Molecular epidemiology of adenoviruses associated with acute lower respiratory disease of children in Buenos Aires, Argentina (1984-1988). DNA restriction analysis was carried out on a sample of 73 adenovirus strains isolated in Buenos Aires from nasopharyngeal aspirates of children with lower acute respiratory infection between 1984 and 1988. Thirty-five isolates (47.9%) were classified as members of subgenus B. Of these, three were identified as a new genome type of Ad3p denominated Ad3p3; five strains corresponded to genome type 7b and two to genome type 7c. The other 25 isolates were identified as the recently recognized genome type 7h. All 6 fatalities recorded within this group of 73 children were associated with infection by Adenovirus genome type 7h. Thirty-seven isolates (50.7%) were classified within subgenus C that corresponded to 9 different genome types denominated 1p (n = 5); 1# (n = 2); 2p (n = 4); 2b (n = 6); 2# (n = 5); 5# (n = 4); 5* (n = 7) and 5+ (n = 2). All genome types of subgenus C were compared with the data reported by Adrian et al. (Archives of Virology 112:235-238, 1990). The Ad1p and Ad1# genome types could be allocated to AV1 genome types D1 and D10, respectively. Ad2b genome type could be allocated to AV2 genome type D25. No counterparts were found for the remaining 6 genomic variants. Only one isolate was identified as Ad4a of subgenus E. The comparison of the results of the present study with those of the molecular characterization of Chilean strains isolated between 1984 and 1987 suggests that the adenovirus strains associated with respiratory disease of children may be common in both countries.


Redox chemistry of complexes of nickel(II) with some biologically important peptides in the presence of reduced oxygen species: an ESR study. The reactions between some Ni(II) oligopeptides (Gly-His-Lys, (Gly)4, Asp-Ala-His-Lys, Gly-Gly-His, beta Ala-His, and serum albumin) and reduced oxygen species have been characterized by spin-trapping experiments using DMPO and Me2SO. Most of the peptides possessed superoxide dismutase- and catalase-like activities leading to the formation of either oxene [NiO]2+ or, in the case of beta Ala-His, hydroxyl radicals. Both these species may affect DNA integrity through distinct mechanisms.


Disorders of neuromuscular transmission due to natural environmental toxins. A variety of natural toxins of animal, plant, and bacterial origin are capable of causing disorders of neuromuscular transmission. Animal toxins include venomous snakes and arthropods, venoms of certain marine creatures, skin secretions of dart-poison frogs, and poisonous fish, shellfish, and crabs. There are plant poisons such as curare, and bacterial poisons such as botulinum toxin. These act at single or multiple sites of the neuromuscular apparatus interfering with voltage-gated ion channels, acetylcholine release, depolarization of the postsynaptic membrane, or generation and spread of the muscle action potential. The specific actions of these toxins are being widely exploited in the study of neuromuscular physiology and pathology. Some toxins have proved to be valuable pharmaceutical agents. Poisoning by natural neurotoxins is an important public health hazard in many parts of the world, particularly in the tropics. Poisoning may occur by a bite or a sting of a venomous animal, or by the ingestion of poisonous fish, shellfish or other marine delicacies. Contaminated food is a vehicle for poisons such as botulinum toxin. Clinically, a cardinal feature in the symptomatology is muscle paralysis with a distribution characteristic of myasthenia gravis, affecting muscles innervated by cranial nerves, neck flexors, proximal limb muscles, and respiratory muscles. Respiratory paralysis may end fatally. This paper reviews from the clinical and pathophysiologic viewpoints, naturally occurring environmental neurotoxins acting at the neuromuscular junction.


Evidence for intramitochondrial complementation between deleted and normal mitochondrial DNA in some patients with mitochondrial myopathy. Twenty-three patients with mitochondrial myopathies and mitochondrial DNA deletions in muscle were studied by means of deletion mapping and sequencing, histochemistry and polarography. Histochemistry showed significantly less focal cytochrome oxidase deficiency relative to number of ragged red fibres when the deletion did not involve reading frames for cytochrome oxidase subunits. Polarography in such patients showed defects exclusively involving complex I, in contrast to the others with larger deletions who generally had more diffuse respiratory chain defects. Analysis of other published histochemical data showed similar findings to our own. It is concluded that translation of a proportion of deleted mitochondrial DNAs occurs in at least some patients with mitochondrial DNA deletions, implying that deleted and normal mitochondrial genomes share transfer RNAs within mitochondria in such cases.


