4 M) and L-NAME (10^–4 M), did not significantly reduce the vasodilatory effect of acetylcholine in the veins; this suggested that the vasodilation was not due to NO. However, in the presence of phenoxybenzamine (10^–7–10^–8 M), L-NNA significantly reduced the vasodilatory effect of acetylcholine in the veins. This unusual response is due to phenoxybenzamine partially inactivating the muscarinic receptor pool in the veins. In addition, the neural NOS inhibitor, vinyl-L-NIO (10^–5 M), significantly reduced the acetylcholine-mediated vasodilation in the presence of phenoxybenzamine. The results show that in toad veins, nitrergic nerves rather than an endothelial NO system are involved in NO-mediated vasodilation.     

Electrophysiological studies of pyridine-sensitive units on the crayfish walking leg

Summary Pyridine-sensitive units located on the walking legs of the crayfishAustropotamobius torrentium were studied by extracellular recording of the action potentials of single afferent fibers. To characterize the sensitivity and specificity of the pyridine receptor, 79 pyridine analogs and other related substances were tested on 70 neurons. The maximum impulse frequency of the response was used to construct dose-response curves. The effectiveness of stimulatory substances was characterized at the half-maximal-response frequency, K_M. The effectiveness rank order of the substances was found to be the same for all units tested. The most effective substances were: pyrazinecarboxamide > 3-acetylpyridine > nicotinamide > pyridine-3-aldoxime, with K_M values of 1.5×10^–6, 4× 10^–6, 10^–5 and 4 x 10^–5 mol/1, respectively. The inferred structural requirements for an optimal stimulatory molecule are that it have a N-containing aromatic ring system with a specific substituent in them position.     

Effects of two microtubule-depolymerizing drugs,vincristine and vinblastine,on porphyrin production by primary neural tissue cultures

A porphyrinogenic effect of 10^–5 to 10^–7 M Vincristine (VC) and Vinblastine (VB) was observed on primary neural tissue cultures prepared from chicken embryo brain in the presence of 10^–3 M delta-aminolevulinic acid. This effect is very pronounced in neurocyte cultures, in contrast with the documented neurite elongation defect. The microtubule disassembly by VC and VB changed the quantity of the porphyrins in the cells and medium of glial cell cultures. The correlation was studied between the depolymerization of the microtubules by VC and VB and the porphyrin overproduction of primary neural tissue cultures, the investigation also extending to 10^–7 M taxol. The direct mediation of nucleotide binding proteins of the adenyl-cyclase complex by GTP liberated from beta-tubulin during the disassembly of the microtubules is presumed.     

Pharmacological study of inhibited frog olfactory bulb glomerular input produced by a puff of air on the olfactory mucosa

The effects of applying 4-aminopyridine (10^–2 M), aminooxyacetic acid (AOAA — 10^–4–10^–3 M), -alanine (10^–3–10^–2 M), and bicuculline (10^–5, 10^–4 M) to the intact frog olfactory bulb were investigated. Having measured inhibition of orthodromic potential postsynaptic components produced either by a puff of air on the olfactory mucosa (OB input inhibition) or by single electrical stimulation of the olfactory nerve (postsynaptic inhibition) or by single electrical stimulation of the olfactory nerve (postsynaptic inhibition), it was found that 4-aminopyridine greatly intensified postsynaptic inhibition but strongly reduced that of OB input; inhibition of the latter was raised by AOAA or bicuculline and decreased by -alanine. These substances failed to exert any consistent, clear-cut effects on postsynaptic inhibition. Findings would support the hypothesis that OB input inhibition produced by a puff of air on the olfactory mucosa could occur as a result of GABA release from glial cells and subsequent binding of GABA to presynaptic GABA_B-receptors in glomeruli.M. V. Lomonosov Moscow State University. Translated from Neirofiziologiya, Vol. 19, No. 1, pp. 12–20, January–February, 1987.     

