Quantification of pain-induced changes in cerebral blood flow by perfusion MRI.

The purpose of this study was to assess if the functional activation caused by painful stimuli could be detected with arterial spin labeling (ASL), which is a non-invasive magnetic resonance imaging (MRI) technique for measuring cerebral blood flow (CBF). Because ASL directly measures blood flow, it is well suited to pain conditions that are difficult to assess with current functional MRI, such as chronic pain. However, the use of ASL in neuroimaging has been hampered by its low sensitivity. Recent improvements in MRI technology, namely increased magnetic field strengths and phased array receiver coils, should enable ASL to measure the small changes in CBF associated with pain. In this study, healthy volunteers underwent two ASL imaging sessions, during which a painful thermal stimulus was applied to the left hand. The results demonstrated that the ASL technique measured changes in regional CBF in brain regions that have been previously identified with pain perception. These included bilateral CBF changes in the insula, secondary somatosensory, and cingulate cortices, as well as the supplementary motor area (SMA). Also observed were contralateral primary somatosensory and ipsilateral thalamic CBF changes. The average change in CBF for all regions of interest was 3.68ml/100g/min, ranging from 2.97ml/100g/min in ipsilateral thalamus to 4.91ml/100g/min in contralateral insula. The average resting global CBF was 54+/-9.7ml/100g/min, and there was no change in global CBF due to the noxious thermal stimulus.

Tamulustoxin: a novel potassium channel blocker from the venom of the Indian red scorpion Mesobuthus tamulus.

We have characterized tamulustoxin, a novel 35-amino-acid peptide found in the venom of the Indian red scorpion (Mesobuthus tamulus). Tamulustoxin was identified through a [125I]toxin I screen, designed to identify toxins that block voltage-activated potassium channels. Tamulustoxin has also been cloned by RT-PCR, using RNA extracted from scorpion venom glands. Tamulustoxin shares no homology with other scorpion venom toxins, although the positions of its six cysteine residues would suggest that it shares the same structural scaffold. Tamulustoxin rapidly inhibited both peak and steady-state currents (18.9 +/- 1.0 and 37 +/- 1.1%, respectively) produced by injecting CHO cells with mRNA encoding the hKv1.6 channel.

A case of supernumerary teeth in the premaxilla, maxillary cuspid, and mandibular premolar regions.

A case of several developing supernumerary teeth is reported. A seven-year-old African-American boy presented with retained primary maxillary central incisors, two impacted mesiodens, and unerupted permanent maxillary central incisors. A dentigerous cyst was removed at the time of surgical removal of the mesiodens. Approximately fourteen months post-extraction, a new panoramic radiograph showed the presence of six previously unidentified developing and unerupted supernumerary teeth, one on each of the maxillary cuspid areas and two on the mandibular premolar regions bilaterally. Practitioners should be aware that supernumerary teeth may develop late. Thus, periodic reevaluation with appropriate radiographs is indicated, especially in patients who have presented with supernumerary teeth.

The contrasting effects of dendrotoxins and other potassium channel blockers in the CA1 and dentate gyrus regions of rat hippocampal slices.

