A new species in the indigenous New Zealand soft scale insect genus Kalasiris Henderson & Hodgson (Hemiptera: Sternorrhyncha: Coccomorpha: Coccidae) on Gahnia setifolia (Cyperaceae).

The genus Kalasiris Henderson & Hodgson (Hemiptera: Sternorrhyncha: Coccomorpha: Coccidae) is currently only known from New Zealand. The adult female and pupa of a new species, K. martini Hodgson & Richmond are described and illustrated below and the possible taxonomic relationships of the genus to other New Zealand genera are discussed.

A post-docking role for active zone protein Rim.

Rim1 was previously identified as a Rab3 effector localized to the presynaptic active zone in vertebrates. Here we demonstrate that C. elegans unc-10 mutants lacking Rim are viable, but exhibit behavioral and physiological defects that are more severe than those of Rab3 mutants. Rim is localized to synaptic sites in C. elegans, but the ultrastructure of the presynaptic densities is normal in Rim mutants. Moreover, normal levels of docked synaptic vesicles were observed in mutants, suggesting that Rim is not involved in the docking process. The level of fusion competent vesicles at release sites was reduced fivefold in Rim mutants, but calcium sensitivity of release events was unchanged. Furthermore, expression of a constitutively open form of syntaxin suppressed the physiological defects of Rim mutants, suggesting Rim normally acts to regulate conformational changes in syntaxin. These data suggest Rim acts after vesicle docking likely via regulating priming.

An open form of syntaxin bypasses the requirement for UNC-13 in vesicle priming.

The priming step of synaptic vesicle exocytosis is thought to require the formation of the SNARE complex, which comprises the proteins synaptobrevin, SNAP-25 and syntaxin. In solution syntaxin adopts a default, closed configuration that is incompatible with formation of the SNARE complex. Specifically, the amino terminus of syntaxin binds the SNARE motif and occludes interactions with the other SNARE proteins. The N terminus of syntaxin also binds the presynaptic protein UNC-13 (ref. 5). Studies in mouse, Drosophila and Caenorhabditis elegans suggest that UNC-13 functions at a post-docking step of exocytosis, most likely during synaptic vesicle priming. Therefore, UNC-13 binding to the N terminus of syntaxin may promote the open configuration of syntaxin. To test this model, we engineered mutations into C. elegans syntaxin that cause the protein to adopt the open configuration constitutively. Here we demonstrate that the open form of syntaxin can bypass the requirement for UNC-13 in synaptic vesicle priming. Thus, it is likely that UNC-13 primes synaptic vesicles for fusion by promoting the open configuration of syntaxin.

UNC-13 is required for synaptic vesicle fusion in C. elegans.

We analyzed the synaptic physiology of unc-13 mutants in the nematode C. elegans. Mutants of unc-13 had normal nervous system architecture, and the densities of synapses and postsynaptic receptors were normal at the neuromuscular junction. However, the number of synaptic vesicles at neuromuscular junctions was two- to threefold greater in unc-13 mutants than in wild-type animals. Most importantly, evoked release at both GABAergic and cholinergic synapses was almost absent in unc-13 null alleles, as determined by whole-cell, voltage-clamp techniques. Although mutant synapses had morphologically docked vesicles, these vesicles were not competent for release as assayed by spontaneous release in calcium-free solution or by the application of hyperosmotic saline. These experiments support models in which UNC-13 mediates either fusion of vesicles during exocytosis or priming of vesicles for fusion.

One GABA and two acetylcholine receptors function at the C. elegans neuromuscular junction.

We describe an electrophysiological preparation of the neuromuscular junction of the nematode C. elegans, which adds to its considerable genetic and genomic resources. Mutant analysis, pharmacology and patch-clamp recording showed that the body wall muscles of wild-type animals expressed a GABA receptor and two acetylcholine receptors. The muscle GABA response was abolished in animals lacking the GABA receptor gene unc-49. One acetylcholine receptor was activated by the nematocide levamisole. This response was eliminated in mutants lacking either the unc-38 or unc-29 genes, which encode alpha and non-alpha acetylcholine receptor subunits, respectively. The second, previously undescribed, acetylcholine receptor was activated by nicotine, desensitized rapidly and was selectively blocked by dihydro-beta-erythroidine, thus explaining the residual motility of unc-38 and unc-29 mutants. By recording spontaneous endogenous currents and selectively eliminating each of these receptors, we demonstrated that all three receptor types function at neuromuscular synapses.

