Characterization of DRP2, a novel human dystrophin homologue.

The currently recognised dystrophin protein family comprises the archetype, dystrophin, its close relative, utrophin or dystrophin-related protein (DRP), and a distantly related protein known as the 87K tyrosine kinase substrate. During the course of a phylogenetic study of sequences encoding the characteristic C-terminal domains of dystrophin-related proteins, we identified an unexpected novel class of vertebrate dystrophin-related sequences. We term this class dystrophin-related protein 2 (DRP2), and suggest that utrophin/DRP be renamed DRP1 to simplify future nomenclature. DRP2 is a relatively small protein, encoded in man by a 45 kb gene localized to Xq22. It is expressed principally in the brain and spinal cord, and is similar in overall structure to the Dp116 dystrophin isoform. The discovery of a novel relative of dystrophin substantially broadens the scope for study of this interesting group of proteins and their associated glycoprotein complexes.

The osmotic effects of electron microscope fixatives.

The reflecting cells on the scales of sprat and herring contain ordered arrays of guanine crystals. The spacing of the crystals within these cells determines the wave bands of the light which they reflect, hence volume changes in the reflecting cells can be observed as color changes directly. This property of the scales is used to show that (a) fixation with osmium tetroxide solutions destroys osmotic activity; (b) fixation with aldehyde solutions does not destroy osmotic activity and does not cause volume changes if the aldehydes are made up in salt or sucrose solutions whose osmolarities, discounting the aldehyde, are about 60% of those to which the cells are in equilibrium in life, and (c) after aldehyde fixation the cells are osmotically active but come to a given volume in salt and sucrose solutions of concentrations only 60% of those which give their volume before fixation. Various possible mechanisms underlying the change of osmotic equilibrium caused by aldehyde fixation are discussed.

Morphology and electrical properties of Schwann cells around the giant axon of the squids Loligo forbesi and Loligo vulgaris.

The first successful dye-fills of Schwann cells around the split giant axon of Loligo show them to be spindle-shaped cells ca. 600 microns long and 20 microns wide lying parallel to the axonal axis. There are some 50,000 Schwann cells per cm2 of axonal membrane. Only a small part (ca. 6% of each Schwann cell membrane) is in contact with the periaxonal space, the remainder is overlain by adjacent Schwann cells, or applied to the basal lamina. The mean membrane potential of the Schwann cells in artificial seawater (ASW) varies from around -40 mV in fresh split-axon preparations to around -60 to -70 mV after 1-2 h; this hyperpolarization is not seen in preparations dissected and maintained in Ca2(+)-free ASW. Electrical- and dye-coupling (abolished by prior octanol treatment) is present between Schwann cells, but is weaker in cells with lower (less negative) membrane potentials. The implications for potassium homeostasis around the axon are briefly discussed.

Immunoreactive human calcitonin-like molecule in the nervous systems of protochordates and a cyclostome, Myxine.

A molecule very closely resembling human calcitonin immunologically and chromatographically was extracted from the nervous systems of several protochordates and a cyclostome, Myxine. The presence of human calcitonin-like molecules in the nervous systems of primitive chordates suggests that they have some function in the nervous system of these species and that the bone-regulating function of the calcitonins may have arisen much later in the vertebrates.

The placenta of the salp (Tunicata: Thaliacea).

The morphology of the mature 'placenta' of the pelagic tunicate Salpa fusiformis is described, and it is shown that two syncytial layers, intimately connected by interdigitating microvilli, separate maternal and embryonic circulations. The central placental layer facing the maternal circulation is bordered by membrane infoldings; the cortical layer facing the embryonic circulation is bordered by extensively branching microvilli. Both layers are of maternal origin, although embryonic leucocytes pass into, and add to, the cortical layer.