Effects of solar radiation,humic substances and nutrients on phytoplankton biomass and distribution in Lake Solumsjö, Sweden

Impacts of solar radiation, humic substances and nutrients on phytoplankton abundance at different depths were investigated in a temperate dimictic lake, Lake Solumsjö. Penetration of solar radiation profiles at different depths, represented as light attenuation coefficient (K _d) were examined. Water sampling and downward irradiance of photosynthetically active radiation (PAR), ultraviolet-A (UV-A, 320–400 nm) and ultraviolet-B (UV-B, 280–320 nm) radiation were performed once a week and at three different times of the day (08.00, 12.00 and 16.00 hrs, local time) between September 13 and November 1, 1999. During the period of investigation, solar radiation above the water surface declined from 474 to 94 mol m^–2 s^–1 for PAR, from 1380 to 3.57 W m^–2 for UV-A and from 13.1 to 0.026 W m^–2 for UV-B, respectively. The attenuation coefficient (K _d) for UV-B radiation ranged from 3.7 to 31 m^–1 and UV-B radiation could not be detected at depths greater than 0.25 m. Humic substances measured at 440 nm ranged from 35.5 to 57.7 Pt mg l^–1. Mean values of biomass, estimated from chlorophyll a, in the whole water column (0–10 m) varied between 2.3 and 5.6 g l^–1 and a diel fluctuation was observed. During stratified conditions, high levels of iron (1.36 mg l^–1) and manganese (4.32 mg l^–1) were recorded in the hypolimnion, suggesting that the thermocline played a major role in the vertical distribution of phytoplankton communities in Lake Solumsjö. The high levels of iron and manganese stimulated the growth of Trachelomonas volvocinopsis in the hypolimnion at a depth of 10 m. Negative impacts of UV-B radiation on phytoplankton in lake Solumsjö are reduced due to the high levels of humic substances and the high degree of solar zenith angle at the latitude studied.     

Role of non-quantum acetylcholine secretion in neural control of the membrane potential in skeletal muscle fibers of rats

Experiments on muscle fibers of the rat diaphragm (in vitro denervation) showed that their three-hour incubation in the cultural medium results in an 8-mV drop in the resting membrane potential (RMP). Addition of 5·10^–8 M carbacholine to the cultural medium, mimicing the effect of non-quantum acetylcholine, delayed depolarization of the denervated muscle. The effect of carbacholine could not be eliminated byd-tubocurarine (5·10^–6 M), a postsynaptic acetylcholine receptor blocker, and by ouabain (1·10^–4 M), and inhibitor of Na⁺, K⁺-ATPase of the membrane. At the same time, the effect could be completely eliminated by Mg²⁺ ions (5·10^–3 M), which blocked Ca²⁺ channels of the membrane, by N-nitroarginine (1·10^–4 M), which inhibited the enzyme NO-synthase, and by hemoglobin (2·10^–5 M), which inactivated the extracellular NO molecules. It is concluded that the released non-quantum acetylcholine can contribute to neural control of RMP of cross-striated muscle fibers via the Ca²⁺-dependent activation of NO synthesis in the sarcoplasm. The NO molecules can play the role of a retrograde signal indicative of the normal functioning of the neuromuscular synapse. The impairment of this link caused by a denervation-induced cessation of the non-quantum secretion can serve as a signal triggering the early changes in the muscle membrane following nerve transection.Neirofiziologiya/Neurophysiology, Vol. 27, No. 1, pp. 67–71, January–February, 1995.     

Multiplicity of chemical mechanisms of regulation of muscle contractions in <Emphasis Type="Italic">Lymnaea stagnalis</Emphasis> L.

Effects of acetylcholine, octopamine, and their antagonists, as well as of glutamic acid were studied on contractions of dorsal longitudinal muscle of the mollusc Lymnaea stagnalis L., evoked by electrical stimulation of n. cervicalis inferior. Acetylcholine and octopamine increased amplitude of contractions evoked by applications at concentrations about 10^–8 M and decreased at concentrations higher than 10^–7 M. Glutamic acid produced only inhibitory effect on the contraction amplitude, which appeared at concentrations beginning from 10^–9 M and higher. The acetylcholine antagonists atropine and d-tubocurarine also decreased amplitude of evoked contractions. Their blocking effect rose with increase of their concentrations in the range from 10^–9 M to 10^–5 M. Specificity of the effect of the studied substances was established in experiments with a combined application of the transmitters and their antagonists. The obtained results indicate multiplicity of chemical mechanisms of regulation of contractions of the dorsal longitudinal muscle in L. stagnalis.Translated from Zhurnal Evolyutsionnoi Biokhimii i Fiziologii, Vol. 41, No. 1, 2005, pp. 44–50.Original Russian Text Copyright © 2005 by Kononenko, Zhukov.     