Slow inactivation in human cardiac sodium channels.

The available pool of sodium channels, and thus cell excitability, is regulated by both fast and slow inactivation. In cardiac tissue, the requirement for sustained firing of long-duration action potentials suggests that slow inactivation in cardiac sodium channels may differ from slow inactivation in skeletal muscle sodium channels. To test this hypothesis, we used the macropatch technique to characterize slow inactivation in human cardiac sodium channels heterologously expressed in Xenopus oocytes. Slow inactivation was isolated from fast inactivation kinetically (by selectively recovering channels from fast inactivation before measurement of slow inactivation) and structurally (by modification of fast inactivation by mutation of IFM1488QQQ). Time constants of slow inactivation in cardiac sodium channels were larger than previously reported for skeletal muscle sodium channels. In addition, steady-state slow inactivation was only 40% complete in cardiac sodium channels, compared to 80% in skeletal muscle channels. These results suggest that cardiac sodium channel slow inactivation is adapted for the sustained depolarizations found in normally functioning cardiac tissue. Complete slow inactivation in the fast inactivation modified IFM1488QQQ cardiac channel mutant suggests that this impairment of slow inactivation may result from an interaction between fast and slow inactivation.

Defective fast inactivation recovery and deactivation account for sodium channel myotonia in the I1160V mutant.

The skeletal muscle sodium channel mutant I1160V cosegregates with a disease phenotype producing myotonic discharges (observed as muscle stiffness) that are worsened by elevated K+ levels but unaffected by cooling. The I1160V alpha-subunit was co-expressed with the beta1-subunit in Xenopus oocytes. An electrophysiological characterization was undertaken to examine the underlying biophysical characteristics imposed by this mutation. Two abnormalities were found. 1) The voltage dependence of steady-state fast inactivation was reduced in I1160V, which resulted in faster rates of closed-state fast inactivation onset and recovery in I1160V compared with wild-type channels. 2) The rates of deactivation were slower in I1160V than in wild-type channels. Using a computer-simulated model, the combination of both defects elicited myotonic runs under conditions of elevated K+, consistent with the observed phenotype of the mutant.

Human Na+ channel fast and slow inactivation in paramyotonia congenita mutants expressed in Xenopus laevis oocytes.

1. Paramyotonia congenita (PC) is a human hereditary disease caused by one or more amino acid substitutions in skeletal muscle sodium channels. Using macropatches, the effect of PC mutations R1448C and T1313M were compared with wild-type (WT) in Xenopus oocytes coinjected with both alpha- and beta-subunits of human skeletal muscle (SkM1) sodium channels. 2. Slow inactivation in either T1313M or R1448C was not different from WT. Fast inactivation in both PC mutants, however, was significantly altered. 3. Commonly used biophysical protocols (such as I-V curves, steady-state inactivation curves, and measurements of inactivation rates) did not uniformly indicate that hyperexcitability should result from T1313M or R1448C. In fact, the only alteration of fast inactivation common to T1313M and R1448C that predicted cellular hyperexcitability was slowed open-state inactivation, compared with WT. 4. To test whether this alteration was sufficient to cause the phenotypic hyperexcitability, we used a novel voltage command that simulated muscle membrane activity. With this protocol, we found that R1448C and T1313M were similar in that they maintained a significantly higher channel availability during high frequency activity, compared with WT.

Interaction between fast and slow inactivation in Skm1 sodium channels.

Rat skeletal muscle (Skm1) sodium channel alpha and beta 1 subunits were coexpressed in Xenopus oocytes, and resulting sodium currents were recorded from on-cell macropatches. First, the kinetics and steady-state probability of both fast and slow inactivation in Skm1 wild type (WT) sodium channels were characterized. Next, we confirmed that mutation of IFM to QQQ (IFM1303QQQ) in the DIII-IV 'inactivation loop' completely removed fast inactivation at all voltages. This mutation was then used to characterize Skm1 slow inactivation without the presence of fast inactivation. The major findings of this paper are as follows: 1) Even with complete removal of fast inactivation by the IFM1303QQQ mutation, slow inactivation remains intact. 2) In WT channels, approximately 20% of channels fail to slow-inactivate after fast-inactivating, even at very positive potentials. 3) Selective removal of fast inactivation by IFM1303QQQ allows slow inactivation to occur more quickly and completely than in WT. We conclude that fast inactivation reduces the probability of subsequent slow inactivation.