Contractile properties of obliquely striated muscle from the mantle of squid (Alloteuthis subulata) and cuttlefish (Sepia officinalis)

The mechanical properties of obliquely striated muscle fibres were investigated using thin slices of mantle from squid Alloteuthis subulata and cuttlefish Sepia officinalis. Brief tetani or twitch stimuli were used as this pattern is likely to occur during jetting of the intact animal. The length­active force relationship for twitches and tetani (0.1s, 50Hz) was similar to that of vertebrate cross-striated fibres. Passive force at the length giving maximum tetanic force was 0.13±0.05P0 (mean ± s.e.m., N=6, where P0 is maximum isometric tetanus force) and increased steeply at longer lengths. Peak force in a brief isometric tetanus (0.2s, 100­150Hz) was 262±16mNmm-2 cross-sectional area of wet tissue (N=6) for squid, and 226±19mNmm-2 (N=7) for cuttlefish. The force­velocity relationship for isotonic shortening during twitches of squid mantle slices was a 'double hyperbolic' relationship as described for cross-striated fibres by Edman. Fitting Edman's equation to the results gave: P*=1.18±0.07, Vmax=2.43±0.11Ltws-1 and 1/G=0.69±0.13 (N=8), where P* is the intercept on the force axis expressed relative to Ptw, peak isometric twitch force, Vmax is the intercept on the velocity axis, Ltw is the length at which Ptw is produced and G is the constant expressing curvature. The large values of 1/G indicate that the force­velocity relationship is not very curved. Maximum power was produced during shortening at 0.45±0.03Ptw (N=8). Maximum power during twitch contraction was 18.3±1.7mWg-1wetmass or, expressed in relative units, (V/Vmax)(P/Ptw), where V is the velocity during shortening and P is the force during shortening, was 0.16±0.01 (N=8), which is higher than that of many cross-striated locomotor muscles.

Locomotion and propagated skin impulses in salps (Tunicata: Thaliacea).

1. Various observations by M. Fedele on the mechanism of forward and reverse locomotion, on the neurogenic origin of the locomotor rhythm and on the coordinated behavior of salp chains are confirmed or extended. Salpa fusiformis was the species chiefly studied. 2. The striated muscle fibers of the body wall exhibit nonpropagative, graded responsivity. The fibers are multiply-innervated. Adjacent fibers are not electrically coupled. 3. Intracellular recordings are reported from a pacemaker and presumed motor neurons in the brain. The locomotor rhythm is exhibited by deafferented and isolated brains. In the intact animal, sensory input can modify the rhythm and alter the firing sequence of the muscles. The rhythm is accelerated by reduction, and inhibited by elevation of the ambient light intensity. 4. The outer skin is a conducting epithelium. The cells conduct action potentials at ca. 17 cm/sec and are connected by gap junctions. Three other independently conducting inner epithelial territories are described. Propagated impulses in the excitable epithelia are believed to enter the nervous system via neurosensory processes in the skin, extending the effective fields of these receptors. 5. Salp chains show coordinated responses but, except in their earliest developmental stages, impulses are probably not through-conducted along the chain, but are relayed from one zooid to the next by an unknown mechanism. 6. Comparisons are drawn between salps and other pelagic tunicates where conducting epithelia have previously been reported.

GABAergic synaptic transmission modulates swimming in the ascidian larva.

To examine the role of the amino acid GABA in the locomotion of basal chordates, we investigated the pharmacology of swimming and the morphology of GABA-immunopositive neurones in tadpole larvae of the ascidians Ciona intestinalis and Ciona savignyi. We verified that electrical recording from the tail reflects alternating muscle activity during swimming by correlating electrical signals with tail beats using high-speed video recording. GABA reversibly reduced swimming periods to single tail twitches, while picrotoxin increased the frequency and duration of electrical activity associated with spontaneous swimming periods. Immunocytochemistry for GABA revealed extensive labelling throughout the larval central nervous system. Two strongly labelled regions on either side of the sensory vesicle were connected by an arc of labelled fibres, from which fibre tracts extended caudally into the visceral ganglion. Fibre tracts extended ventrally from a third, more medial region in the posterior sensory vesicle. Two rows of immunoreactive cell bodies in the visceral ganglion extended neurites into the nerve cord, where varicosities were seen. Thus, presumed GABAergic neurones form a network that could release GABA during swimming that is involved in modulating the time course and frequency of periods of spontaneous swimming. GABAergic and motor neurones in the visceral ganglion could interact at the level of their cell bodies and/or through the presumed GABAergic fibres that enter the nerve cord. The larval swimming network appears to possess some of the properties of spinal networks in vertebrates, while at the same time possibly showing a type of peripheral innervation resembling that in some protostomes.