1. The effects of potassium channel blocking compounds on synaptic transmission in the CA1 and dentate gyrus regions of the rat hippocampus were examined by means of simultaneous field potential recording techniques in brain slices. 2. 4-Aminopyridine (4-AP) enhanced the excitatory postsynaptic potential (e.p.s.p.) and induced multiple population spike responses in both regions. EC50 values were 6.7 microM in the CAI (n = 5) and 161.7 microM (n = 5) in the dentate gyrus. 3. Tetraethylammonium (TEA) increased the amplitude and induced broadening of the population spike in both regions. In the dentate gyrus (n = 5) a single slow spike response was introduced (EC50 12.8 mM) and in the CA1 region (n = 5) the response was transformed into two wide spikes (EC50 2.6 mM). 4. In the CA1 region all of the dendrotoxins (toxin I, toxin K, alpha-Dtx and delta-Dtx) induced multiple population spikes and enlarged e.p.s.p. responses. Potentials recorded simultaneously in the dentate gyrus exhibited comparatively minor enhancements. The EC50 value for toxin 1 in the CA1 was calculated to be 237 nM (n = 4). Estimated EC50 values were obtained for alpha-Dtx (1.1 microM, n = 3), toxin K (411 nM, n = 4) and delta-Dtx (176 nM, n = 3). 5. In the presence of toxin 1, DL-2-amino-5-phosphonovaleric acid (APV) induced slight reduction of the late e.p.s.p. phase (n = 3). 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX) abolished all population spikes leaving a late slow positive waveform (n = 3). Co-application of APV and CNQX abolished all postsynaptic responses. 6. Charybdotoxin (CbTx) was significantly less potent than the dendrotoxins and had mixed actions in the CA1 region (n = 3). Again the dentate gyrus exhibited reduced sensitivity (n = 3). 7. In the presence of mast cell degranulating peptide (MCDP), enhancement of the CA1 field potential response (n = 5) was greater than that observed in the dentate gyrus (n = 5). 8. The results show that some potassium channel modulators can profoundly enhance CA1 region synaptic responses in the absence of notable changes in dentate gyrus excitability. Selective enhancement of defined synaptic pathways by potassium channel modulators may prove to have considerable therapeutic potential.

The relative potencies of dendrotoxins as blockers of the cloned voltage-gated K+ channel, mKv1.1 (MK-1), when stably expressed in Chinese hamster ovary cells.

1. The mKv1.1 voltage-gated K+ channel has been expressed stably in Chinese hamster ovary cells and whole-cell currents recorded by the patch-clamp method. 2. A range of structurally related peptide toxins (dendrotoxins) from the venom of green mamba (Dendroaspis angusticeps) and black mamba (Dendroaspis polylepis polylepis) snakes were tested for mKv1.1 channel blocking activity. Their potencies were compared based on EC50s derived from their respective concentration-inhibition relationships. 3. The rank order of potency, thus determined was: Toxin K > 7-dendrotoxin(7-Dtx) > delta-Dtx > Toxin I = alpha-Dtx > beta-Dtx. 4. Block was independent of voltage and no effects of the toxins on the kinetics of activation were observed. These results are consistent with a mechanism involving the block of closed channels. 5. A wide range of activity was observed even between toxins with an extremely high degree of sequence homology. Toxin K, in particular was an exquisitely potent blocker of the mKv1.1 channel, having an EC50 of 30 pM compared with 1.8 nM for delta-Dtx in spite of 95% sequence identity.

Studies on the blocking action of human Kv3.4 inactivation peptide variants in the mouse cloned Kv1.1 K+ channel.

1. Whole-cell patch clamp recordings were made from Chinese hamster ovary (CHO) cells stably expressing homomeric mouse Kv1.1 (delayed rectifier K+; mKv1.1) channels. The effects of internal application of a number of different peptides, based on part of the amino terminal sequence of the human Kv3.4 channel subunit (hKv3.4), were examined in order to determine their influence on N-type inactivation. 2. For the native hKv3.4 peptide, the association rate constant (kon) increased with membrane depolarization, whilst the dissociation rate constant (koff) had little dependence on voltage. This resulted in the apparent dissociation constant (KD) of the hKv3.4 peptide tending to increase with depolarization. 3. In general, kon increased and apparent KD decreased with positive charge of the hKv3.4 peptide variants; in peptides lacking a hydrophobic amino terminal this correlation was not maintained. In contrast, the rate of dissociation of the variant peptides (koff) was independent of net charge. 4. The blocking activity of the hKv3.4 peptide was not dependent on a disulphide bridge between cysteine residues C6 and C24 and the presence of cysteine residues in the hKv3.4 peptide was not a prerequisite for rapid inactivation. All cysteine-substituted variants, especially at C6, showed a more rapid recovery from inactivation than the hKv3.4 peptide. Substitutions at C24, and not C6, reduced kon. 5. The present results concerning the action of the mammalian hKv3.4 channel inactivation particle on mKv1.1 channels complement earlier models for the invertebrate Shaker K+ channel. It is proposed that the hydrophobic amino terminal region of the hKv3.4 inactivation peptide blocks the channel pore, whilst the adjacent positively charged region interacts with negative charges on the channel protein.

