The tubular vacuolation process in amphibian skeletal muscle

The exposure of amphibian muscle to osmotic shock through the introduction and subsequent withdrawal of extracellular glycerol causes vacuolation in the transverse tubules. Such manoeuvres can also electrically isolate the transverse tubules from the surface (detubulation), particularly if followed by exposures to high extracellular [Ca2+] and/or gradual cooling. This study explored factors influencing vacuolation in Rana temporaria sartorius muscle. Vacuole formation was detected using phase contrast microscopy and through the trapping or otherwise of lissamine rhodamine dye fluorescence within such vacuoles. The preparations were also examined using electron microscopy, for penetration into the transverse tubules and tubular vacuoles of extracellular horseradish peroxidase introduced following the osmotic procedures. These comparisons distinguished for the first time two types of vacuole, open and closed, whose lumina were respectively continuous with or detached from the remaining extracellular space. The vacuoles formed close to and between the Z-lines, but subsequently elongated along the longitudinal axis of the muscle fibres. This suggested an involvement of tubular membrane material; the latter appeared particularly concentrated around such Z-lines in the electron-micrograph stereopairs of thick longitudinal sections. Open vacuoles formed following osmotic shock produced by extracellular glycerol withdrawal from a glycerol-loaded fibre at a stage when one would expect a net water entry to the intracellular space. This suggests that vacuole formation requires active fluid transport into the tubular lumina in response to fibre swelling. Closed vacuoles only formed when the muscle was subsequently exposed to high extracellular [Ca2+] and/or gradual cooling following the initial osmotic shock. Their densities were similar to those shown by open vacuoles in preparations not so treated, suggesting that both vacuole types resulted from a single process initiated by glycerol withdrawal. However, vacuole closure took place well after formation of open vacuoles, over 25 min after glycerol withdrawal. Its time course closely paralleled the development of detubulation reported recently. It was irreversible, in contrast to the reversibility of open vacuole formation. These findings identify electrophysiological detubulation of striated muscle with closure of initially open vacuoles. The reversible formation of open vacuoles is compatible with some normal membrane responses to some physiological stresses such as fatigue, whereas irreversible formation of closed vacuoles might only be expected in pathological situations as in dystrophic muscle.This revised version was published online in September 2005 with corrections to the Cover Date.     

Changes in sizes of the adrenaline-containing vesicles and their cores in frog cardiac sympathetic nerves after pharmacological treatments

Summary The sizes of adrenergic vesicles and their cores, as made visible by an acrylic aldehyde in sodium dichromate fixative, have been measured in electron micrographs of the sympathetic nerves amongst the frog's ventricular muscle. The animals were either normal or previously treated with drugs expected to affect the catecholamine content of the heart. The sympathetic nerves contain two overlapping populations of vesicles. A graphical method was used to separate these and determine the mean diameter of each population. The distribution of vesicles between the large and small populations is variable in normal animals. No changes could be detected in the experimental animals. The mean size of the large vesicles is variable in normal animals. No changes could be detected in the experimental animals. Four injections of 5-hydroxydopamine caused a 5.8% increase in the diameter of the small vesicles. No other treatment produced significant changes in vesicle size. Four injections of 5-hydroxydopamine caused a 50% increase in the diameter of the cores of the small vesicles. Two injections of reserpine caused a 20% reduction in the diameter of the visible cores in the small vesicles, and 34% of the vesicles lost their cores entirely. One injection of 6-hydroxydopamine or ten injections of -methyl-tyrosine caused small reductions in small core diameter. It is postulated that core formation in adrenergic nerves under these conditions is not solely dependent on their catecholamine content, but on this and another factor which may be part of the storage complex.     

Do ‘evaluation potentials’ reflect cognitive assessment?

Summary Event-related potentials were recorded when a subject evaluated the outcome of a simple TV game as successful/unsuccessful, where the goal was specified randomly as one of two areas on the screen. The evaluation potential elicited by the outcome was consistently larger for unsuccessful outcomes, regardless of the location of the goal.     