Locomotor adaptations of some gelatinous zooplankton.

Swimming behaviour and locomotor adaptations are described in chaetognaths, larvacean tunicates, some cnidaria, and thaliacean tunicates. The first two groups swim by oscillating a flattened tail, the others by jet propulsion. In chaetognaths, the locomotor muscle fibres are extensively coupled and relatively sparsely innervated, they exhibit compound spike-like potentials. The motoneurons controlling the rhythmic activity of the locomotor muscle lie in a ventral ganglion whose organization is briefly described. Rhythmic swimming bursts in larvaceans are similarly driven by a caudal ganglion near the base of the tail, but each caudal muscle cell is separately innervated by two sets of motor nerves, as well as being coupled to its neighbours. The external epithelium is excitable, and linked to the caudal ganglion by the axons of central cells. Mechanical stimulation of the epithelium evokes receptor potentials followed by action potentials and by bursts of rapid swimming. The trachyline medusa Aglantha and the small siphonophore Chelophyes also show rapid escape responses; in Aglantha these are driven by a specialized giant axon system lacking in other hydromedusae, and in Chelophyes. Slow swimming in Aglantha apparently involves a second nerve supply to the same muscle sheets used in rapid swimming, whereas in Chelophyes slow swimming results from the activity of the smaller posterior nectophore. Slow swimming in siphonophores is more economical than the rapid responses. In the hydrozoan medusa Polyorchis (as in Chelophyes) action potentials in the locomotor muscle sheet change in shape during swimming bursts, and their duration is related to the size of the medusa; they are not simply triggers of muscular contraction. The two groups of thaliacean tunicates are specialized differently. Doliolum is adapted for single rapid jet pulses (during which it achieves instantaneous velocities of 50 body lengths s-l), whilst salps are adapted for slow continuous swimming. The cost of locomotion is greater in Doliolum. Few gelatinous zooplankton show special adaptations both for rapid escape movements, and for slow sustained swimming, those that do deserve further study.

On the respiratory flow in the cuttlefish sepia officinalis.

The respiratory flow of water over the gills of the cuttlefish Sepia officinalis at rest is produced by the alternate activity of the radial muscles of the mantle and the musculature of the collar flaps; mantle circular muscle fibres are not involved. Inspiration takes place as the radial fibres contract, thinning the mantle and expanding the mantle cavity. The rise in mantle cavity pressure (up to 0.15 kPa), expelling water via the siphon during expiration, is brought about by inward movement of the collar flaps and (probably) mainly by elastic recoil of the mantle connective tissue network 'wound up' by radial fibre contraction during inspiration. Sepia also shows a second respiratory pattern, in which mantle cavity pressures during expiration are greater (up to 0.25 kPa). Here, the mantle circular fibres are involved, as they are during the large pressure transients (up to 10 kPa) seen during escape jetting. Active contraction of the muscles of the collar flaps is seen in all three patterns of expulsion of water from the mantle cavity, electrical activity increasing with increasing mantle cavity pressures. Respiratory expiration in the resting squid Loligo vulgaris is probably driven as in Sepia, whereas in the resting octopus Eledone cirrhosa, the mantle circular musculature is active during expiration. The significance of these observations is discussed.

Ciliary Feeding Assisted by Suction From the Muscular Oral Hood of Phoronid Larvae.

Phoronid larvae have an oral hood upstream from a postoral encircling array of ciliated tentacles. Cilia move water over the hood and between the tentacles, from anterior to posterior. When an algal cell or other particle in this current contacts a tentacle, the neighboring part of the hood lifts, and the particle is drawn toward the mouth. The correlation between movements of hood and particles indicates that the particle moves with water entering the enlarged space beneath the hood. Each lift of the hood is preceded by contact between a particle and a tentacle. A hood lift follows contact with a particle anywhere along the length of a tentacle, and clearance rates are thus proportional to the total length of tentacles deployed and the velocity of the current past the tentacles. After being detained at the ciliary bands of tentacles, particles are transported by the hood lift at speeds exceeding measured transport along the frontal ciliated surfaces of other larval forms. Faster transport may aid capture of faster prey. The larva's feeding mechanism is unique to the phylum Phoronida. Larvae of brachiopods, bryozoans, hemichordates, and echinoderms have similar ciliary bands producing feeding currents, but none are known to transport food toward the mouth by suction produced by muscle contractions.