Effects of aspartate,substance P,and serotonin on neuronal activity in the central gray substance:In vitro experiments

Effects of aspartate (2 · 10^–5 M), substance P (10^–7–10^–8 M), and serotonin (5-hydroxytryptamine, 5-HT; 5 · 10^–5 M) on the background activity of neurons in the central gray substance (CGS) were studied on slices of the rat midbrain. Aspartate and substance P (transmitters of nociceptive signals), and 5-HT (modulator of transmission of nociceptive influences) were found either to facilitate or to depress the activity of CGS neurons. The predominant effect of substance P or 5-HT applications to neurons of the dorsal CGS part was facilitation, and to neurons of the ventral CGS part, inhibition. The effects of aspartate application on studied CGS neurons were of varying nature, but inhibitory effects were found to prevail.The findings support our earlier hypothesis that assigned the studied neurons to spontaneously discharging inhibitory CGS interneurons, which control the activity of efferent CGS neurons. The role of tested substances in the regulation of CGS neuronal activity and the antinociceptive CGS effects is discussed.Neirofiziologiya/Neurophysiology, Vol. 25, No. 5, pp. 354–362, September–October, 1993.     

A yeast biosensor for glucose determination

Summary A yeast potentiometric biosensor for glucose determination is described. After induction of glycolytic enzyme synthesis a cell suspension of the yeast Hansenula anomala is retained in calcium alginate gel on the surface of a glass electrode. This biosensor gives a Nernstian response in glucose concentration of 5·10^–4–5·10^–3 mol/l with a response time of 5 min and a life-time of at least 2 months. Mannose and fructose are the only significantly interfering substances. The biosensor was used for measurement of glucose concentration in urine with results comparable to those obtained by a photometric enzymatic method.     

Interactions between prostaglandin E2 andd-ala2-met-enkephalinamide on adenylate cyclase activity in the guinea-pig superior cervical ganglion

Crude membrane fractions, obtained from superior cervical ganglia of normal and sympathectomized guinea-pigs, have been used to investigate the role of prostaglandin E₂ andd-ala²-met-enkephalinamide in the modulation of ganglionic adenylate cyclase as well as their functional interrelationship. In ganglia from normal animals the enzyme activity was stimulated and inhibited, respectively, by the prostaglandin (10^–4M) and by the opiate pentapeptide (10^–4M), while little or no effects were observed in denervated preparations. When the two substances were tested in combination, a supra-additive stimulation of adenylate cyclase activity was obtained both in normal and denervated ganglia. In the latter preparation the opiate increased prostaglandin E₂ specific binding, suggesting that the mechanism of supra-additivity could involve interactions at receptors level. Furthermore, the supra-additive stimulation of adenylate cyclase activity by the combination of the two drugs was obtained in a narrow range of concentrations since at low prostaglandin E₂ doses (10^–7–10^–6M) or at very high doses of the opiate (10^–3M), only the inhibitory effect ofd-ala²-met-enkephalinamide was evidenced.     

Stimulatory effect of genistein and daidzein on protein synthesis in osteoblastic MC3T3-E1 cells: Activation of aminoacyl-tRNA synthetase