Blockade of two voltage-dependent potassium channels, mKv1.1 and mKv1.2, by docosahexaenoic acid.

The effects of the polyunsaturated fatty acid, docosahexaenoic acid, were examined on two single cloned potassium channels, mKv1.1 and mKv1.2, stably expressed in Chinese hamster ovary cells using whole-cell patch clamp techniques. Docosahexaenoic acid produced a time- and dose-dependent, reversible block of mKv1.1 and mKv1.2. Interestingly, docosahexaenoic acid increased the rate of activation of mKv1.2 leading to an enhancement of current amplitude at short intervals following activating the voltage step. This phenomenon was not seen in the case of mKv1.1. Intracellular administration of docosahexaenoic acid did not block either type of channel. These findings suggest that docosahexaenoic acid inhibits mKv1.1 and mKv1.2 channels by acting at an extracellular site and by an open-channel blocking mechanism.

The modulation of the rate of inactivation of the mKv1.1 K+ channel by the beta subunit, Kv beta 1 and lack of effect of a Kv beta 1 N-terminal peptide.

The coexpression of the rat Kv beta 1 subunit with the mouse Kv1.1 (mKv1.1) K+ channel in Chinese hamster ovary cells caused an increase in the rate of inactivation of whole-cell current. Current decayed in a bi-exponential fashion with a fast voltage-dependent and a slower voltage-independent component. The inactivating current component accounted for around 40% of the total outward current. In contrast to previous studies using K+ channel alpha subunits, peptides based on the N-terminal of the Kv beta 1 subunit were unable to mimic the action of the entire subunit. The findings indicate differences between the inactivation induced by the Kv beta 1 subunit and the N-type inactivation mechanism associated with certain rapidly-inactivating cloned K+ channel alpha subunits.

Cysteine-modifying reagents alter the gating of the rat cloned potassium channel Kv1.4.

The effects of cysteine-modifying reagents on the gating of rat cloned Kv1.4 channels expressed in HEK-293 cells were examined using the whole-cell patch-clamp technique. Cells transfected with Kv1.4 expressed a rapidly inactivating K+ current with a mid-point of activation of -31 mV and a slope factor of 5 mV measured with tail current protocols in 35 mM Rb+ external solutions. The cysteine-specific oxidizing agents 2,2'-dithiobis-5-nitropyridine (DTBNP, 50 microM) and chloramine-T (CL-T, 500 microM) removed inactivation of Kv1.4. These effects were reversed by the reducing agent dithiothreitol (DTT, 10mM). In addition, DTBNP and CL-T also slowed Kv1.4 deactivation and increased the voltage sensitivity of deactivation. The action of cysteine-modifying reagents on Kv1.4 suggests that redox state affects channel gating, with oxidation tending to stabilize the open state of the channel, both by removing inactivation and slowing deactivation.

On the mechanism of 4-aminopyridine action on the cloned mouse brain potassium channel mKv1.1.

1. This study used the whole-cell patch clamp technique to investigate the mechanism of action of the K+ channel blocker 4-aminopyridine (4-AP) on the cloned K+ channel mouse Kv1.1 (mKv1.1) expressed in Chinese hamster ovary cells. 2. Cells transfected with mKv1.1 expressed a non-inactivating, delayed rectifier-type K+ current. 4-AP induced a dose-, voltage- and use-dependent block of mKv1.1. 3. 4-AP blockade of mKv1.1 was similar whether 4-AP was administered extracellularly (IC50 = 147 microM) or intracellularly (IC50 = 117 microM). 4. Inclusion of the first twenty amino acids of the N-terminus sequence of the Shaker B K+ channel ('inactivation peptide') in the patch electrode transformed mKv1.1 into a rapidly inactivating current. The time constant of decay for the modified current was dependent on the concentration of inactivation peptide, and under these conditions extracellular 4-AP had a reduced potency (IC50 values of 471 and 537 microM for 0.5 and 2 mg ml-1 inactivation peptide, respectively). 5. A permanently charged analogue of 4-AP, 4-aminopyridine methiodide (4-APMI), was found to block mKv1.1 when applied inside the cell, but was without effect when administered externally. 6. Decreasing the intracellular pH (pHi) to 6.4 caused an increase in 4-AP potency (IC50 = 76 microM), whereas at pHi 9.0, the 4-AP potency fell (IC50 = 295 microM). Conversely, increasing extracellular pH (pHo) to 9.0 caused an increase in 4-AP potency (IC50 = 93 microM), whereas at pHo 6.4, 4-AP potency decreased (IC50 = 398 microM). 7. Taken together, these findings support the hypotheses that the uncharged form of 4-AP crosses the membrane, and that it is predominantly the cationic form which acts on mKv1.1 channels intracellularly, possibly at or near to the binding site for the inactivation peptide.