Mediation of macrophage effector function by lymphokine

The progressive effect of lymphokine contact on macrophages in vitro has been studied using various quantitative cytochemical techniques. Changes in the physiology of the macrophages have been seen rapidly after lymphokine contact. These appear to correlate with the functional effect of inhibition of migration. After more prolonged contact with lymphokine however, the macrophages exhibit different changes to their physiology and reach a state of enhanced cytochemical activity which has been termed activation. By comparing the rapid effect of lymphokine to the changes seen after prolonged contact it is suggested that one can rationalize the apparent paradoxical effects of these soluble mediators which appear initially to turn off the macrophage and subsequently activate the same cell. In doing so an hypothesis is put forward as to how this mediator might work in vivo.     

The synaptonemal complex — the chaperone of crossing over

During meiosis homologous chromosomes pair and exchange homologous chromosome segments. The synaptonemal complex (SC) forms between paired chromosomes. The role of the SC in the process of reciprocal exchange of flanking markers is a matter of debate. I propose a dual pathway for reciprocal exchange of flanking markers (REFM). In the first, SC-independent, path, two half-nodules and an independent REFM protein combine to form a functional recombination nodule (RN). The RN binds to paired chromosomes and accomplishes reciprocal exchange of flanking markers. In the other, SC-dependent, pathway half-nodules occur at pairing initiation sites. Half-nodules move along the SC as it forms. Assisted by an SC-bound REFM protein, half-nodules combine to form functional RNs. I propose that different organisms rely to different extents on the two pathways, and hence rely to different extents on the SC.accepted for publication by H. C. Macgregor     

A new sequence variant ofColeus blumei viroid 1 from theColeus blumei cultivar ‘Rainbow Gold’

Summary A viroid was isolated from symptomlessColeus blumei cultivar (cv.) Rainbow Gold plants using the bidirectional PAGE method for analysis of small circular RNA molecules. The viriod was transmitted to viroid-free plants ofColeus blumei cv. Scarlet Dragonfly by mechanical inoculation. Cloning and sequencing revealed that the viriod from theColeus cv. Rainbow Gold is closely related to theColeus blumei viriod 1-BvA (CbVd 1-BvA) isolated from theColeus cv. Bienvenue. Therefore, new viroid sequence variant has been namedColeus blumei viriod 1-RG (CbVd 1-RG).Coleus blumei viriod 1-RG consists of 251 nucleotides, 140 G + C, 111 A + U with a GC content of 55.4%. The most stable rod-like secondary structure of this viroid has 53 G:C, 29 A:U and 5 G:U base pairs with a minimum free energy (at 25°C) of – 81.2 kcal/mol (– 339.4 kJ/mol). The right terminal domain shows a high sequence similarity to the corresponding domain of hop latent viroid (HLVd).The sequence data reported here have been submitted to the EMBL database and have been assigned the accession numbers X95291 (for CbVd 1-RG) and X57284 (for CbVd 1-BvA).     

Oculomotor response to rapid head oscillation (0.5–5.0 Hz) after prolonged adaptation to vision-reversal

Summary This study examines long-term (up to 27 days) effects of maintained vision reversal on (i) smooth visual tracking with head still, (ii) oculomotor response to actively generated head oscillation and (iii) spontaneous saccades. Dove prism goggles produced horizontal, but not vertical (sagittal plane), vision reversal. Eye movements were recorded by EOG; head movements by an electro-magnetic search coil.Both visual tracking and saccade dynamics remained unchanged throughout. In contrast, both the ocular response to active head osculations (goggles off and subject looking at a stationary target) and associated retinal image blur showed substantial and retained adaptive changes, akin to those previously found in the vestibulo-ocular reflex as tested in darkness at 0.17 Hz.However, several additional unexpected results emerged. First, in the fully adapted state smooth eye movements tended to be of reversed phase in the range 0.5–1.0 Hz (in spite of normal vision during tests), but of normal phase from about 2 Hz and above (in spite of negligible visual tracking in this upper range). Second, after permanent removal of the inverting goggles, this peculiar frequency response of the fully adapted state quickly (36 h) reverted to a dynamically simpler condition manifest as retained (2–3 weeks) attenuation of gain (eye vel./ head vel.) which, as in control conditions, was monotonically related to frequency. From these two findings it is inferred that the fully adapted state may have comprised two separate components: (i) A simple element of monotonic and long-lasting gain attenuation and (ii) a complex, frequency labile, element which could be quickly rejected. Dynamic characteristics of the putative complex element were estimated by vectorial subtraction of the simple one from that of the fully adapted condition. The outcome suggests that the inferred complex condition might represent a predictive element.Two further findings are reported: (i) Substantially different vectors of the adapted response were obtained with normal and reversed vision at 3.0 Hz head oscillation, indicating a novel visual influence acting above the cut-off frequency for visual tracking. (ii) During head oscillation in the vertical sagittal plane (in which vision was not reversed) there was never any image blur, indicating high geometric specificity in the adaptive process.Supported by Canadian Medical Research Council Operating Grant No. MT5630     

