Comparative activity of selected antiviral compounds against clinical isolates of varicella-zoster virus

Sixteen freshly isolated varicella-zoster virus (VZV) strains were evaluated in vitro, in parallel with two reference strains expressing a functional thymidine kinase (TK⁺) (Oka and YS) and two thymidine kinase-deficient mutants (TK^–) (07–1 and YS-R), for their susceptibility to a broad range of antiviral compounds. The following compounds were included: acyclovir (ACV), brivudine (BVDU), sorivudine (BVaraU), other BVDU congeners such as BTDU, CTDU, CVDC and CVDU, ganciclovir (GCV), FIAC, araT, araA, araC, foscarnet (PFA), phosphonoacetic acid (PAA), the acyclic nucleoside phosphonates HPMPC, cHPMPC, HPMPA, cHPMPA, HPMPc³A, PMEA and PMEDAP, the N⁷-isomeric acyclic nucleoside analogue N⁷ AP, penciclovir (PCV), compounds 882C87 and H2G and two oxetanocin derivatives OXT-A and OXT-G. Fourteen of the 16 clinical isolates displayed the following order of decreasing selectivity against VZV: BVaraU > BVDU > CVDU CVDC > H2G > N⁷AP } CTDU BTDU OXT-G 882C87 > ACV > FIAC araT > HPMPC cHPMPC HPMPA HPMPc³A cHPMPA > PCV GCV OXT-A > PMEDAP PMEA > PFA PAA araA > araC. Two VZV strains (isolated from the cerebrospinal fluid of an AIDS patient) that were shown to have a truncated TK were clearly resistant to all the compounds that need the viral TK for their phosphorylation, while sensitivity to the acyclic nucleoside phosphonates, PFA, PAA, OXT-A and araA, remained unchanged. A slight (5- and 10-fold) increase was noted in the 50 % inhibitory concentration of N⁷AP and OXT-G, respectively, for the TK^– VZV strains as compared to the TK⁺ VZV strains. Ganciclovir and FIAC also showed a marked decrease in their activity against these two strains, but this was not as pronounced as for the other viral TK-dependent drugs. From our results, it appears that although acyclic nucleoside phosphonates may not have as favourable a therapeutic index as drugs requiring the viral TK, they should be considered for the treatment of TK^– VZV life-threatening infections that are resistant to the viral TK-dependent drugs     

Structure of the intramural nerves of the rat bladder

The bladder of adult female rats receives 16,000 axons (i.e., is the target of that many ganglion neurons) of which at least half are sensory. In nerves containing between 40 and 1200 axons cross-sectional area is proportional to number of axons; >99% of axons are unmyelinated. A capsule forms a seal around nerves and ends abruptly where nerves, after branching, contain 10 axons. A single blood vessel is present in many of the large nerves but never in nerves of <600 axons. The number of glial cells was estimated through the number of their nuclei. There is a glial nucleus profile every 76 axonal profiles. Each glial cell is associated with many axons and collectively covers 1,000 m of axonal length. In all nerves a few axonal profiles contain large clusters of vesicles independent of microtubules. The axons do not branch; they alter their relative position along the nerve; they vary in size along their length; none has a circular profile. All the axons are fully wrapped by glial cells and never contact each other. The volume of axons is larger than that of glial cells (55%–45%), while the surface of glial cell is twice as extensive as that of axons; there are 2.27 m² of axolemma and 4.60 m² of glial cell membrane per gram of nerve. Of the mitochondria of a nerve 3/4 are in axons and 1/4 in glial cells.     