Pharmacology of a cloned potassium channel from mouse brain (MK-1) expressed in CHO cells: effects of blockers and an 'inactivation peptide'.

1. Chinese hamster ovary cells (CHO), maintained in cell culture, were stably transfected with DNA for the MK-1 voltage-activated potassium channel, previously cloned from a mouse brain library. 2. Voltage-activated currents were recorded by the whole cell patch clamp method. In CHO cells transfected with the vector only, there were no significant outward voltage activated currents. However, large outward voltage-activated potassium currents were always observed in those cells which had been transfected with the vector containing the DNA encoding for MK-1. 3. These potassium currents activated from -40 mV, and reversed at the potassium equilibrium potential. The half-maximal conductance of MK-1 was at -10 mV and had a slope factor of 11 mV when fitted with a Boltzmann function. There was only very slight (< 10%) inactivation of MK-1 even at very large positive voltages. 4. MK-1 was reversibly blocked by: 4-aminopyridine (4-AP, 0.1-4 mM), Toxin I 10-100 nM), mast cell degranulating peptide (1 microM), tetraethylammonium (TEA, 4-10 mM), tedisamil (100 microM), quinine (100 microM) and ciclazindol (100 microM); all applied to the outside of the cell from a 'U tube' rapid perfusion system. 4-AP may block closed as well as open MK-1 potassium channels. 5. A synthetic 20 amino acid peptide derived from the N-terminus sequence of the Shaker B potassium channel (the 'inactivation peptide') produced dramatic inactivation of MK-1 when applied to the inside, but not the outside of the cell. Reducing peptide concentration or 'degrading' the peptide produced less inactivation. 6. The block of MK-1 by the synthetic inactivation peptide was quite different in time dependence from block by internal TEA (0.4-4 mM), which probably blocks much more quickly but less potently than the peptide.

Inactivation of enveloped viruses by anthraquinones extracted from plants.

To determine the extent of antiviral activity present in a number of plant extracts, hot glycerin extracts were prepared from Rheum officinale, Aloe barbadensis, Rhamnus frangula, Rhamnus purshianus, and Cassia angustifolia and their virucidal effects were tested against herpes simplex virus type 1. All the plant extracts inactivated the virus. The active components in these plants were separated by thin-layer chromatography and identified as anthraquinones. A purified sample of aloe emodin was prepared from aloin, and its effects on the infectivity of herpes simplex virus type 1 and type 2, varicella-zoster virus, pseudorabies virus, influenza virus, adenovirus, and rhinovirus were tested by mixing virus with dilutions of aloe emodin for 15 min at 37 degrees C, immediately diluting the sample, and assaying the amount of infectious virus remaining in the sample. The results showed that aloe emodin inactivated all of the viruses tested except adenovirus and rhinovirus. Electron microscopic examination of anthraquinone-treated herpes simplex virus demonstrated that the envelopes were partially disrupted. These results show that anthraquinones extracted from a variety of plants are directly virucidal to enveloped viruses.

M-current noise and putative M-channels in cultured rat sympathetic ganglion cells.