Contraction and relaxation in the absence of a sarcoplasmic reticulum: muscle fibres in the small pelagic tunicate Doliolum.

Previous ultrastructural observations suggested that Doliolum muscle fibres apparently lacked both sarcoplasmic reticulum and transverse tubular-system. External Ca2+ is required for contraction, caffeine does not evoke contraction, nor does it increase intracellular Ca2+ level. Ryanodine at 50 microM has no effect on electrically-evoked contractions. Further, electrical stimulation in external solutions lacking Na+ leads to sustained contracture. We conclude that intracellular Ca2+ stores are absent in these rapid obliquely-striated fibres, and that reduction in internal Ca2+ levels following contraction depends upon Na(+)-Ca2+ exchange across the sarcolemma.

Excitation-contraction coupling in isolated locomotor muscle fibres from the pelagic tunicate Doliolum which lack both sarcoplasmic reticulum and transverse tubular system.

In the locomotor muscle of the pelagic tunicate Doliolum, both the sarcoplasmic reticulum (SR) and the transverse-tubular (T-tubular) system are absent. The mechanism of excitation-contraction (E-C) coupling was studied in single muscle fibres enzymatically dissociated from Doliolum denticulatum. Whole cell voltage clamp experiments demonstrated an inward ionic current associated with membrane depolarisation. This current was blocked by 5 mmol.l(-1)Co(2+), a calcium current blocker, and suppressed by nifedipine, a specific L-type calcium channel blocker. An increase in the external K(+) concentration to 200 mmol.l(-1) (K(+)-depolarisation) induced a rise in the intracellular Ca(2+) level detected with fluo-3, a Ca(2+)-sensitive dye. However, when 5-10 mmol.l(-1) Co(2+) or 10-15 micro mol.l(-1) nifedipine was present in the external solution, K(+)-depolarisation did not induce a rise in the intracellular Ca(2+) level. Externally applied 5-10 mmol.l(-1) caffeine or 20 micro mol.l(-1) ryanodine had no effect on the intracellular Ca(2+) level. K(+)-depolarisation induced a rise in the intracellular Ca(2+) level in the presence of caffeine or ryanodine. Replacement of external Na(+) with Li(+) increased intracellular Ca(2+) levels. Our results show that contraction of the locomotor muscle in Doliolum is solely due to the influx of Ca(2+) through L-type calcium channels, and that relaxation is due to extrusion of Ca(2+) by Na(+)/Ca(2+) exchange across the sarcolemma.

Epithelial action potentials in embryos of the Australian lungfish.

The epithelial cells of the skin of embryonic Australian lungfish (Neoceratodus forsteri) between stages 30 and 39, are mechanosensitive and excitable, showing overshooting action potentials 400-800 ms long with a rapid rise followed by a slower repolarization (usually with a shoulder on the repolarizing phase), which propagate at around 10 mm s-1. This skin impulse system is very similar to that found in embryonic and larval Amphibia; the significance of this similarity is discussed.

Energy storage by passive elastic structures in the mantle of sepia officinalis.

The passive elastic properties of the mantle of the cuttlefish Sepia officinalis have been characterized in experiments on intact mantle and on pieces cut from the mantle. The mantle was found to be very compliant over a wide range of circumferential strains, corresponding to a change in mantle circumferential strain of 0.45. Beyond this range of strain, the mantle was much stiffer, in both the circumferential direction, 0.542+/-0.025 MPa (mean +/- s.e.m., N=51) and through the thickness of the mantle wall, 0.152+/-0.041 MPa (N=11). Almost 80 % of the work done on the tissue during compression in the circumferential direction was recovered during elastic recoil of the tissue; this elastic work could contribute to refilling the mantle after a jet. Our estimates of the work done during a cycle of jetting and refilling show that such elastic work is small (approximately 1 %) compared with the contractile work done by the circular muscle fibres. However, although the elastic work is almost negligible in the overall energy budget, it is probably sufficient to power refilling of the mantle.