Characterization of the Ca2+ current in isolated terminals of crustacean peptidergic neurons.

Ca2+ currents (ICa) were recorded from the neurosecretory terminals of the crab X-organ-sinus gland under voltage-clamp conditions. ICa was detectable at command potentials above -40mV, with maximum currents at approximately +20mV. No differences were observed between current-voltage (I/V) relationships from holding potentials of -50 or -90mV, indicating that there were no low-voltage-activated Ca2+ channels present in the terminals. The decay of ICa was best fitted with a single exponential, the extent of inactivation over 50 ms averaging 53%. The rate of decay of ICa was reduced by the substitution of Ca2+ with Sr2+ in the external solution and was eliminated by substitution with Ba2+. The effect of varying prepulse potential on the amplitude of ICa at +20mV was tested. ICa declined with increasing prepulse depolarization up to +20mV and then showed partial recovery at more depolarized prepulse potentials. Inactivation curves in solutions containing Sr2+ and Ba2+ showed much less current-dependent inactivation. Removing Ca2+ chelators from the internal solution significantly increased ICa decay. ICa was insensitive to nifedipine at a concentration of 1 mumol l-1. Pretreatment of the isolated sinus gland containing the intact terminals with a combination of omega-conotoxin (omega-Ctx) GVIA, omega-Ctx MVIIC and omega-agatoxin IVA had no effect on the levels of K+-induced peptide release.

Calcium- and barium-dependent exocytosis from the rat insulinoma cell line RINm5F assayed using membrane capacitance measurements and serotonin release.

Electrophysiological measurements of cell capacitance (Cm) and biochemical assays of [3H] serotonin ([3H]5-hydroxytryptamine or [3H]5-HT) release were combined to study the control of secretion in rat insulinoma RINm5F cells. Depolarizing pulses produced Cm changes (DeltaCm), indicative of exocytosis, with the same voltage and Ca2+ dependency as the inward Ca2+ currents (ICa). Ba2+ was able to substitute for Ca2+ in stimulating exocytosis, but not endocytosis. However, both the relative potency and kinetics of Ca2+-versus Ba2+-triggered exocytosis differed significantly. 5-HT synthesis and uptake were demonstrated in RINm5F cells. This allowed the use of [3H]5-HT to study hormone release from cell populations. [3H]5-HT was released in a depolarization-, Ca2+- and time-dependent manner. Ba2+ also substituted for Ca2+ in depolarization-induced [3H]5-HT release. Thapsigargin, used to deplete Ca2+ stores, had no effects on Ca2+-triggered Cm increases, but Ca2+-triggered [3H]5-HT release was abolished. Ba2+-triggered [3H]5-HT release, however, was only slightly affected by Ca2+ store depletion. Ba2+ was found to act directly as a secretagogue of [3H]5-HT in intact cells, but not in Cm measurements of voltage-clamped cells, suggesting that cell depolarization is a prerequisite for this action.

Regulation of calcium currents and secretion by magnesium in crustacean peptidergic neurons.

The effect of varying the external Mg2+ concentration on Ca2+ currents through voltage-operated Ca2+ channels has been examined with the patch-clamp technique in acutely isolated neuronal somata from the X-organ-sinus gland (XOSG) of the crab, Cardisoma carnifex. Neurons from this neurosecretory system were selected for morphology associated with crustacean hyperglycemic hormone (CHH) content. In parallel, the effects of Mg2+ concentration on K(+)-evoked secretion of CHH from isolated, intact XOSGs have been assayed by ELISA. At physiological Ca2+ levels the high-voltage-activated Ca2+ currents were attenuated with increasing Mg2+ concentration, with 50% inhibition at approximately 75 mM. Mg2+ block was voltage-dependent, relief from block occurring with increasing depolarization. Thus, in 24 mM Mg2+ inhibition of the Ca2+ current was approximately 55% at -10 mV. Secretion of CHH varied almost linearly with the log of Mg2+ concentration; in 2.4 mM Mg2+ it was double that in 24 mM Mg2+ and almost completely inhibited in 100 mM. Thus, Mg2+ produces a parallel inhibition of Ca2+ currents and CHH secretion and may play a role as a physiological modulator of neuronal activity and secretion in the XOSG of these crabs.