1. Whole-cell recordings of M-currents and single-channel recordings have been made in cultured rat sympathetic ganglion (SCG) neurones using the patch clamp technique. 2. Muscarine caused a reduction in macroscopic M-current relaxations, induced by voltage steps, and a concomitant reduction in whole-cell current noise. Power spectra of the muscarine-sensitive component of current noise were fitted with two Lorentzian components corresponding, on average, to 162 and 15 ms. The longer time constant was very similar to that of deactivation tail currents measured at the same potential. 3. The single-channel conductance at -30 mV was estimated from power density spectra and whole-cell current-variance relationships to be 1-2 pS. 4. Putative single M-channels, activated by depolarization, were identified in cell-attached and outside-out patches from cultured SCG neurones. In particular, the ensemble average of a small amplitude channel (estimated to be ca4 pS in physiological [K+]) in a cell-attached patch, exhibited a similar time dependence to whole-cell M-current.

Gnotobiotic, athymic mice; a possible system for the study of the role of bacteria in human amoebiasis.

Two groups of 12 + 14 gnotobiotic, athymic mice were intracaecally injected with Entamoeba histolytica strain HK9 and NIH:200, respectively. Two groups of 16 and 15 mice were given amoebae together with a pure strain of Escherichia coli and a further two groups of 16 and 27 were given amoebae with a pure strain of Clostridium perfringens. Batches of 3-7 mice from each group were killed at intervals of 1-4 weeks. All the mice given NIH:200 alone were found to be infected with trophozoites. Of those given HK9 alone, 20% of the first and 57% of the second group to be examined were infected. Groups of mice given either strain of amoeba monocontaminated with E. coli were all found to be infected at post-mortem examination with no apparent clinical signs and little histological change. The group given HK9 and C. perfringens, although all were infected, failed to produce clinical signs or histological lesions, though some died expectedly. In the group given NIH:200 with C. perfringens the amoebae showed a change of activity and there was evidence of both caecal and liver lesions after 120 days. The usefulness of the system in studying the effect of individual species of bacteria on invasive amoebae is discussed.

Anterior chamber lens implantation after vitreous loss.

Vitreous loss is a serious complication of cataract surgery. Following vitreous loss it is common practice to perform a primary implantation of an anterior chamber lens (AC-IOL). We retrospectively analysed 642 consecutive cases of cataract extraction performed between 1983 and 1986 with special attention to those patients in whom vitreous loss occurred and an AC-IOL was placed. There were 27 such cases, and 24 of these were available for follow-up. Eighteen (75%) had visual acuity of 20/40 or better. All six patients (25%) who had a visual acuity of less than 20/40 in the operated eye had a functional visual acuity of 20/200 or less. Complications that occurred in this group are discussed. We are concerned that the complications associated with vitreous loss and with AC-IOLs may be acting in concert to cause visually disabling results.

Vitreous loss rates in extracapsular cataract surgery by residents.

Vitreous loss is a serious complication of cataract surgery. It has been suggested that high rates of vitreous loss may be an inevitable consequence when residents are learning extracapsular cataract extraction (ECCE). The authors retrospectively analyzed all (n = 936) cataract operations done by second- and third-year residents in a single Veterans Administration hospital from 1982 through 1988. Between 1982 and 1985, the incidence of vitreous loss was 10.3%. In 1985, a new program of resident surgical education was introduced, and the incidence from 1985 through 1988 declined to 3.2% (P less than 0.001). Statistical analysis confirms that this decrease cannot be attributed to any individual surgeon, class of residents, or year of surgery. The authors believe that an educational program including practice surgery, graded responsibility, and experienced assistance may be responsible in part for dramatically reducing the rate of this serious complication during surgery done by the beginning resident.

Signal transduction mechanisms in cultured CNS neurons and clonal pituitary cells.