Are proteasomes involved in the formation of the kinetochore?

Proteasomes catalyse the degradation of proteins responsible for the regulation of mitosis enabling the cell to complete cell division. We have studied the effect of an inhibitor of the chymotrypsin-like activity of the proteasome on the trilaminar structure of the kinetochore in HeLa cells. Whereas a role for the proteasome in the degeneration of the kinetochore was predicted, we found instead that the inhibitor strongly retarded kinetochore development. We observed different developmental stages of the kinetochore from the fibrous ball of a prekinetochore to the mature kinetochore in one cell. The data presented here support the proposition that proteasomes are involved in kinetochore formation.accepted for publication by H. C. MacgregorDedicated to the memory of Prof. Dr. Daniel Mazia, a fatherly friend.     

Chromaticity of synaptic inputs to H1 horizontal cells in carp retina: analysis by voltage-clamp and spectral adaptation

Summary Cone photoreceptor inputs to H1 horizontal cells (H1 HCs) in carp retina were studied by measuring light-modulated currents (I_L) to monochromatic stimuli (460, 533, 688 nm) under a voltage-clamp condition. By using double-barrelled micro-electrodes H1 HCs were voltage-clamped whilst perfusing with dopamine to uncouple the cells. The I_L of the H1 HCs driven by each cone input was segregated by selective chromatic adaptation, and differences in the kinetics of the I_L of the H1 HCs were revealed. Thus, all together, three types of I_L were observed: (1) a fast outward current to the long-wavelength stimulus; (2) a slow outward current to the middle-wavelength stimulus; and (3) a delayed inward current that followed the peak of slow outward current to the short-wavelength stimulus. The reversal potentials of the three currents were estimated to be at least 20 mV more positive than the dark resting potential by extrapolation of the I_L-V curve. These observations are consistent with the idea that the H1 HCs receive sign-inverting, conductance decreasing synaptic input(s) from at least one other cone mechanism, in addition to the main conventional EPSP type synaptic input from red-sensitive cones.     

The influence of display characteristics on active pursuit and passively induced eye movements

Summary A series of experiments has been conducted on human subjects to examine the effect of the movement of small targets located in the peripheral visual field on oculomotor response. Subjects were presented with either a single centrally positioned target or a pair of targets displaced at angles of ±5°, ±10° and ±20° from centre. Target movement was in the horizontal plane, the paired targets always moving in unison. The stimulus waveform consisted of either a sinusoidal or random target motion encompassing a frequency range from 0.1 to 4 Hz with an angular displacement of ±3.5°. Subjects made two types of response. First they were instructed to follow the single target or the centre point of the paired targets. In this active pursuit condition the gain of slow-phase eye velocity progressively decreased as the moving targets were moved from the central position to the most peripheral location (±20°). Secondly, subjects were required passively to ignore the target movement by staring blankly ahead. During this passive response nystagmic eye movements were induced for which the slowphase eye velocity also decreased with increasing target eccentricity, but the gains were always less than those induced during active pursuit. The frequency characteristics of the passive response were very similar to those of the active response, breaking down at frequencies beyond 1 Hz. The ability to suppress the passive response was also investigated by the presentation of a tachistoscopically illuminated earth-fixed target. The response was found to decline as the interval between presentations of the fixation target was decreased from 3000 ms to 100 ms. It is suggested that the passive response originates from a basic velocity drive to the oculomotor system resulting from image movement across the retina. This velocity drive may be cancelled with adequate fixation but must be enhanced to accomplish desired eye velocity during active pursuit.     

