Axoplasmic RNA species synthesized in the isolated squid giant axon

Isolated squid stellate nerves and giant fiber lobes were incubated for 8 hr in Millipore filtered sea water containing [³H]uridine. The electrophoretic patterns of radioactive RNA purified from the axoplasm of the giant axon and from the giant fiber lobe (cell bodies of the giant axon) demonstrated the presence of RNA species with mobilities corresponding to tRNA and rRNA. The presence of labeled rRNAs was confirmed by the behavior of the large rRNA component (31S) which, in the squid, readily dissociates into its two constituent moyeties (17S and 20S). Comparable results were obtained with the axonal sheath and the stellate nerve. In all the electrophoretic patterns, additional species of radioactive RNA migrated between the 4S and the 20S markers, i.e. with mobilities corresponding to presumptive mRNAs. Chromatographic analysis of the purified RNAs on oligo(dT)cellulose indicated the presence of labeled poly(A)⁺ RNA in all tissue samples. Radioactive poly(A)⁺ RNA represented approximately 1% of the total labeled RNA in the axoplasm, axonal sheath and stellate nerve, but more than 2% in the giant fiber lobe. The labeled poly(A)⁺ RNAs of the giant fibre lobe showed a prevalence of larger species in comparison to the axonal sheath and stellate nerve. In conclusion, the axoplasmic RNAs synthesized by the isolated squid giant axon appear to include all the major classes of axoplasmic RNAs, that is rRNA, tRNA and mRNA.Special Issue dedicated to Prof. Holger Hydén.     

Phosphorylation of K+ channels in the squid giant axon. A mechanistic analysis

Protein phosphorylation is an important mechanism in the modulation of voltage-dependent ionic channels. In squid giant axons, the potassium delayed rectifier channel is modulated by an ATP-mediated phosphorylation mechanism, producing important changes in amplitude and kinetics of the outward current. The characteristics and biophysical basis for the phosphorylation effects have been extensively studied in this preparation using macroscopic, single-channel and gating current experiments. Phosphorylation produces a shift in the voltage dependence of all voltage-dependent parameters including open probability, slow inactivation, first latency, and gating charge transferred. The locus of the effect seems to be located in a fast 20 pS channel, with characteristics of delayed rectifier, but at least another channel is phosphorylated under our experimental conditions. These results are interpreted quantitatively with a mechanistic model that explains all the data. In this model the shift in voltage dependence is produced by electrostatic interactions between the transferred phosphate and the voltage sensor of the channel.     

Permeability of the squid giant axon to organic cations and small nonelectrolytes

Summary The permeability of the Na channel of squid giant axon to organic cations and small nonelectrolytes was studied. The compounds tested were guanidinium, formamidinium, and¹⁴C-labeled urea, formamide, thiourea, and acetone. Permeability was calculated from measurements of reversal potential and influx on internally perfused, voltage clamped squid axons. The project had two objectives: (1) to determine whether different methods of measuring the permeability of organic cations yield similar values and (2) to see whether neutral analogs of the organic cations can permeate the Na channel. Our results show that the permeability ratio of sodium to a test ion depends upon the ionic composition of the solution used. This finding is consistent with the view put forward previously that the Na channel can contain more than one ion at a time. In addition, we found that the uncharged analogs of permeant cations are not measurably permeant through the Na channel, but instead probably pass through the lipid bilayer.     

COMPARISON OF THE STOMACH CONTENTS OF SOUTHERN ELEPHANT SEALS, MIROUNGA LEONINA, AT MACQUARIE AND HEARD ISLANDS

Abstract: There are three major breeding populations of southern elephant seals centered on Macquarie Island, Kerguelen-Heard Islands and South Georgia-Antarctic Peninsula. The composition of the diet differs between these populations based on published data from Signy Island and data presented here from Macquarie and Heard Islands. These differences in diet appear to be linked to the location at which seals were sampled ranging from the least Antarctic (Macquarie Island) to the most Antarctic (Signy Island). The major food remains consisted of cephalopod beaks and fish eye lenses. More benthic material was found at Heard Island than at Macquarie Island. The diet at Macquarie Island differed between summer and winter and between young animals and adults. The difficulty in collecting dietary samples of southern elephant seals near their main foraging areas makes the study of the feeding ecology of this species extremely difficult in comparison with other Southern Ocean species.     

Design of a Robotized Magnetic Platform for Targeted Drug Delivery in the Cochlea

Inner ear disorders' treatment remains challenging due to anatomical barriers. Robotic assistance seems to be a promising approach to enhance inner ear treatments and, more particularly, lead to effective targeted drug delivery into the human cochlea. In this paper we present a combination of a micro-macro system that was designed and realized in order to efficiently control the navigation of magnetic nanoparticles in an open-loop scheme throughout the cochlea, considering that the magnetic particles cannot be located in real time.In order to respect the anatomical constraints, we established the characteristics that the new platform must present then proceeded to the design of the latter. The developed system is composed of a magnetic actuator that aims to guide nanoparticles into the cochlea. Mounted on a robotic manipulator, it ensures its positioning around the patient's head. The magnetic device integrates four parallelepiped-rectangle permanent magnets. Their arrangement in space, position and orientation, allows the creation of an area of convergence of magnetic forces where nanoparticles can be pushed/pulled to. To ensure the reachability of the desired orientations and positions, a 3 DOF robot based on a Remote Centre of Motion (RCM) mechanism was developed. It features three concurrent rotational joints that generate a spherical workspace around the head. The control of the latter is based on kinematic models.A prototype of this platform was realized to validate the actuation process. Both magnetic actuator and robotic manipulator were realized using an additive manufacturing approach. We also designed a virtual human head with a life-size cochlea inside. A laser was mounted on the end effector to track the positioning of the actuator. This permitted to experimentally prove the capacity of the robotic system to reach the desired positions and orientations in accordance with the medical needs.This promising robotic approach, makes it possible to overcome anatomical barriers and steer magnetic nanoparticles to a targeted location in the inner ear and, more precisely, inside the cochlea.     