The experimental accessibility of monolayer culture has been used to study signal transduction mechanisms in primary CNS neurons and clonal pituitary cells. Here we review results on two signals representative of the emerging diversity of mechanisms discovered in all species studied thus far. One is mediated by micromolar concentrations of the amino acid GABA at postsynaptic membranes throughout the mammalian CNS and involves transient activation of Cl- ion channels whose distribution of conducting periods accounts for the millisecond time course of the signal. This signal serves to depress the probability that the target cell will trigger an action potential. The signal intensifies as the postsynaptic membrane is depolarized and can be modulated by clinically important drugs, primarily through changes in channel kinetics. The other signal involves nanomolar concentrations of the peptide TRH, which stimulates secretion of prolactin from clonal "GH3" pituitary cells. Intracellular recordings of GH3B6 cells show that TRH triggers a complex electrical response lasting several minutes. The response consists of Ca2+-activated K+ conductance followed by Ca2+-action potential activity. Whole-cell patch recordings, which rapidly dialyze the cell, can eliminate the TRH-induced changes in membrane excitability. Inclusion of aqueous lysates of the GH3B6 clone or the soluble second messenger factors inositol trisphosphate (IP3) or protein kinase (PKC) can restore various aspects of the change in membrane excitability. Thus, TRH alters ion conductance mechanisms through a second messenger cascade likely to involve IP3-mediated mobilization of Ca2+ from the endoplasmic reticulum and transient translocation of PKC from cytoplasm to plasma membrane. These synaptic and extrasynaptic signals reflect some of the diversity of transduction mechanisms involved in intercellular communication.

Potentiation of gamma-aminobutyric-acid-activated chloride conductance by a steroid anaesthetic in cultured rat spinal neurones.

1. Intracellular recordings from cultured rat spinal cord neurones demonstrated that Cl(-)-dependent responses to GABA (gamma-aminobutyric acid) (but not glycine) were increased in amplitude and duration by the steroid anaesthetic alphaxalone (3 alpha-hydroxy-5 alpha-pregnane-11,20-dione) at submicromolar concentrations that produced little or no effect on passive electrical properties. The non-anaesthetic 3 beta-hydroxy analogue was without effect on GABA-evoked responses. 2. Under voltage clamp, membrane currents evoked by GABA were potentiated by alphaxalone without change in the reversal potential for the GABA-evoked response. Fluctuation analysis of GABA-evoked currents suggested that the mean open-time of GABA-activated channels was prolonged from 30 to 74 ms in the presence of the anaesthetic. 3. Higher concentrations of alphaxalone, similar to those reported during surgical anaesthesia, increased membrane conductance in the absence of exogenously applied GABA. Under voltage clamp, current responses to alphaxalone reversed at the same potential as did responses to GABA, suggesting that they result from increased Cl- conductance. 4. Alphaxalone responses were reduced by the GABA antagonist bicuculline. Fluctuation analysis of current responses to the anaesthetic suggest that they result from the activation of ion channels of long (100 ms) open-time and elementary conductance indistinguishable from that of channels activated by GABA (20 pS). Taken together, these findings indicate that the steroid anaesthetic is able to directly activate Cl- conductance normally activated by GABA in spinal neurones. 5. The actions of the steroid at GABA-receptor-Cl(-)-channel complexes are similar to those produced by the anaesthetic barbiturates (e.g. pentobarbitone), although obtained at 50-100-fold lower concentrations. These effects on the inhibitory Cl(-)-conductance mechanism may be partly responsible for the depressant actions of alphaxalone on the mammalian central nervous system.

Chloride conductances in central neurons.

Two types of chloride conductances--gamma-aminobutyric acid (GABA)-activated and Ca2+-voltage dependent--were studied in tissue-cultured central neurons. Cultured hippocampal neurons contain identified GABA-mediated perisomatic synaptic connections. Activation of these synapses evokes, in voltage-clamped neurons, an inhibitory postsynaptic current (IPSC) that rises within 3 to 5 msec and decays exponentially with a time constant of 15 to 20 msec. The decay of the IPSC is voltage-dependent, and is altered by drugs known to act on GABA receptors. The kinetics of the IPSC appears to be determined by that of the GABA-activated Cl- ion channel. Ca2+ and voltage-dependent Cl- conductance is best seen in cells where K conductances are blocked. It usually follows a Ca spike and is blocked by Ca antagonists. Analysis of tail currents in voltage clamped hippocampal, hypothalamic and spinal cord neurons indicates that the current decays slowly (200 msec) and passively and reverses a function of [E]C1 (equilibrium potential for Cl-). Chloride conductance functions to suppress excitability of these neurons. The pharmacological and physiological properties of this conductance in vivo are not as yet known.