Haematological findings in cats naturally infected with feline immunodeficiency virus

The types and prevalence of haematological abnormalities occurring in FIV infected cats were determined. In addition, the role of FIV infection per se in influencing haematological values was examined by analysing results between infected and non-infected cats which had been allocated to similar clinical disease groups. FIV-positive cats were grouped as asymptomatic carriers (AC), cats with AIDS-related complex (ARC) or AIDS. FIV-negative cats were placed into matched groups using the same criteria and designated as healthy, ARC or AIDS. Healthy FIV-negative cats also formed the reference ranges for peripheral blood and bone marrow. Anaemia was no more frequent in sick (ARC and AIDS) FIV-positive cats than sick (ARC and AIDS) FIV-negative cats. However, it was observed more frequently in FIV-positive cats than FIV-negative cats in the absence of concurrent disease, suggesting a direct effect of FIV infection. Bone marrow was affected by FIV infection; as evidenced by anaemic FIV-positive cats having proportionally less Type I reticulocytes than anaemic FIV-negative cats. In addition, FIV-positive cats demonstrated proportionally fewer mature erythroid cells in their marrow. This implied that FIV may cause a decreased life span or maturation arrest of the erythroid cell line. Lymphopenia and eosinopenia were seen more frequently in AC FIV-positive cats than healthy FIV-negative cats, suggesting the direct involvement of FIV. Thus, although FIV infection affected some haematological findings in AC cats, it appeared that haematological abnormalities in sick FIV-positive cats may be due as much to the disease state as to the virus specifically. Apart from the subjective assessment that bone marrow of FIV-positive cats appeared hypercellular, there were no pathognomonic features for FIV infection.     

Localization of epitopes and functional effects of two novel monoclonal antibodies against skeletal muscle myosin

Summary Two skeletal myosin monoclonal antibodies, raised against human skeletal myosin, were used to study the correlation between function, primary and tertiary structure of S-1 prepared from rabbit skeletal myosin. The heavy chain of S-1 is cleaved into three fragments by trypsin—27 kDa, 50 kDa and 20 kDa—aligned in this order from the N-terminus. The epitope of the first antibody was assigned to the N-terminal 1–23 amino acid stretch of S-1, since it reacted with the 27 kDa N-terminal tryptic fragment of S-1 but not with a derivative of the 27 kDa fragment, which lacks the above amino acid stretch. The epitope of the second antibody was assigned to the 3 kDa N-terminal region of the central 50 kDa domain of S-1. This assignment was based on proteolytic and photochemical cleavage of S-1 and on the labelling of its N-terminus by a specific antibody. The antibodies were visualized binding to the myosin head on electron micrographs of rotary-shadowed complexes of antibodies with myosin. Measurements on the micrographs indicated that the distances between the head-tail junction of myosin and the anti-27 K and anti-50 K epitopes are 14 nm and 17 nm, respectively. Both antibodies have a high affinity to S-1. The affinity of the anti-50 K to S-1 decreased upon actin binding, while that of the anti-27 K was not affected by binding of S-1 to F-actin. The anti-50 K antibody inhibited the K⁺ (EDTA) and the actin-activated ATPase activity of S-1, while the anti-27 K had no effect. The results indicate that either the epitope of the anti-50 K is near to the actin or to the ATP-binding sites of S-1, or that there is communication, expressed as propagated conformational changes, between these sites and the epitope.     