Stretch receptors in urodele limb muscles.

Non-encapsulated, fine beaded nerve endings were found histologically on some muscle fibres in a number of limb muscles in newts and axolotls. They were present in newt muscles that had been chronically de-efferented, and in which no efferent activity survived, and were therefore likely to be sensory. They were located only on muscle fibres on or near the outside surface of the muscle. These small-diameter muscle fibres were characterised histochemically by low lipid, SDH and phosphorylase content; ultrastructurally by low glycogen content, and relatively large myofilaments poorly delimited by a sparse SR. There were many of this type (Type 1) that did not support sensory endings. A few endings occurred on another larger-diameter type of fibre (Type 2) whose properties were opposite to those listed above for Type 1. There was virtually no specialization of muscle fibre structure beneath the sensory endings. Physiological experiments involving ramp-and-hold and sinusoidal stretch applied to the muscle whilst recording single-unit afferent responses in m.ext. dig. III of axolotls showed unit responses very similar to those known from muscle spindles, particularly those of the frog.

Activation of locomotor and grasping spine muscle fibres in chaetognaths: a curious paradox.

Chaetognath muscle fibres resemble vertebrate muscle fibres in having an abundant sarcoplasmic reticulum (SR) and analogues of the transverse (T) tubular system. but contraction is regulated differently. In intact chaetognaths electrically-evoked contractions of the striated locomotor muscles were largely or totally blocked by d-tubocurarine, by surgical removal of the ventral ganglion and by Co2 +. Contractions of single cells enzymatically dissociated from locomotor muscles were likewise blocked by Co2+, they twitched once only after calciseptine, showed neither contractures nor elevated intracellular Ca2+ with caffeine, and ryanodine did not block contractions. Whole cell voltage-clamped locomotor muscle cells displayed a typical inward rectified Ca2 + current that was sensitive to the Ca2+ channel blockers nifedipine and calciseptine and showed voltage-dependent activation with a threshold at approximately-25 mV and a peak inward current at approximately + 10 mV. In contrast, whole cell voltage-clamped cells from the muscles operating the grasping spines of the head showed an initial very rapid and rapidly-inactivating inward current abolished by tetrodotoxin (TTX), followed by a slower and slowly-inactivating inward current blocked by calciseptine. The relation between these observations and the unusual 'vertebrate-like' structure of the muscle cells is discussed.

Contractile properties and ultrastructure of three types of muscle fibre in the dogfish myotome.

Three main types of fibre can be differentiated in the adult dogfish myotome at the immediate post-anal level. An outer band of muscle consists of 80-90 pale multiply innervated fibres (superficial fibres). These fibres are 80-90 micron in diameter, lack M-lines and have a low Ca2+-activated myosin ATPase activity. Volume densities of myofibrils (Vv(my,f] and mitochondria (Vv(mt,f] are respectively 76 and 9.5%. Beneath this layer are around 8000 red multiply innervated fibres. These have an average diameter of 25-40 micron. Vv(my,f) and Vv(mt,f) are 62 and 21% respectively, and M-lines are present. Around 11 000 white focally innervated twitch fibres lie beneath the red fibre zone. White fibres with an average diameter of 80-120 micron have a high Ca2+-activated myosin ATPase activity and Vv(my,f) and Vv(mt,f) are 78 and 5% respectively. Contractile properties of single skinned fibres were determined at 12 degrees C. Maximum Ca2+ activated tensions (kN m-2) and unloaded contraction speeds (muscle lengths s-1) were 49 and 0.5 for superficial, 70 and 1.4 for red and 180 and 4.4 for white muscle fibres. Superficial fibres have not been reported in other elasmobranchs with the exception of the closely related nursehound (Scyliorhinus stellaris L.) It is suggested that they are specialized for sustained force generation, having a tonic (postural) rather than a locomotor role.