Macromolecular structure of axon membrane and action potential conduction in myelin deficient and myelin deficient heterozygote rat optic nerves

Summary The macromolecular structure of the axon membrane in optic nerves from 25-day-old male littermate control and myelin deficient (md) rats and 16-month-old md heterozygotic rats was examined with quantitative freeze-fracture electron microscopy.The axon membrane of control optic nerves displayed an asymmetrical partitioning of intramembranous particles (IMPs); P-fracture faces of myelinated internodal axon membrane were more particulate than those of pre-myelinated axons (1600 1100 m^–2, respectively), while relatively few IMPs (150 m^–2) were present on external faces (E-faces) of internodal or pre-myelinated axon membrane. Amyelinated axons of md optic nerves also exhibited an asymmetrical partitioning of IMPs; protoplasmic membrane face (P-face) IMP densities, taken as a group, exhibited a wide range (600–2300 m^–2) and, in most regions, E-faces displayed a relatively low IMP density (175 m^–2). Axons of > 0.4 m diameter exhibited significantly greater mean P-face IMP density than axons < 0.4 m diameter. Aggregations of E-face IMPs (350 m^–2) were occasionally observed along amyelinated axon membrane from md optic nerves.Optic nerves from md heterozygote rats exhibit myelin mosaicism, permitting examination of myelinated and amyelinated axon membrane along the same tract. The axon membrane exhibits different ultrastructure in these two domains. Myelinated internodal axon membrane from md heterozygote optic nerves exhibits similar P- and E-face IMP densities to those of control internodal axolemma (1800 and 140 m^–2, respectively). Amyelinated axons in the heterozygote exhibit a membrane structure similar to amyelinated axons in md optic nerve. P-face IMP density of large diameter (> 0.4 m) amyelinated axons from md heterozygote optic nerves is significantly greater than that of small calibre (< 0.4 m) axons. In most regions, amyelinated axon membrane exhibits a relatively low E-face IMP density (200 m^–2); however, focal aggregations (400 m^–2) of E-face particles are present.Electrophysiological recordings demonstrate that amyelinated axons in md optic nerves support the conduction of action potentials. Compound action potentials in md optic nerves exhibit a monophasic configuration, even at 20-days postnatal, similar to that of pre-myelinated optic nerve of 7-day-old normal rats. Moreover, conduction velocities in the amyelinated 20-day-old md optic nerve are similar to those displayed by pre-myelinated axons from 7-day-old optic nerves. These results are consistent with persistence of action potential conduction in md axons, despite the absence of myelination in the optic nerves of the md mutant.     

Complete nucleotide sequence of Oat necrotic mottle virus: a distinct Tritimovirus species (family Potyviridae) most closely related to Wheat streak mosaic virus

Summary. The RNA genome (9346 nucleotides) of Oat necrotic mottle virus (ONMV) was cloned and sequenced. Complete genome comparisons indicated that ONMV, currently classified as a rymovirus, was most closely related (73% nucleotide, 79% amino acid identity) to the tritimovirus Wheat streak mosaic virus (WSMV). ONMV encoded a single polyprotein, with proteinase cleavage sites very similar to those of WSMV. Pairwise comparison of ONMV and WSMV cistrons revealed that P3 was most conserved (79% nucleotide, 86% amino acid), whereas HC-Pro was most divergent (67% nucleotide, 69% amino acid). In contrast, the ONMV sequence was distantly related (40% nt, 26% amino acid identity) to that of the rymovirusRyegrass mosaic virus, with highest sequence conservation noted within the NIb cistron (47% nucleotide, 41% amino acid identity). These results firmly establish that ONMV is not a rymovirus but is instead a distinct species of the genus Tritimovirus.     

Pharmacological modulation of plasminogen activator secretion by P388D1 cell line

P388D1 is a murine macrophage cell line which spontaneously secretes plasminogen activator (PA; activated function) and lysozyme (LYS; constitutive function). Compounds which decrease PA secretion without affecting LYS secretion have potential as down-regulators of macrophage function and, hence, of the immune system. Glucocorticoids (e.g., dexamethasone, IC₅₀<0.01 M) and auranofin (IC₅₀=1 M) are positive in this model. In contrast, cyclooxygenase inhibitors (indomethacin, ibuprofen and piroxicam, all at 1 M) boost PA secretion; lipoxygenase inhibitors (REV-5901, NDGA and piriprost, all at 10 M) have little or no effect.Dexamethasone, but not auranofin, induces a urokinase-inhibitory activity which elutes between 0.13 and 0.19M NaCl upon anion exchange HPLC (TSK-DEAE-5-PW). Fibrin overlay following SDS-PAGE of the HPLC peak reveals a urokinase-inhibitory band at 90 Kd.     