Synergistic activation of rat supraoptic neurosecretory neurons by noxious and hypovolemic stimuli

Summary The effects of saphenous nerve stimulation on discharge activity of supraoptic neurosecretory (NS) cells were studied in anesthetized rats. Of 112 supraoptic neurosecretory cells, 62 exhibited a phasic discharge pattern. The nerve stimulation transiently excited 46 of these 62 phasic units, as well as 35 of the 50 remaining non-phasic units. No appreciable blood pressure change was noted using PSTHs with 1-ms resolution. Though the nerve stimulation also evoked a flexor reflex of the ipsilateral hind limb, blockage of the hind limb movement with gallamine did not alter the amplitude of the supraoptic cell excitation. The threshold of the nerve stimulation was higher for the excitation than for the flexor reflex. Effects of hypovolemic and hyperosmotic stimuli on discharge activity of phasic cells during saphenous nerve stimulation were studied to find a possible interaction between these stimuli. Hemorrhage potentiated the transient excitation evoked by the nerve stimulation in all of the 8 phasic cells tested, while no such effect was seen after an injection of hypertonic sodium chloride solution in the 7 phasic cells tested. These electrophysiological data suggest that hypovolemic and noxious stimuli potentiate VP secretion in a synergistic manner but that hyperosmotic and noxious stimuli do not.Supported by grants from the Ministry of Education, Science and Culture, Japan     

A comparative immunohistological study of cerebellar enolases

Summary A comparative immunohistological study of the neurone-specific enolase and enolase, demonstrates the exclusive neuronal localization of enolase and its absence from glial cells. In contrast, enolase is located in astroglial cells. The validity of enolase as a neuronal marker and enolase as an astrocytic marker, is confirmed both by a double labelling technique, using antibodies to and to revealed with fluorescence or peroxidase in the same tissue sections, and by immunoelectronmicroscopy.     

Ultrastructural and X-ray microanalytical studies of EGTA- and detergent-treated heart muscle

Summary The efficacy of the EGTA-treatment for producing a model of selectively skinned cardiac muscle has been questioned. This paper deals with ultrastructural evidence designed to test whether small ions can gain access to the myofibrillar space of the heart after EGTA-treatment. Lanthanum has been employed because of its widespread use as an extracellular marker and because its presence can be unequivocally demonstrated by X-ray microanalysis. The results of both standard transmission electron microscopy and X-ray microanalysis reveal that, despite some deterioration of the ultrastructure after EGTA-treatment at 2° C for 24 h, lanthanum is still apparently excluded from the intracellular spaces. Parallel runs with detergent treatments, such as Triton X-100 and the alkaloid saponin, demonstrate that La³⁺ is deposited on the contractile proteins in readily detectable amounts in these circumstances. Even in areas of the sections devoid of visible electron-opaque deposits, recognizable by eye in standard transmission images, X-ray microanalysis frequently revealed the presence of La³⁺.It is concluded that the sarcolemma persists as a selective permeability barrier to small ions such as La³⁺ and LaEGTA after EGTA-treatment. These findings are complementary to the mechanical behaviour of chemically treated cardiac muscle.     

Modulation of corticospinal excitability and intracortical inhibition during motor imagery is task-dependent

Previous studies have clearly shown that motor imagery modulates corticospinal excitability. However, there is no clear evidence for the modulation of intracortical inhibition (ICI) during imagined task performance. The aim of this study was to use transcranial magnetic stimulation (TMS) to assess changes in corticospinal excitability and ICI during the imagined performance of two types of task. In Experiment 1, eight subjects performed phasic depression of a computer mouse button using the dominant index finger in time with a 1 Hz auditory metronome. Single and paired pulse magnetic stimuli were delivered at rest, and during the on and off phases of actual and imagined task performance. Motor evoked potentials (MEPs) were recorded from FDI and APB. In Experiment 2, eight subjects performed phasic isometric abduction of the dominant thumb in time with a 1 Hz auditory metronome. As before, single and paired pulse magnetic stimuli were delivered at rest, and during the on and off phases of actual and imagined task performance. In both experiments, the conditioning stimulus intensity was set to produce 50% inhibition at rest, to enable both increases and decreases in ICI during task performance to be detected. No significant temporal or spatial modulation of MEP amplitude or ICI was observed in Experiment 1. In contrast, MEP amplitude was significantly greater, and ICI significantly lower during the on phase of imagined task performance in Experiment 2. These results are most likely related to the higher levels of target muscle activation required during actual task performance and the greater anatomical distance between target and control muscles in Experiment 2. These task characteristics may influence the observed degree of corticospinal excitability and ICI modulation.     

