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Neuronal (N)-type Ca2+channel-selective ω-conotoxins have emerged as potential new drugs for the treatment of chronic pain. In this study, two new ω-conotoxins, CVIE and CVIF, were discovered from a Conus catuscDNA library. Both conopeptides potently displaced 125I-GVIA binding to rat brain membranes. In Xenopus laevisoocytes, CVIE and CVIF potently and selectively inhibited depolarization-activated Ba2+currents through recombinant N-type (α1B-b/α2δ1/β3) Ca2+channels. Recovery from block increased with membrane hyperpolarization, indicating that CVIE and CVIF have a higher affinity for channels in the inactivated state. The link between inactivation and the reversibility of ω-conotoxin action was investigated by creating molecular diversity in β subunits: N-type channels with β2asubunits almost completely recovered from CVIE or CVIF block, whereas those with β3subunits exhibited weak recovery, suggesting that reversibility of the ω-conotoxin block may depend on the type of β-subunit isoform. In rat dorsal root ganglion sensory neurons, neither peptide had an effect on low-voltage-activated T-type channels but potently and selectively inhibited high voltage-activated N-type Ca2+channels in a voltage-dependent manner. In rat spinal cord slices, both peptides reversibly inhibited excitatory monosynaptic transmission between primary afferents and dorsal horn superficial lamina neurons. Homology models of CVIE and CVIF suggest that ω-conotoxin/voltage-gated Ca2+channel interaction is dominated by ionic/electrostatic interactions. In the rat partial sciatic nerve ligation model of neuropathic pain, CVIE and CVIF (1 nM) significantly reduced allodynic behavior. These N-type Ca2+channel-selective ω-conotoxins are therefore useful as neurophysiological tools and as potential therapeutic agents to inhibit nociceptive pain pathways.

The synthetic α-conotoxin Vc1.1 is a small disulfide bonded peptide currently in development as a treatment for neuropathic pain. Unlike Vc1.1, the native post-translationally modified peptide vc1a does not act as an analgesic in vivo in rat models of neuropathic pain. It has recently been proposed that the primary target of Vc1.1 is the α9α10 nicotinic acetylcholine receptor (nAChR). We show that Vc1.1 and its post-translationally modified analogs vc1a, [P6O]Vc1.1, and [E14γ]Vc1.1 are equally potent at inhibiting ACh-evoked currents mediated by α9α10 nAChRs. This suggests that α9α10 nAChRs are unlikely to be the molecular mechanism or therapeutic target of Vc1.1 for the treatment of neuropathic pain.

The effects of verapamil and related phenylalkylamines on neuronal excitability were investigated in isolated neurons of rat intracardiac ganglia using whole-cell perforated patch-clamp recording. Verapamil (≥10 μM) inhibits tonic firing observed in response to depolarizing current pulses at 22°C. The inhibition of discharge activity is not due to block of voltage-dependent Ca2+channels because firing is not affected by 100 μM Cd2+. The K+channel inhibitors charybdotoxin (100 nM), 4-aminopyridine (0.5 mM), apamin (30–100 nM), and tetraethylammonium ions (1 mM) also have no effect on firing behavior at 22°C. Verapamil does not antagonize the acetylcholine-induced inhibition of the muscarine-sensitive K+current (M-current) in rat intracardiac neurons. Verapamil inhibits the delayed outwardly rectifying K+current with an IC50value of 11 μM, which is approximately 7-fold more potent than its inhibition of high voltage-activated Ca2+channel currents. These data suggest that verapamil inhibits tonic firing in rat intracardiac neurons primarily via inhibition of delayed outwardly rectifying K+current. Verapamil inhibition of action potential firing in intracardiac neurons may contribute, in part, to verapamil-induced tachycardia.

Brevetoxin-3 (PbTx-3), produced by marine dinoflagellates (Ptychodiscus brevis), is a lipophilic 11-ring polyether molecule that binds with high affinity to site 5 of the voltage-sensitive sodium (Na+) channel. The effects of PbTx-3 and its derivatives were studied in cell-attached membrane patches on neurons dissociated from neonatal rat nodose ganglia by the patch-clamp technique. PbTx-3 (30–500 nM) produced a shift in activation to more negative membrane potentials whereby single-channel activity was observed under steady-state conditions (maintained depolarization at −50 mV). The unitary current-voltage relationship is linear, which exhibits a reversal potential of approximately +60 mV. Two unitary current amplitudes could be observed in the presence of PbTx-3, with slope conductances of 10.7 pS and 21.2 pS. PbTx-3 inhibits the inactivation of Na+channels and prolongs the mean open time of these channels. Unitary Na+currents could be blocked by 1 μM tetrodotoxin (TTX) added to the pipette solution, which indicates that the single-channel currents are caused by the opening of TTX-sensitive Na+channels. The PbTx-3 molecule is proposed to have multiple active centers (A-ring lactone, C-42 of R side chain) interacting with the Na+channel binding site. Modification of the molecular structure of PbTx-3 at these centers produced derivatives (PbTx-6, 2,3,41,43-tetrahydro-PbTx-3, 2,3,27,28,41,43-hexahydro-PbTx-3 and 2,3-dihydro-PbTx-3 A-ring diol), which were less potent than PbTx-3 in producing similar effects on Na+channel kinetics. PbTx-3 and its derivatives may provide insight into the mechanics of voltage-sensitive Na+channel gating.