Length-dependent effects of osmotic compression on skinned rabbit psoas muscle fibers

The goal of this study was to characterize the interrelationship between sarcomere length and interfilament spacing in the control of Ca²⁺ sensitivity in skinned rabbit psoas muscle fibers. Measurements were made at sarcomere lengths 2.0, 2.7 and 3.4 m. At 2.7 m the fiber width was reduced by 17% relative to that at 2.0 m and the pCa₅₀ for force development was increased by 0.3 pCa units. In the presence of 5% Dextran T-500 the fiber width at sarcomere length 2.0 m was also decreased by 17% and the Ca²⁺ sensitivity was increased to the same value as at 2.7 m. In contrast, at sarcomere length 2.7 m the addition of as much as 10% Dextran T-500 had no effect on Ca²⁺ sensitivity. At sarcomere length 3.4 m there was an additional 7% compression and the Ca²⁺ sensitivity was increased slightly (0.1 pCa units) relative to that at 2.7 m. However at 3.4 m the addition of 5% Dextran T-500 caused the Ca²⁺ sensitivity to decrease to the level seen at 2.0 m. Given that the skinning process causes a swelling of the filament lattice it is evident that the relationship between sarcomere length and Ca²⁺ sensitivity observed in skinned fibers may not always be applicable to intact fibers. These data are consistent with measurements of Ca²⁺ in intact fibers which indicate that there might be a decline in Ca²⁺ sensitivity at long sarcomere lengths.     

The AC impedance of necturus gallbladder epithelium

The impedance of Necturus gallbladder epithelium was determined using sine wave currents of 1 Hz to 30 kHz. In control Ringer's solution the impedance locus exhibited a simple semicircle with minute shift of the high frequency end along the real axis and a minute depression of the center below the real axis (average 0.9±0.7°). Neglecting the slight suppression, the impedance of 1 cm² of epithelium can be represented by an electrical analogue consisting of a parallel RC element of 115±26 and 5.16±0.9 F in series with a small resistor of 5.3±1.3. In agreement with experimental results obtained under ionic or osmotic substitutions, the applicability of this simple RC analogue to gallbladder epithelium under control conditions can be explained by the influence of the paracellular shunt and by assuming the time constants of the apical and basal cell membranes to be comparable. Based on these data and on voltage divider measurements obtained with microelectrodes the capacitances of the apical and basal cell membrane can be estimated to be 7 and 18 F/cm². The latter value agrees well with estimates of the surface folding obtained from electronmicrographs, if the specific cell membrane capacitance is assumed to be 1 F/cm² as in other cell membranes.     

Fibre types inLimulus telson muscles: morphology and histochemistry

Summary Using a variety of techniques, we have demonstrated the presence of at least two fibre types inLimulus median telson levator muscle. By light and electron microscopy, large (21 56 m² mean cross-sectional area) fibres have A-bands of 4.1 m, one-half I bands of 2.15 m and Z lines 0.5 m in width. Few mitochondria are found in these fibres, which comprise 54% of those present in a given microscope field and which occupy 82% of the total cross-sectional area. Small fibres (484 m² mean cross-sectional area) have A bands of 6.3 m, one-half I bands of 3.1 m and Z lines between 0.5 and 1.0 m in width and are rich in mitochondria. Although small fibres comprise nearly one-half (46%) of the fibres in a field, they occupy only 18% of the total cross-sectional area.Histochemical staining for alkaline-stable myofibrillar ATPase activity and mitochondrial reduced -nicotinamide adenine nucleotide (-NADH) tetrazolium reductase activity confirms the presence of two fibre types. The large fibres react positively for the myofibrillar ATPase activity and negatively for the mitochondrial enzyme activity. The reverse is seen with the small fibres. Some fibres of intermediate size, having intermediate staining characteristics, were also observed. Native gel electrophoresis of both myofibrillar and purified myosin preparations supports the observed differences in myofibrillar ATPase activity in that two myosin isozymes are resolved on pyrophosphate gels. Although the thick filaments isolated from unstimulated small fibres are longer (>6.0 m) than those isolated from unstimulated large fibres (4.26 m), all have a similar appearance with respect to the arrangement of myosin heads on their surfaces, and similar diameters. The implications of the observed heterogeneity of fibre types is discussed with reference to previously reported phenomena inLimulus telson muscle, including changes in length of thick filaments on fibre stimulation and the shape of the length-tension curve obtained from fibre bundles.     

