Pre-clinical safety assessment of biotechnologically produced lacto-N-tetraose (LNT).

Lacto-N-tetraose (LNT) is a human milk oligosaccharide with average concentrations ranging from 0.74 to 1.07 g/L in breastmilk, depending on the lactation stage. In this study, the preclinical safety of LNT produced by the Escherichia coli K-12 E2083 production strain was assessed. LNT was negative in both the bacterial reverse mutation assay and the in vitro micronucleus assay, demonstrating the absence of genotoxic potential for this substance. In the OECD 408 guideline compliant 90-day oral toxicity study rat, LNT did not induce any adverse effects in any treatment group up to and including the highest dose tested, and no LOAEL could be determined. Therefore, the no-observed-adverse effect level (NOAEL) is set at the highest dose level tested, i.e. a dietary level of 5 % (w/w), corresponding to ≥2856 mg/kg bw/day and ≥3253 mg/kg bw/day for males and females, respectively. This might be an underestimation of the NOAEL, caused by the range of dose levels tested. The results obtained in the current study are in good agreement with available data generated using other biotechnologically produced LNT batches and therefore support its safe use as a food ingredient.

Anatomical and physiological background permitting spatial odor sensation in stylommatophoran molluscs.

Earlier experiments demonstrated that in order to place protracted tentacles and thereby olfactory receptors in an appropriate position for optimal perception of odor stimuli extraordinary complex movements are required. Until recently both large scale tentacle movements and patterned tentacle movements have been attributed to the concerted involvement of the tentacle retractor muscle and muscles of tegumentum. Recently the existence of three novel muscles in the posterior tentacles of Helix has been discovered. The present review, based on experimental data obtained by our research group, outlines the anatomy, physiology and pharmacology of these muscles that enable the tentacles to execute complex movements observed during foraging both in naïve and food-conditioned snails. Our findings are also compared as far as possible with earlier and recent data obtained on innervation characteristics and pharmacology of molluscan muscles.

PACAP Modulates Acetylcholine-Elicited Contractions at Nicotinic Neuromuscular Contacts of the Land Snail.

In this study, we investigate the potentiating effect of PACAP27 on cholinergic neuromuscular transmission in the recently discovered flexor muscles of the land snail, Helix pomatia. Using immunohistochemistry, we show that PACAP and PAC1 receptors are present in nerve fibers innervating the flexor muscles but not in the muscle itself. We also observed that PACAP27 exerts both pre- and postsynaptic effects on the cholinergic synapse and performed tests using a broad spectrum of chemicals in order to explore the possible intracellular pathways through which PACAP mediates its stimulatory effect. Our pharmacological data demonstrate that PACAP27 presynaptically enhances the release of acetylcholine by activating the adenylate cyclase-cAMP-PKA pathway. Postsynaptically, PACAP27 was found to enhance muscle contractility by PKC-mediated signaling pathway resulting in an increased Ca(2+) release from intracellular stores. These findings suggest that regulation of Ca(2+) release may contribute to the stimulatory effect of PACAP. Our data are the first demonstration of the potentiating effect of PACAP27 at the molluscan excitatory neuromuscular contact.

Nicotinic acetylcholine receptors containing the α7-like subunit mediate contractions of muscles responsible for space positioning of the snail, Helix pomatia L. tentacle.

Three recently discovered tentacle muscles are crucial to perform patterned movements of upper tentacles of the terrestrial snail, Helix pomatia. The muscles receive central and peripheral excitatory cholinergic innervation lacking inhibitory innervation. Here, we investigate the pharmacology of acetylcholine (ACh) responses in muscles to determine the properties of the ACh receptor (AChR), the functional availability of which was assessed using isotonic contraction measurement. Using broad spectrum of nicotinic and muscarinic ligands, we provide the evidence that contractions in the muscles are attributable to the activation of nAChRs that contain the α7-like subunit. Contractions could be evoked by nicotine, carbachol, succinylchloride, TMA, the selective α7-nAChR agonist choline chloride, 3-Bromocytisine and PNU-282987, and blocked by nAChR selective antagonists such as mytolon, hexamethonium, succinylchloride, d-tubocurarine, hemicholinium, DMDA (decamethonium), methyllycaconitine, α-Bungarotoxin (αBgTx) and α-Conotoxin IMI. The specific muscarinic agonist oxotremorine and arecoline failed to elicit contractions. Based on these pharmacological properties we conclude that the Na+ and Ca2+ permeable AChRs of the flexor muscle are nicotinic receptors that contain the α7-like subunit. Immunodetection experiments confirmed the presence of α7- or α7-like AChRs in muscle cells, and α4-AChRs in nerves innervating the muscle. These results support the conclusion that the slowly desensitizing αBgTx-sensitive responses obtained from flexor muscles are produced by activation of α7- like AChRs. This is the first demonstration of postsynaptic expression and an obligatory role for a functional α7-like nAChR in the molluscan periphery.

Novel peripheral motor neurons in the posterior tentacles of the snail responsible for local tentacle movements.

Three flexor muscles of the posterior tentacles of the snail Helix pomatia have recently been described. Here, we identify their local motor neurons by following the retrograde transport of neurobiotin injected into these muscles. The mostly unipolar motor neurons (15-35 µm) are confined to the tentacle digits and send motor axons to the M2 and M3 muscles. Electron microscopy revealed small dark neurons (5-7 µm diameter) and light neurons with 12-18 (T1 type) and 18-30 µm diameters (T2 type) in the digits. The diameters of the neurobiotin-labeled neurons corresponded to the T1 type light neurons. The neuronal processes of T1 type motor neurons arborize extensively in the neuropil area of the digits and receive synaptic inputs from local neuronal elements involved in peripheral olfactory information processing. These findings support the existence of a peripheral stimulus-response pathway, consisting of olfactory stimulus-local motor neuron-motor response components, to generate local lateral movements of the tentacle tip ("quiver"). In addition, physiological results showed that each flexor muscle receives distinct central motor commands via different peritentacular nerves and common central motor commands via tentacle digits, respectively. The distal axonal segments of the common pathway can receive inputs from local interneurons in the digits modulating the motor axon activity peripherally without soma excitation. These elements constitute a local microcircuit consisting of olfactory stimulus-distal segments of central motor axons-motor response components, to induce patterned contraction movements of the tentacle. The two local microcircuits described above provide a comprehensive neuroanatomical basis of tentacle movements without the involvement of the CNS.

Morphology, ultrastructure and contractile properties of muscles responsible for superior tentacle movements of the snail.

Bending, twitching and quivering are different types of tentacle movements observed during olfactory orientation of the snail. Three recently discovered special muscles, spanning along the length of superior tentacles from the tip to the base, seem to be responsible for the execution of these movements. In this study we have investigated the ultrastructure, contractile properties and protein composition of these muscles. Our ultrastructural studies show that smooth muscle fibers are loosely embedded in a collagen matrix and they are coupled with long sarcolemma protrusions. The muscle fibers apparently lack organized SR and transverse tubular system. Instead subsarcolemmal vesicles and mitochondria have been shown to be possible Ca2+ pools for contraction. It was shown that external Ca2+ is required for contraction elicited by high (40 mM) K+ or 10-4 M ACh. Caffeine (5 mM) induced contraction in Ca2+-free solution suggesting the presence of a substantial intracellular Ca2+ pool. High-resolution electrophoretic analysis of columellar and tentacular muscles did not reveal differences in major contractile proteins, such as actin, myosin and paramyosin. Differences were observed however in several bands representing presumably regulatory enzymes. It is concluded that, the ultrastructural, biochemical and contractile properties of the string muscles support their special physiological function.