The effect of genistein and daidzein on protein synthesis in osteoblastic MC3T3-E1 cells in vitro was investigated to determine a cellular mechanism by which the isoflavones stimulate bone formation. Cells were cultured for 48 h in -minimal essential medium containing either vehicle, genistein (10^–7–10^–5 M) or daidzein (10^–7–10^–5 M). The 5,500 g supernatant of cell homogenate was used for assay of protein synthesis with [³H]leucine incorporation in vitro. The culture with genistein or daidzein caused a significant elevation of protein synthesis in the cell homogenate. The effect of genistein (10^–5 M) or daidzein (10^–5 M) in elevating protein synthesis was significantly prevented, when cells were cultured for 48 h in a medium containing either actinomycin D (10^–7 M) or cycloheximide (10^–6 M) in the absence or presence of isoflavones. Moreover, when genistein (10^–7–10^–5 M) or daidzein (10^–6 and 10^–5 M) was added to the reaction mixture containing the cell homogenate obtained from osteoblastic cells cultured without isoflavone, protein synthesis was significantly raised. This increase was markedly blocked by the addition of cycloheximide (10^–7 M). In addition, [³H]leucyl-tRNA synthetase activity in the cytosol of osteoblastic cells was significantly increased by the addition of genistein (10^–6 and 10^–5 M) or daidzein (10^–5 M) into the enzyme reaction mixture. The present study demonstrates that genistein or daidzein can stimulate protein synthesis in osteoblastic MC3T3-E1 cells. The isoflavones may have a stimulatory effect on osteoblastic bone formation due to increasing protein synthesis.     

Ion and sugar permeabilities of lecithin bilayers: Comparison of curved and planar bilayers

Summary Na⁺ and sugar permeabilities of egg lecithin bilayers were measured using curved bilayers and planar bilayers as represented by single-bilayer vesicles and black lipid films, respectively. The Na⁺ permeability coefficient measured with single-bilayer vesicles at 25°C is (2.1±0.6)×10^–13 cm sec^–1. Because of technical difficulties it has been impossible to measure ionic permeabilities of values lower than about 10^–10 cm sec^–1 in planar (black) lipid bilayers using tracer methods. Thed-glucose andd-fructose permeabilities were measured with both curved and planar bilayers. The permeability coefficients measured with vesicles at 25°C are (0.3±0.2)×10^–10 cm sec^–1 for glucose and (4±1)×10^–10 cm sec^–1 ford-fructose; these are in reasonable agreement with the corresponding values obtained for planar (black) lipid bilayers which are (1.1±0.3)×10^–10 cm sec^–1 ford-glucose and (9.3±0.3)×10^–10 cm sec^–1 ford-fructose, respectively.This paper is dedicated to the memory of Walther Wilbrandt,cuius nomini nullum par elogium.     

Regulation of Adenylyl Cyclase Signaling System in Cell Cultures of Infusoria Dileptus anser and Tetrahymena pyriformis by Peptides of Insulin Superfamily

Hormone-sensitive adenylyl cyclase signaling system (ACS) provides transduction of a wide spectrum of hormonal signals in cells of the higher eucaryotes. At the same time, ACS in the lower eucaryotes at present is practically not studied. We studied regulatory effects on ACS of the infusoria Dileptus anser and Tetrahymena pyriformis of peptide hormones of the higher eukaryotes—insulin, IGF-1, and relaxin, whose action on ACS of the higher eucaryotes was the subject of our earlier studies. The action of these hormones at concentrations of 10^–10–10^–8 M on the AC activity in infusoria had clearly stimulating character, the dose–effect curves being of a bell-shaped form with a maximum of the stimulating effect of the hormones at concentrations of 10^–9–10^–8 M. the shape of the curves and the value of the stimulating effect of the peptide hormones depended substantially on the level of the AC basal activity in homogenates of infusorian cell cultures. All the hormones (10^–8 M) stimulated GTP-binding activity of G-proteins. It was shown by the example of relaxin that its stimulating effect on GTP-binding in infusorian cells was dose-dependent and increased in the range of hormone concentrations from 10^–10 to 10^–8 M to reach its maximum at concentrations of 10^–8–10^–7 M. In the presence of suramin, an inhibitor of heterotrimeric G-proteins, the stimulating effects of the hormones on the GTP-binding and the AC activity decreased essentially or were absent completely. This indicates that the heterotrimeric G-proteins are ones of components of the signaling cascade that mediates regulatory effects of the hormones of the insulin group on the AC activity in infusorian cell cultures. Based on the obtained data, it is suggested that the basic molecular mechanisms of regulation of ACS by insulin and the related peptides that are similar to those found in the higher vertebrates already begin to be formed as early as at the level of the lower eucaryotes.     