Plasma membrane structure at the axon hillock, initial segment and cell body of frog dorsal root ganglion cells

Summary Analysis of the plasmalemma of frog dorsal root ganglion cells by freeze-fracture demonstrates regional differences in the distribution of intramembranous particles. Although P-face particles are distributed rather uniformly, the E-face particle concentration at the cell body (300 m^–2) is much lower than that at the axon hillock (900 m^–2), proximal initial segment (1000 m^–2), or intermediate portion of the initial segment (800 m^–2). The particle concentrations in the latter regions approach that at the node of Ranvier and, moreover, particle size analysis reveals that the E-face particles, like those at the node, include a large number that are 10 nm or more in diameter. Thin sections reveal patches of a dense undercoating on the cytoplasmic surface of the axolemma in some regions of the initial segment but not the axon hillock. It is concluded from these results that the axon hillock and the initial segment of dorsal root ganglion cells have some of the structural characteristics of the node of Ranvier.     

Interaction of G-actin with thymosin β4 and its variants thymosin β9 and thymosin β 9 met

Summary Thymosin ₄ is a major actin sequestering peptide in vertebrate cells and plays a role in the regulation of actin monomer/polymer ratio. Thymosin ₉ and thymosin ₉ ^met are minor variants of thymosin ₄. The possible function of these peptides has been investigated by comparing the actin binding properties of these -thymosins. Thymosin ₉ and thymosin ₉ ^met were found to inhibit polymerization of ATP-actin with identical K _d s of 0.7–0.8 M (as compared to 2±0.3 M for thymosin ₄); like thymosin ₄, they bound to ADP-G-actin with a 100-fold lower affinity than to ATP-G-actin. The interaction of thymosin ₄ and thymosin ₉ ^met with G-actin was weakened 20-fold upon oxidation of methionine-6 into methionine sulfoxide. Binding of thymosin ₄ to G-actin was accompanied by a 15% increase in the fluorescence intensity of actin tryptophans, and a 10 nm emission blue shift. Methionine-6 played an important role in this effect. The fluorescence change was used to monitor the kinetics of thymosin ₄ binding to G-actin in the stopped-flow. The reaction was bimolecular, with association and dissociation rate constants of 1.5 M^-1 s^-1 and 2s^-1 respectively, under physiological conditions. The possible physiological significances of methionine-6 oxidation and of the relatively slow binding kinetics in regulating thymosin ₄ function in vivo is discussed.     