Anticalmodulin drugs block the sodium gating current of squid giant axons

Summary The effects of calmodulin (CaM) antagonists (W-7, W-5, trifluoperazine, chlorpromazine, quinacrine, diazepam, propericyazine and carmidazolium) on the sodium and potassium channels were studied on the intracellularly perfused and voltage-clamped giant axon of the squid. It was found that the drugs are more potent blockers of the sodium current than of the potassium current. The drugs also reduce the sodium gating current. The blockage of the sodium and gating current can be explained by assuming that the drugs interact with the sodium gating subunit in one of its closed states. The site of action is probably the intracellular surface of the axolemma where presumably a Ca²⁺-calmodulin complex can be formed.     

A naphthyl analog of the aminostyryl pyridinium class of potentiometric membrane dyes shows consistent sensitivity in a variety of tissue,cell, and model membrane preparations

The fast potentiometric indicator di-4-ANEPPS is examined in four different preparations: lipid vesicles, red blood cells, squid giant axon, and guinea pig heart. The dye gives consistent potentiometric responses in each of these systems, although some of the detailed behavior varies. In lipid vesicles, the dye displays an increase in fluorescence combined with a red shift of the excitation spectrum upon hyperpolarization. Similar behavior is found in red cells where a dual wavelength radiometric measurement is also demonstrated. The signal-to-noise ratio of the potentiometric fluorescence response is among the best ever recorded on the voltage-clamped squid axon. The dye is shown to be a faithful and persistent monitor of cardiac action potentials with no appreciable loss of signal or deterioration of cardiac activity for periods as long as 2 hr with intermittent illumination every 10 min. These results, together with previously published applications of the dye to a spherical lipid bilayer model and to cells in culture, demonstrate the versatility of di-4-ANEPPS as a fast indicator of membrane potential.     

Membrane differentiations in spermatozoa of the squid,Loligo pealeii

Regional differences in the structure of the plasma membrane and acrosome membrane of squid spermatozoa were studied by freeze-fracture and thin section electron microscopy. In regions of close apposition the plasma membrane and acrosome membrane are adjoined to one another by regularly spaced linkages. These linkage sites, overlie a set of fibers located at the inner face of the acrosomal membrane. The acrosomal fibers terminate in a layer of granular material located at the base of the acrosome. Detergent treatment of sperm releases the fibers and granular material as an interconnected complex. Freeze-fracture replicas reveal a random arrangement of intramembranous particles in the plasma membrane over the sperm head and linear aggregates of intramembranous particles in the acrosomal membrane. Several regional differences in the structure of the flagellar plasma membrane are present. The thickness of the glycocalyx is progressively reduced distally along the flagellum. Freeze-fracture replicas show evenly spaced linear arrays of intramembranous particles which extend parallel t o the flagellar long axis. Examination of spermatozoa extracted to disrupt flagellar geometry suggest that the dense fiber-doublet microtubule complexes are attached to the plasma membrane. The possible functional role of these membrane differentiations and their relationship t o membrane structures in mammalian spermatozoa are discussed.     

The effects of post-stroke upper-limb training with an electromyography (EMG)-driven hand robot

Loss of hand function and finger dexterity are main disabilities in the upper limb after stroke. An electromyography (EMG)-driven hand robot had been developed for post-stroke rehabilitation training. The effectiveness of the hand robot assisted whole upper limb training was investigated on persons with chronic stroke (n = 10) in this work. All subjects attended a 20-session training (3–5 times/week) by using the hand robot to practice object grasp/release and arm transportation tasks. Significant motor improvements were observed in the Fugl-Meyer hand/wrist and shoulder/elbow scores (p < 0.05), and also in the Action Research Arm Test and Wolf Motor Function Test (p < 0.05). Significant reduction in spasticity of the fingers as was measured by the Modified Ashworth Score (p < 0.05). The training improved the muscle co-ordination between the antagonist muscle pair (flexor digitorum (FD) and extensor digitorum (ED)), associated with a significant reduction in the ED EMG level (p < 0.05) and a significant decrease of ED and FD co-contraction during the training (p < 0.05); the excessive muscle activities in the biceps brachii were also reduced significantly after the training (p < 0.05).     

Cartesian stiffness for wrist joints: analysis on the Lie group of 3D rotations and geometric approximation for experimental evaluation

This paper is concerned with the analysis and the numerical evaluation from experimental measurements of the static, Cartesian stiffness of wrist joints, in particular the human wrist. The primary aim is to extend from Euclidean spaces to so(3), the group of rigid body rotations, previous methods for assessing the end-point stiffness of the human arm, typically performed via a robotic manipulandum. As a first step, the geometric definition of Cartesian stiffness from current literature is specialised to the group so(3). Emphasis is placed on the choice of the unique, natural, affine connection on so(3) which guarantees symmetry of the stiffness matrix in presence of conservative fields for any configuration, also out of equilibrium. As the main contribution of this study, a coordinate-independent approximation based on the geometric notion of geodesics is proposed which provides a working equation for evaluating stiffness directly from experimental measurements. Finally, a graphical representation of the stiffness is discussed which extends the ellipse method often used for end-point stiffness visualisation and which is suitable to compare stiffness matrices evaluated at different configurations.