Expansion and neural differentiation of embryonic stem cells in adherent and suspension cultures

The embryonic stem cell line, S25, is a genetically modified line that allows lineage selection of neural cells (M. Li, L. Lovell-Badge, A. Smith (1998) Current Biology 8: 971–974). Here, the growth parameters of this cell line were analysed. Serial passaging in adherent conditions enabled these cells to grow rapidly (average specific growth rates of 0.035 h^–1) and generate high viable cell densities (above 90%). The aggregation of the S25 cells into embryoid bodies (EBs) was also studied, indicating limited cell growth (maximum cell densities of 2.7×10⁵ cells ml^–1) and a high variability of aggregate size (70–400 m after 8 d). Enzymatic dissociation of EBs with 1% (v/v) trypsin gave highest cell viability (91%) and density (1.4×10⁴ cells ml^–1) and the cells thus obtained are able to differentiate into neurons.     

Wall appositions induced by ionophore A 23187, CaCl2, LaCl3, and nifedipine in characean cells

Summary The formation of wall appositions (plugs) by ionophore A 23187, CaCl₂, LaCl₃, and nifedipine was studied in mature internodal cells of characeaen algae. CaCl₂ at concentrations above 10^–2M induces thick fibrillar plugs without callose inNitella flexilis. InChara corallina andNitella flexilis ionophore A 23187 (1.25×10^–5 to 5×10^–5M) and LaCl₃ (7.5×10^–5 to 2.5×10^–4M) cause flat appositions which contain callose and have a more granular structure. Plug formation by ionophore A 23187, CaCl₂, and LaCl₃ is pH-dependent and occurs beneath the alkaline regions of the cell. Nifedipine (10^–4 to 10^–5M) induces plugs inNitella flexilis after previous injury. These callose-containing wall appositions consist of a heterogeneous granular core which is covered by a fibrillar layer. The results of this work are compared with previous studies on wound wall formation and chlortetracycline (CTC)-induced plug formation which reveal that abundant coated vesicles occur only when a thick fibrillar wall layer is formed. Neither LaCl₃ nor nifedipine inhibit the formation of CaCl₂- or CTC-plugs. The unusual effects of these substances, which normally act as Ca²⁺ antagonists and therefore should prevent and not induce plug formation, are discussed. It is suggested that La³⁺ mimicks the effects of calcium and that nifedipine binding to the Ca²⁺ channels is altered in the alkaline regions of characean internodes and allows an influx of Ca²⁺.Abbreviations AFW artificial fresh water - CTC chlortetracycline - DCMU dichlorphenyldimethylurea - DMSO dimethylsulfoxide - EGTA ethyleneglycoltetraacetic acid - MES 2-(N-morpholino) ethanesulfonic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - TAPS N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid     

Methodology for assessing respiration and cellular incorporation of radiolabeled substrates by soil microbial communities

A method is described for determining biodegradation kinetics of both naturally occurring and xenobiotic compounds in surface and sub-surface soil samples. The method measures both respiration and uptake into cellular biomass of¹⁴C-labeled substrates. The estimation of biomass incorporation entailed removal of cells from soil particles by washing the soil with a polyvinyl-pyrrolidone/pyrophosphate solution and H₂O₂. After separation of the cells and the soil particles by centrifugation, the cells were trapped on membrane filters for liquid scintillation counting. Mass balances were easily obtained. The technique was used to measure metabolic activity in soil profiles, including unsaturated and saturated zones. First order rate constants (K₁) were in the range of 10^–3–10^–2 hour^–1 for amino acid metabolism and 10^–5–10^–4 hour^–1 for m-cresol metabolism. Saturation kinetics were observed for amino acids and m-cresol. m-Cresol K₁ values for uptake often exceeded those for respiration by greater than a factor of ten. V_max values were low (amino acids, 10¹–10² ng g^–1 hour^–1; m-cresol, 10^–1 ng g^–1 hour^–1), whereas K_m values were quite high (amino acids, 10³–10⁴ ng g^–1; m-cresol 10³–10⁵ ng g^–1). Saturation was not observed in many horizons even at 10⁵ ng g^–1 dry soil. Frequently, respiration obeyed saturation kinetics whereas uptake was first order. It is concluded that measuring only kinetics of respiration may lead to severe underestimations of biodegradation rates.     