Freeze-fracture study of the mechanoreceptive digital corpuscles of mice

Summary The freeze-fracture replication technique was used to study the mechanoreceptive digital corpuscles in toe pads of mice. The axon terminal plasmalemma had intramembranous particles (IMPs) at a density of 2367 ± 517 m^–2 (mean ±s.e.m.) in the P-face and 84 ± 4 m^–2 in the E-face. Particles were 10 ± 1.8 nm in diameter in the P-face and 10 ± 1.5 nm (mean ±s.d.) in the E-face. Particle-rich and particle-free areas were noted in the P-face. The lamellar cell plasmalemma had IMPs at a density of 3359 ± 224 m^–2 in the P-face and 265 ± 95 m^–2 in the E-face. Particles were 10 ± 1.4 nm in diameter in the P-face and 10 ± 1.6 nm in the E-face. Non-terminal unmyelinated fibres in the connective tissue compartment of toe pads were also examined: the P-faces of the axolemma and Schwann cell plasmalemma had IMPs at a density of 1356 ± 283 m^–2 and 1514 ± 514 m^–2, respectively, while the E-face of these membranes had only a few particles. Particles were 9 ± 1.2 nm and 10 ± 1.6 nm in diameter in the P-faces of axon and Schwann cell plasmalemmata, respectively.The results show that the IMPs in terminal axolemma and in lamellar cell plasmalemma have a much higher density than those of non-terminal axons or Schwann cells in myelinated and unmyelinated fibres. In addition, IMPs in the terminal axolemma are larger than those in non-terminal axolemma except for the nodal axolemma. It can be said that plasmalemmata of both the axon terminals and lamellar cells of digital corpuscles are specialized in terms of IMPs, suggesting that they have specific physiological properties in mechanoreceptive functions including mechano-electric transduction.     

Enzymatic Microreactor Using Si, Glass and EPON SU-8

This article describes the design, fabrication and initial testing of an enzymatic microreactor using Si, glass, epoxy and Perspex components. The microreactor geometry was defined by a photostructured EPON SU-8 spacer. This gave two chambers, an upstream chamber (vol. 2.7 l) which was packed with control pore glass (CPG) and a downstream chamber (vol. 1.5 l) which contained the detectors. Eleven EPON SU-8 columns separated the chambers and prevented the CPG from passing. Glucose detection via glucose oxidase immobilized to the CPG was used as a model system. Conversion efficiencies between 15 and 60% at flow rates of 150 and 26 1 min^–1 respectively were obtained. Overall the system was found to be robust and to have a lifetime of at least one month.     

Efficacy of the two-microelectrode voltage clamp technique in crayfish muscle

Crayfish muscle fibres of different dimensions were voltage clamped and white noise current was injected into the fibres at various distances from the voltage clamp current electrode. The clamp current was measured and power spectral densities were calculated. This method revealed the efficacy of the voltage clamp in these fibres. In large fibres (l=1.8–2.0 mm; =100–180m) a space clamp was achieved only for a band width f=40Hz. At a distance of 100m from the clamp electrodes f was 250–500Hz. In fibres of medium size (l=1.0–1.3mm; =60–120m) f was about 80Hz and about 800 Hz at a distance of 100m. In experiments with very small muscle fibres (l=400–600m; =30–50m) f was more than 500Hz. The improvement of the space clamp for the smaller muscle fibres resulted mainly from the reduced total membrane capacity,c _m, of these fibres. The limitations of the space clamp could be derived from the impedance properties of the fibres. The band width of the space clamp correlated with the band width for which the square of the absolute impedance, |Z _p|², of the muscle fibre could be described by a simple RC-model. This correlation was demonstrated in a model circuit.Power density spectra of membrane current fluctuations were measured also. To optimize the resolution of these measurements the contribution of instrumental noise was minimized. The effects of instrumental noise are discussed.This investigation was supported by the Deutsche Forschungs-gemeinschaft     

ω-Grammotoxin blocks action-potential-induced Ca2+ influx and whole-cell Ca2+ current in rat dorsal-root ganglion neurons