Activated charcoal is as effective as fuller's earth or bentonite in paraquat poisoning

Summary In vitro investigations have shown that the adsorption capacity of activated charcoal (Kohle-Compretten, Ultracarbon, E. Merck, Darmstadt, FRG) is just as high as that of Fuller's earth (Surrey powder, Laporte Industries Ltd., Luton, GB) or Bentonite BP W.B. (Steetley Minerals Ltd., Milton Keynes, GB). Fuller's earth (Fullererde) from another manufacturer has had very poor adsorption properties and is thus not suitable for the treatment of paraquat poisoning. Animal experiments have shown that the curative effect of activated charcoal given 0.5, 1, 2, and 3 h after ingestion of 200 and 300 mg paraquat/kg body weight is equally as good or even better than that of Fuller's earth or Bentonite BP W.B.. Activated charcoal is a substitute of equal value to these mineral soils.     

A new approach for evaluation of the in vitro haemolytic potential of a solution of a new medicine

In the process of developing an intravenously injectable drug, its haemolytic potential must be considered. There are no Regulatory Guidelines for this kind of test. Many authors have set up different models, attempting to obtain early information about the behaviour of test compounds when injected into the bloodstream.In the present work, an in vitro static model is presented, which takes into account the injection rate (R _inj.) of the drug, and the blood flow rate (Q _v) of the vein in which the drug must be injected. From the relationship between these two parameters, the C_max, expressed as mg/ml, can be calculated. This latter parameter allows us to calculate the drug concentration which, at any moment during injection, comes into contact with a known aliquot of new' blood passing through the injection site. Furthermore, a dynamic test has been developed, which simulates an injection into the blood flow using a tubing system and infusion pumps set for the same R _ini. and Q _v values used in static test. Two injectable drugs, Valium® and Lanoxin®, and a commonly used vehicle, propylene glycol, have been tested by both the methods. These compounds have also been tested with another in vitro method (Prieur et al. 1973), in which a volumetric blood-to-test solution ratio of 1:1 is adopted for every drug tested, with neither R _inj. nor Q _v being taken into account. Results of the haemolytic potential obtained with the three tests have been compared.A good correlation has been observed between the static and the dynamic tests, whereas Prieur's model, which uses a drug-to-blood ratio which is far higher than in vivo, has been shown to give false positive results.It is concluded that a test for the evaluation of the haemolytic potential of drugs must take into account the pharmacodynamic characteristics of the formulation intended to be injected, and at least the blood flow rate. The proposed static test has been demonstrated to be an easy and reliable method of obtaining a true picture of the in vivo situation.     

Spontaneous and induced host-range mutants of cyanophage N-1

Summary The porcine paramyxovirus LPM recognizes , but not , anomers of sialic acid containing structures, specifically sialyl ( 2,3) lactose. The virus specificity is directed to the sialyl residue and to the C 4 axial OH and the C 6 CH2OH of the galactose present in this structure.     

Psilocybin-induced contraction of nearby visual space

Using apparent fronto-parallel plane (AFP) monitoring techniques, the relative stability of the abathic plane, i.e. Euclidean visual space, was investigated in 16 volunteers with a median age of 23.5 years under 160 g/kg psilocybininduced ergotropic arousal. Handwriting area and pressure were also measured in the same subjects.Drug-induced contraction of nearby visual space was inferred from changes of AFP curvature and tilt, as well as from increased handwriting area at drug peak. The rising horizon (Rennert) in the drawings of schizophrenics is also considered a manifestation of the contraction of visual space and is described in terms of an arousal-dependent transformation of constancies. The projection of central nervous system activity as experience out there is also discussed as an arousal-dependent learned constancy.