Effects of L-alanine and L-alanine peptides on the chemotaxis of tetrahymena: Evolutionary conclusions

L-alanine and its peptides (L-Ala-2–6) do not attract or repulse Tetrahymena in a 10^–8M concentration. In 10^–10M concentration there is a consistent repellent effect. Twenty four hours after L-alanine or L-alanine-peptides' pretreatment (imprinting) the progeny generation of the cells react differently to the same materials. L-Alanine, L-alanine penta- and hexapeptide in both concentrations are chemoattractant, while L-alanine tetrapeptide is repellent. L-Alanine dipeptide is inert in 10^–10M and repellent at 10^–8M concentrations, while L-alanine tripeptide is strongly repellent at 10^–10M and attractant at 10^–8M concentrations. This means, that the first encounter (imprinting) with an exogeneous amino acid or peptide is decisive to the later reaction of the protozoan cell. The chain length is important in the imprinting, however the reaction is not consistent. The experiments call the attention to the significance of imprinting in the receptor and hormone evolution.     

Blocking action of calmidazolium and chlorpromazine on outward potassium current dependent on extra-cellular calcium ions

The effects of the calmodulin antagonists, calmidazolium (R 24571) and chlorpromazine on delayed outward potassium current at the somatic membrane were investigated in non-identified intracellularly perfused neurons isolated fromHelix pomatia. Voltage was clamped at the membrane. Extracellular application of these substances produced effective depression of the outward current. This effect even occurred at test substance concentrations of 10^–9–10^–8 M. Block-ade of delayed outward current was produced mainly as a result of suppressing the potassium current component dependent on intracellular potassium ions (I_k(Ca/in)). The possibility that the receptor for intracellular calcium responsible for modulating this current may be of a calmodulin-like nature is discussed.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 19, No. 3, pp. 356–361, May–June, 1987.     

Activation of the cAMP-generating system by vasoactive intestinal polypeptide (VIP) in the human laryngeal malignant cell line HEp-2

In the presence of 3-isobutyi-l-methylxanthine, VIP produced a dose-related (3×10^–9–10^–7 M) increase (g-fold) in cAMP production in isolated HEp-2 cells incubated at 15°C in KRP buffer. Among the peptides structurally related to VIP, including secretin (10^–7 M), pancreatic glucagon (10^–6 M), PHI, somatostatin-14 (10^–6 M), hpGRF (10^–8–4×10^–M), GIP (2×10^–7 M), only PHI (3×10^–7 M and above) is able to activate the cAMP-generating system in HEp-2 cells, but at 102 times lower potency. Under the same conditions, histamine (10^–3 M) was also ineffective, while PGE 2 (10^–7–10^–4 M) increased (0-fold) basal cAMP levels in HEp-2 cells. The VIP effect is related to the interaction os the peptide on VIP recognition sites (12SI-VIP-binding capacity ), coupled to the membrane-bound adenylate cyclase . The results indicate that the transformed laryngeal cell line HEp-2 possessesa receptor-cAMP system preferentially activated by VIP (relative potencies: VIP > PHI other peptides of the secretin family), and suggest that this neuropeptide could modulate biological functions in normal laryngeal epithelia in man.     

Action of tetrodotoxin on the contractile responses of isolated guinea-pig urinary bladder preparation to X-irradiation and to electrical stimulation

Summary Tetrodotoxin (10^–8 to 10^–6 g/ml) blocked the contractile responses of isolated guinea-pig urinary bladder preparation to electrical (25 and 100 Hz) neural but not to transmural stimulation and to X-irradiation (50 kV, 20 kR/min, 20 kR). The irradiation had no influence on the bladder responses to electrical and hormonal (acetylcholine and histamine) stimulation. It is concluded that the X-ray-induced contraction is of myogenic origin and that it is possibly not related to the electro-mechanical coupling system.