Field-potential stimulation of rat dorsal-root ganglion (DRG) neurons evoked action-potential-mediated transient increases in intracellular free calcium concentration ([Ca²⁺]_i) as measured by indo-1-based microfluorimetry. Field-potential-evoked [Ca²⁺]_i transients were abolished by tetrodotoxin, and their dependence on stimulus intensity exhibited an abrupt threshold. -Conotoxin GVIA (-CgTx, 100 nM) inhibited action-potential-mediated Ca²⁺ influx by 79%, while nitrendipine (1 M) had little effect. -Grammotoxin SIA (-GsTx, 267 nM), a peptide toxin purified from the venom of the tarantula spider, Grammostola spatulata, blocked action-potential-mediated Ca²⁺ influx as effectively as did -CgTx, suggesting that -GsTx blocks N-type Ca²⁺ channels. In contrast to block by -CgTx, the block produced by -GsTx reversed upon washout of the peptide. -GsTx (270 nM) blocked 80%, and -CgTx (1 M) blocked 64%, of whole-cell Ca²⁺ current (I _Ca) elicited by step depolarization to 0 mV from a holding potential of –80 mV. -GsTx completely occluded inhibition of I _Ca by -CgTx. However, when applied after -CgTx, -GsTx produced an additional inhibition of 27%, indicating that -GsTx also blocked a non-N-type Ca²⁺ channel. BayK8644 (1 M) elicited an increase in I _Ca in the presence of maximally effective concentrations of -GsTx, suggesting that -GsTx does not block L-type channels. Thus, -GsTx displays a selectivity for Ca²⁺ channel subtypes which should prove useful for studying Ca²⁺ channels and Ca²⁺-channel-mediated processes.     

Identification and quantitation of cortisol metabolites in rat liver homogenate and -slices

Summary Cortisol-1, 2-H³ was incubated with rat liver homogenate and/or rat liver slices in the presence of a NADPH-generating system. The following metabolites could be identified in adult male rats: -cortol, allo--cortol, 3-allo--cortol, 20-hydroxy-cortisol, 11, 17, 20, 21-tetrahydroxy-5-pregnan-3-one, 3-allotetrahydrocortisol, tetrahydrocortisol, trace amounts of allotetrahydrocortisol and two highly polar metabolites only partly identified. In female rats only tetrahydrocortisol, allotetrahydrocortisol and allodihydrocortisol could be detected in significant amounts.The radioactive metabolites mentioned above were localized and quantitated on paper chromatograms by a 4-radiochromatogram scanner. A nearly perfect correlation was found between these results so obtained and those given by liquid-scintillation counting of each metabolite after its elution from the paper.Part of this work was supported by grant n° 695 of the National Fonds voor Wetenschappelijk Geneeskundig Onderzoek.Stagiair of the Nationaal Fonds voor Wetenschappelijk Onderzoek.     

Kinetic and pharmacological properties of high- and low-threshold calcium channels in primary cultures of rat hippocampal neurons

The kinetic, permeability and pharmacological properties of Ca currents were investigated in primary cultures of rat hippocampal neurons. The low-voltage-activated (LVA) Ca current turned on positive to –60mV and fully inactivated in a voltage-dependent way. This current was depressed by nickel (Ni, 40 M) and amiloride (500 M) and was insensitive to -conotoxin (-CgTx) (4 M) and to the Ca agonist Bay K 8644 (5 M). The high-voltage-activated (HVA) Ca current turned on positive to –40 mV and inactivated slowly and incompletely. This current was much less sensitive than the LVA current to Ni and amiloride but more sensitive to cadmium. CgTx blocked only partially this current (about 50%) in an irreversible way. Bay K 8644 had a clear agonistic action almost exclusively on the -CgTx-resistant HVA current component. The present results suggest that the HVA channels, quite homogeneous for their kinetic properties and sensitivity to holding potentials, can be pharmacologically separated in two classes: (i) -CgTx-sensitive and Bay-K-8644-insensitive (-S/BK-I) and (ii) -CgTx-insensitive and Bay-K-8644-sensitive (-I/BK-S), the latter displaying a stronger Cadependent inactivation.     