Characterization of the Ca2+ current in freshly dissociated crustacean peptidergic neuronal somata.

1. Freshly dissociated neuronal somata of the crab (Cardisoma carnifex) X-organ were studied in the whole cell patch-clamp configuration. To characterize the Ca2+ currents in these somata, recordings were made under conditions designed to suppress K+ and Na+ currents. 2. In 52 mM external Ca2+ the threshold for activation of Ca2+ currents was above -40 mV, with peak amplitudes occurring around +10 to +20 mV. The full component of the current was available for activation at -50 mV because no current increase was observed when the holding potential was increased to -90 mV. These characteristics of the current characterize it as a high-voltage activated (HVA) current. 3. The Ca2+ current was almost completely (60-90%) inactivated within 200 ms at maximal current potentials (+10 to +20 mV). The decay was best described by a double-exponential function with a fast and slow component of inactivation (tau f = 12 ms and tau s = 64 ms). Both Sr2+ and Ba2+ substitutions reduced the rates of inactivation. 4. In double-pulse experiments, plots of variable prepulse potential versus test pulse current produced a U-shaped curve with test pulse currents showing maximal inactivation at potentials that produced maximal Ca2+ influx during the prepulse. Tail currents also displayed a U-shaped inactivation curve. The extent of current-dependent inactivation was sequentially reduced by Sr2+ and Ba2+ substitutions. These data suggest that inactivation in crab somata is predominantly Ca2+ dependent. The remaining inactivation of Ba2+ currents suggests that there is also a component of voltage-dependent inactivation in the somata. 5. Part of the inactivated Ca2+ current could be recovered during short (4-10 ms) hyperpolarizing pulses to -130 mV. The absolute extent of recovery from inactivation was greatest for currents carried by Ca2+ rather than Sr2+ or Ba2+. When voltage-dependent inactivation was dominant (Ba2+ currents), the relative amount of current recovered was greater. The data suggest that hyperpolarizing pulses are more effective in removing voltage-dependent inactivation, but also allow some recovery from Ca(2+)-dependent inactivation. 6. In the crab saline, which contained 24 mM Mg2+, the amplitudes of currents carried by 52 mM Ca2+, Sr2+ and Ba2+ were similar. Removing the Mg2+ from the saline augmented both the Ba2+ and Sr2+ currents relative to the Ca2+ current.(ABSTRACT TRUNCATED AT 400 WORDS)

A lipoxygenase pathway of arachidonic acid metabolism mediates FMRFamide activation of a potassium current in an identified neuron of Helisoma.

The neuropeptide FMRFamide causes a presynaptic inhibition of neurotransmitter release from neuron B5 of Helisoma. In this study we demonstrate that one of FMRFamide's actions is to activate an outwardly rectifying potassium current. Arachidonic acid also activates an outward current in B5. The phospholipase A2 inhibitor, 4-bromophenacylbromide (BPB), and nordihydroguaiaretic acid (NDGA), an inhibitor of arachidonic acid metabolism, but not indomethacin, block FMRFamide's activation of the potassium current. Taken together these data demonstrate that one of FMRFamide's presynaptic actions is to activate a potassium current through a lipoxygenase pathway of arachidonic acid metabolism.

Activation of a peptidergic synapse locally modulates postsynaptic calcium influx.