Human cytochrome P-450, UDP-glucuronosyltransferase and sulphotransferase activities have been measured in the cell line Hep G2 following treatment of cells with 3-methylcholanthrene or phenobarbital. 3-Methylcholanthrene treatment caused a 20–30-fold increase in the O-deethylation of 7-ethoxycoumarin. The glucuronidation and sulphation of the product 7-hydroxycoumarin were increased 36 and 7 fold, respectively. In comparison, phenobarbital treatment did not increase these activities significantly. However, phenobarbital-inducible proteins were identified on ‘Western blots’ using antibodies to a rat liver phenobarbital inducible P-450 form. The molecular masses of the proteins did not coincide with those expected for cytochromes P-450. However, characteristic of P-450 forms, the synthesis of these proteins was suppressed by 3-methylcholanthrene treatment. The Hep G2 cell line represents a potentially useful model for studying the regulation of human P-450 genes.

Digestion of total DNA from Candida albicansisolates with the restriction enzyme HinfI generated simple, readily interpretable band patterns on ethidium bromide-stained gels. The procedure, which was highly reproducible, was used to detect restriction fragment length polymorphisms in 47 isolates of this species.

1Single cell photometry and whole‐cell patch clamp recording were used to study caffeine‐induced intracellular Ca2+signals and membrane currents, respectively, in endothelial cells freshly dissociated from rabbit aorta.2Caffeine (5 mM) evoked a transient increase in [Ca2+]iin fura‐2‐loaded endothelial cells. Pretreatment of cells with lO μMryanodine did not alter resting [Ca2+]ibut irreversibly inhibited the caffeine‐induced rise in [Ca2+]i. The caffeine‐induced increase in [Ca2+]iwas not attenuated by the removal of extracellular Ca2+and did not stimulate the rate of Mn2+quench of fura‐2 fluorescence.3Bath application of caffeine evoked a dose‐ and voltage‐dependent outward current. The rate of onset and amplitude of the caffeine‐evoked outward current increased with higher caffeine concentrations and membrane depolarization. The relationship between caffeine‐evoked current amplitude and membrane potential was non linear, suggesting that the channels underlying the current are voltage‐sensitive.4In the absence of extracellular Ca2+, the amplitude of the caffeine‐evoked outward current was reduced by approximately 50% but the duration of the current was prolonged compared to that observed in the presence of external Ca2+. Ca2+‐free external solutions produced an unexpected increase in both the frequency and amplitude of spontaneous transient outward currents (STOCs).5Inclusion of heparin (10 μg ml−1) in the patch pipette abolished the acetylcholine (ACh)‐induced outward current but failed to inhibit either STOCs or the caffeine‐evoked outward current in native endothelial cells. In the absence of extracellular Ca2+, heparin did not affect either STOCs or the caffeine‐induced outward current.6Externally applied tetraethylammonium ions (TEA, 3–10 mM) reversibly inhibited unitary Ca2+‐activated K+currents and STOCs in endothelial cells but failed to inhibit completely the outward current evoked by 20 mM caffeine.7Bath application of 0.1 mM zinc ion (Zn2+), a chloride channel blocker, did not affect unitary currents or STOCs but reduced the amplitude of the caffeine‐evoked current by >75% compared to control. Replacement of extracellular NaCl with Na gluconate also reduced the amplitude of the caffeine‐induced outward current. Bath application of 0.1 mM Zn2+and 10 mM TEA completely blocked the caffeine‐evoked outward current in endothelial cells.8Caffeine‐induced Ca2+release from intracellular stores evokes a transient rise in [Ca2+]iwhich is correlated with a large, transient outward current. The ionic dependence and inhibition of the caffeine‐sensitive current by TEA and Zn2+suggests that Ca2+‐activated K+and Cl−conductances contribute to the caffeine response in rabbit aortic endothelial cells. Single cell photometry and whole‐cell patch clamp recording were used to study caffeine‐induced intracellular Ca2+signals and membrane currents, respectively, in endothelial cells freshly dissociated from rabbit aorta. Caffeine (5 mM) evoked a transient increase in [Ca2+]iin fura‐2‐loaded endothelial cells. Pretreatment of cells with lO μMryanodine did not alter resting [Ca2+]ibut irreversibly inhibited the caffeine‐induced rise in [Ca2+]i. The caffeine‐induced increase in [Ca2+]iwas not attenuated by the removal of extracellular Ca2+and did not stimulate the rate of Mn2+quench of fura‐2 fluorescence. Bath application of caffeine evoked a dose‐ and voltage‐dependent outward current. The rate of onset and amplitude of the caffeine‐evoked outward current increased with higher caffeine concentrations and membrane depolarization. The relationship between caffeine‐evoked current amplitude and membrane potential was non linear, suggesting that the channels underlying the current are voltage‐sensitive. In the absence of extracellular Ca2+, the amplitude of the caffeine‐evoked outward current was reduced by approximately 50% but the duration of the current was prolonged compared to that observed in the presence of external Ca2+. Ca2+‐free external solutions produced an unexpected increase in both the frequency and amplitude of spontaneous transient outward currents (STOCs). Inclusion of heparin (10 μg ml−1) in the patch pipette abolished the acetylcholine (ACh)‐induced outward current but failed to inhibit either STOCs or the caffeine‐evoked outward current in native endothelial cells. In the absence of extracellular Ca2+, heparin did not affect either STOCs or the caffeine‐induced outward current. Externally applied tetraethylammonium ions (TEA, 3–10 mM) reversibly inhibited unitary Ca2+‐activated K+currents and STOCs in endothelial cells but failed to inhibit completely the outward current evoked by 20 mM caffeine. Bath application of 0.1 mM zinc ion (Zn2+), a chloride channel blocker, did not affect unitary currents or STOCs but reduced the amplitude of the caffeine‐evoked current by >75% compared to control. Replacement of extracellular NaCl with Na gluconate also reduced the amplitude of the caffeine‐induced outward current. Bath application of 0.1 mM Zn2+and 10 mM TEA completely blocked the caffeine‐evoked outward current in endothelial cells. Caffeine‐induced Ca2+release from intracellular stores evokes a transient rise in [Ca2+]iwhich is correlated with a large, transient outward current. The ionic dependence and inhibition of the caffeine‐sensitive current by TEA and Zn2+suggests that Ca2+‐activated K+and Cl−conductances contribute to the caffeine response in rabbit aortic endothelial cells.