Effects of vector cutting on its recombination with the chromosomal immunoglobulin gene in hybridoma cells

We have analyzed the effects of linearizing vector DNA on the frequency and pathway of its recombination with the homologous chromosomal gene. The pSV2neo vector bearing a 4.3-kb fragment encoding the mouse immunoglobulin heavy chain constant (C) region was cut either at sites within the C segment or outside C and then transferred to hybridoma cells bearing a mutant gene. The frequency of recombinant cells producing normal was then measured. For most cut sites, whether in regions of homology or of nonhomology, linearization of the transferred DNA enhanced the recombination frequency between the vector and chromosomal genes. When the vector was either uncut or cut at SacI in the region of homology, G418-resistant m⁺ recombinants were found to have integrated the vector by a single reciprocal homologous crossover; the enzyme site (SacI) used for cutting was present in the recombinants. By contrast, when the vector had been linearized at Pvul or SfiI in the region of nonhomology, vector integration involved nonhomologous crossovers, either between transferred DNA molecules or between transferred and chromosomal DNA, and the vector cut sites were absent in these recombinants. Some recombinants were found to have an unaltered as well as recombinant gene, suggesting that the nonhomologous recombination process might have involved sister chromatids.     

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.     

Facilitation by 5-hydroxytryptamine of ATP-activated current in rat pheochromocytoma cells

The effects of 5-hydroxytryptamine (5-HT) on an inward current activated by extracellular ATP were investigated in rat pheochromocytoma PC12 cells. Under whole-cell voltage-clamp conditions 5-HT (10 M) reversibly enhanced the amplitude of the current activated by 30 M ATP. The enhancement may not be due to an increase in the number of functional channels because the current activated by 300 M ATP was not remarkably augmented compared with the current activated by 30 M ATP. The current enhancement by 100 M 5-HT was less obvious than that by 10 M 5-HT. When the current kinetics were compared, activation of the ATP-evoked current was accelerated to the same extent by either 10 or 100 M 5-HT, whereas deactivation was largely more accelerated by 100 M 5-HT. Propranolol (10 M), a 5-HT₁ receptor antagonist, or LY53857 (10 M), a 5-HT₂ receptor antagonist, exerted an agonistic effect: the ATP-activated current was facilitated by these compounds. Metoclopramide (10 M), a 5-HT₃ receptor antagonist, neither facilitated the ATP-activated current, nor blocked the current facilitation by 5-HT. Guanosine 5-O-(2-thiodiphosphate) (GDP[S]) (2 mM), the non-hydrolysable analog of guanosine 5-triphosphate (GTP), or K-252a (2 M), a protein kinase inhibitor, did not affect the facilitation by 5-HT of the ATP-activated current when they were included in the intracellular solution. The ATP-activated current pre-facilitated by 10 M dopamine was not enhanced by 10 M 5-HT. Similarly, the pre-facilitation by 5-HT attenuated the current enhancement by dopamine. The results suggest that 5-HT facilitates the ATP-activated channels through receptors that are not readily classified into conventional subclasses of 5-HT receptors. The reciprocal masking between the current facilitation by 5-HT and that by dopamine, combined with their sensitivities to the compounds involved in the intracellular solution, indicates that the facilitation by 5-HT may share not all, but some, common cellular mechanism with that by dopamine.     

Nuclease treatment results in high specific purification of Creutzfeldt-Jakob disease infectivity with a density characteristic of nucleic acid-protein complexes

Summary Representative preparations of partially purified Creutzfeldt-Jakob disease (CJD), including disaggregated density gradient fractions, were treated with a variety of nucleases. RNases as well as exhaustive digestions with micrococcal nuclease did not significantly diminish infectivity, but resulted in an 7,000-fold specific purification of infectivity with respect of nucleic acid. Protected nucleic acids included species of up to 2,000 bases in length. After nuclease treatment, infectivity co-migrated with nucleic acid-protein complexes at a density of 1.27 g/cm³ in sucrose. Substantial specific protein purification were also achieved in the gradient step ( 11,000-fold), where 70% the host Gp34 (prion protein) as well as other free proteins separated from infectivity. These CJD purifications are better than those previously attained in scrapie, and may be useful for further studies of non-host protein and nucleic acid species. The data are consistent with the hypothesis that CJD-like agents are composed of nucleic acid-protein complexes.