We examined the synaptic connection between Phe-Met-Arg-Phe-NH2 (FMRFamide)-immunoreactive neurone VD4 and its target neurone P1, both found in the central nervous system of the pond snail Helisoma trivolvis. The major FMRFamide-like peak in neurone VD4 appears to be FMRFamide itself, based on its high performance liquid chromatography (HPLC) elution time and immunoreactivity before and after oxidation, but small peaks are also present at the elution times of Phe-Leu-Arg-Phe-NH2 (FLRFamide) and Gly-Asp-Pro-Phe-Leu-Arg-Phe-NH2 (GDPFLRFamide). The modulatory actions of the neuropeptides found in neurone VD4 were tested on the postsynaptic target cell P1. Bath application of both the tetrapeptides FMRFamide and FLRFamide at a concentration of 10(-5) mol l-1 reduced the macroscopic voltage-sensitive calcium current of neurone P1 in culture; FMRFamide by 45% and FLRFamide by 51%. Bath application of the heptapeptide GDPFLRFamide (10(-5) mol l-1) reduced the calcium current by only 8%. We reconstructed the synaptic connection between VD4 and P1 in culture. Action-potential-evoked calcium transients in neurites growing from P1 cells in culture were monitored using Fura-2. Addition of FMRFamide, FLRFamide or GDPFLRFamide reduced the magnitude of the calcium transient in P1. Stimulation of VD4 mimicked the effects of peptide application and caused localized reductions in the action-potential-evoked calcium transients in P1 at the points of contact between the neurites of neurones VD4 and P1. These results suggest that neurone VD4 modulates the calcium influx of neurone P1 through the release of endogenous FMRFamide-related peptides and that this modulatory action is restricted to sites of synaptic interaction.

Evidence for the presence, synthesis, immunoreactivity, and uptake of GABA in the nervous system of the snail Helisoma trivolvis.

In the present study several techniques were employed to test the hypothesis that gamma-aminobutyric acid (GABA) is a neurotransmitter in the central nervous system (CNS) of the pond snail Helisoma trivolvis (Mollusca, Pulmonata). First, by using chromatographic techniques, the presence of GABA and its differential distribution among the ganglia constituting the CNS was demonstrated. Second, de novo synthesis of 3H-GABA from 3H-glutamate was shown by the CNS. Levels of both endogenous and newly synthesized GABA were greatest in the buccal, cerebral, and pedal ganglia. Third, indirect immunohistochemistry of wholemounts revealed a central network of GABA-like immunoreactive neurons. With the possible exceptions of two pairs of fibers in nerve trunks, all projections from GABA-immunoreactive neurons were confined to the CNS, suggesting a predominantly central role for GABA. Stained neurons were found on the dorsal surface of the buccal ganglia and throughout the cerebral and pedal ganglia. No GABA-immunoreactive cell bodies were observed in the parietal, pleural, or visceral ganglia. Finally, uptake of 3H-GABA was examined autoradiographically in sectioned ganglia. A pattern of radiolabelled cells was observed that closely resembled the distribution of GABA-immunoreactive neurons. The data described above fulfill several criteria necessary to establish GABA as a transmitter in the nervous system of Helisoma. Taken together with previously obtained pharmacological evidence demonstrating that GABA acts on Helisoma central neurons, GABA is considered to be a strong candidate for a neurotransmitter in Helisoma.

Attempted isolation of viruses from myasthenia gravis thymus.

A systematic study of thymus homogenates and cell suspensions from 13 patients with myasthenia gravis (MG) of recent onset, and 6 non-myasthenic controls, has failed to detect or isolate virus by cell culture with 'rescue techniques', electron microscopy, or intracerebral inoculation into neonatal mice. These results do not support the case for persistent viral infection in the thymus, and impose constraints on hypotheses of a viral aetiology of MG.

Peptidergic modulation of a neuromuscular junction in Aplysia: bioactivity and immunocytochemistry.

Three endogenous peptides were assayed for bioactivity at an Aplysia neuromuscular junction. Evoked contractions were enhanced by Phe-Met-Arg-Phe-NH2 (FMRFamide) and suppressed by arginine vasotocin; small cardioactive peptide B (SCPB) also enhanced contractions at low concentrations, but caused suppression at higher doses. In accordance with their putative roles as neuromodulators, immunocytochemistry revealed FMRFamide-like and SCPB-like fibers on the muscle surface.

Hairy cell leukaemia occurring during phenytoin (diphenylhydantoin) treatment.

We describe the case of a patient who developed hairy cell leukaemia (leukaemic reticuloendotheliosis) during phenytoin treatment. Hairy cells were identified by fluorescence, phase contrast and electron microscopy; they contained tartrate-resistant acid phosphatase activity, formed rosettes with mouse but not sheep erythrocytes and bore monoclonal surface immunoglobulin. Because of the association of pseudo- and true lymphomas with phenytoin it is possible that this lymphoproliferative disorder arose as a result of the treatment with phenytoin, possibly in conjunction with sulthiame.