The administration of interferon-alpha/beta to female nude (nu/nu) mice caused significant changes in the levels of the cytosolic hepatic glutathione transferases. Antibodies raised against rat subunits, Ya, Yc, Yb1, Yb2, and Yk, and the subunits of the human transferases, mu (YbYb), lambda (YfYf), and epsilon (B1B1) all reacted with enzymes in the mouse and were used to demonstrate suppression and induction of transferase levels. Western blot analysis followed by semiquantitation by laser scanning showed the Ya, Yb1, Yb2, Yc, Yk, mu, and B1 subunits to be suppressed by 11, 11, 44, 30, 12, 14, and 47%, respectively, by interferon treatment. In contrast to these findings, the Yf subunit was induced 5-7-fold. A concomitant 220% increase was observed in the specific activity of the hepatic cytosol for ethacrynic acid, a substrate for the Yf subunit. Changes in the levels of transferase enzymes in normal and tumor cells may have significant implications when cytotoxic drugs are used in combination with interferons in cancer therapy. The Yf subunit, an enzyme found in human tumors and in placenta (Polidoro, G., Di Mio, C., Del Boccio, G., Zulli, P., and Fererici, G. (1980) Biochem. Pharmacol. 29, 1677-1680) has also been shown to be elevated in hepatic preneoplastic lesions (Kitahara, A., Satoh, K., Nishimura, K., Ishikawa, T., Ruike, K., Sato, K., Tsuda, H., and Ito, N. (1984) Cancer Res. 44, 2698-2703). These data indicate that the Yf subunit represents a potentially important interferon-inducible gene product.

The repetitive DNA sequence poly[d(GT).d(CA)], (polyGT) can be used to generate DNA finger-prints that distinguish different yeast genera. In this study we demonstrate that the probe can also be used to distinguish individual strains of clinical isolates of Candida albicans. Isolates were fingerprinted by probing Southern blots of restriction enzyme-cleaved DNA samples with radioactively labelled polyGT. The discrimination between strains was clearer than can be achieved by direct visualization of ethidium bromide stained gels and was comparable to that achieved with previous DNA probes. However, the advantage of this probe is that it is not limited to Candida species since polyGT sequences appear to be ubiquitous in eukaryotes. Fingerprints were also generated from Southern blots using a commercial (AMBIS) radioanalytical imaging computer system. A proprietary software package (MICRO PM) was effective in discriminating between the C. albicans strains. These preliminary results indicate the potential value of this probe for discrimination between Candida isolates in epidemiological studies of candidosis.

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Digestion of total DNA from Candida albicans isolates with the restriction enzyme HinfI generated simple, readily interpretable band patterns on ethidium bromide-stained gels. The procedure, which was highly reproducible, was used to detect restriction fragment length polymorphisms in 47 isolates of this species.