Effects of ethanol and other drugs on excitatory and inhibitory neurotransmission in the crayfish. 1. Crayfish exposed to 434 mM ethanol (EtOH) showed signs of hyperactivity within 0.5-2 h, at which times crayfish hemolymph EtOH concentration had reached 60-90 mM. 2. A 10-min exposure to 60-90 mM EtOH reduced presynaptic inhibition of excitatory postsynaptic currents (EPSCs) at the crayfish opener neuromuscular junction (NMJ) in vitro but did not significantly alter excitatory neurotransmission. The same concentrations of EtOH did not alter other potentials or currents associated with inhibition at this synapse, such as presynaptic inhibitory potentials (PIPs), inhibitory postsynaptic potentials (IPSPs), and inhibitory postsynaptic currents (IPSCs). 3. Intermediate EtOH concentrations (120-180 mM) applied for 10 min in vitro reduced the amplitude of excitatory postsynaptic potentials (EPSPs) by decreasing the membrane resistance of opener muscle fibers and by reducing the amplitude of EPSCs. 4. High EtOH concentrations (434 mM) applied for 10 min in vitro had yet greater depressive effects on measures of postsynaptic properties described above. The time course of EPSCs was also significantly reduced. In addition, presynaptic properties such as action-potential (AP) amplitude and frequency of spontaneous release of neurotransmitter were reduced by 434 mM EtOH. 5. Presynaptic inhibition, gamma-aminobutyric acid (GABA; 250-500 microM), muscimol (50 microM), and baclofen (75 microM) all reduced the depolarizing afterpotential of APs in the excitor axon and reduced EPSPs in opener muscle fibers. GABA (500 microM) and baclofen (75 microM) significantly reduced presynaptic AP amplitudes, whereas presynaptic inhibition, GABA (250 microM), and muscimol (50 microM) had no effect on AP amplitude. Bicuculline (250-500 microM), a GABAA antagonist, did not entirely eliminate presynaptic inhibition, whereas picrotoxin (50 microM), another GABAA antagonist, completely removed presynaptic inhibition. Thus presynaptic inhibitory mechanisms may involve both GABAA and GABAB receptors on the opener excitor axon. 6. Our data suggest that the behavioral hyperactivity seen at hemolymph EtOH concentrations of 60-90 mM is not accompanied by a change in excitatory synaptic transmission observed at the opener NMJ. Rather, crayfish hyperactivity may be due to depressive effects of EtOH on inhibitory synapses in the CNS similar to the disinhibition evoked by EtOH at the opener NMJ.


Synaptic connections between nonspiking afferent neurons and motor neurons underlying phase-dependent reflexes in crayfish. 1. This paper analyzes the synaptic connections made by nonspiking afferent neurons of the thoracocoxal muscle receptor organ (TCMRO) with basal limb motor neurons in the crayfish. The T fiber, a dynamically sensitive afferent, monosynaptically excites promotor motor neurons. Evidence suggests that both tonic graded chemical transmission and electrical synaptic transmission may be involved, depending on the motor neuron under consideration. 2. In preparations in the active state (spontaneously producing reciprocal motor patterns), the T fiber also inhibits promotor motor neurons in a phase-dependent manner. This inhibitory pathway is probably indirect, because it involves additional synaptic delay. 3. The statically sensitive S fiber also excites promotor motor neurons, but phase-dependent inhibition of promotor motor neurons by the S fiber was not seen. 4. The T fiber excites a subclass of remotor motor neurons (group 1) by a combination of direct chemical input and electrical input. This connection underlies the positive feedback reflex that excites these remotor motor neurons, in a phase-dependent manner, on stretch of the TCMRO during the active state. In inactive preparations, this connection remains subthreshold. 5. Central synaptic outputs of group 1 remotor motor neurons can also inhibit promotor motor neurons. This pathway may contribute to the phase-dependent reflex inhibition of promotor motor neurons that occurs in the active state.


Calcium homeostasis in dissociated embryonic neurons: a flow cytometric analysis. 1. Ca2+ homeostasis in freshly dissociated neurons from embryonic rat hypothalamus, cortex, and brain stem was investigated with flow cytometry. Cells were dissociated from embryonic brain by enzymatic and mechanical means and were incubated with the acetoxymethylester derivative of the Ca(2+)-sensitive dye indo-1. Neurons hydrolyzed and retained the dye as determined by the intensity of fluorescence emission, whereas similarly treated cultured astrocytes gave very low-level fluorescence. 2. The fluorescence of the indo-1 dye was measured at two wavelengths (405 and 485 nm) for each cell. Data were collected only from those cells (presumptive neurons) with high levels of fluorescence. Methods were developed to calibrate the level of intracellular free calcium ([Ca2+]i) as the ratio of fluorescence at 410 and 485 nm. The level of intracellular free Ca2+ was then calculated for each neuron. 3. A wide distribution of resting [Ca2+]i was found, with a median of approximately 90 nM. After addition of ionomycin to cells in Ca(2+)-free medium, there was a transient increase in [Ca2+]i, suggesting that all embryonic neurons had internal Ca2+ stores. The presence of active calcium extrusion mechanisms was demonstrated with the use of ionomycin in Ca(2+)-containing medium and with metabolic inhibitors. Furthermore, incubation in sodium-free medium resulted in a transient increase in [Ca2+]i and a reduced ability to eliminate elevated [Ca2+]i from the cytoplasm, suggesting that calcium homeostasis was dependent on the activity of the Na(+)-Ca2+ exchange mechanism. 4. Depolarization with K+ or veratrine increased [Ca2+]i in approximately 20% of the cells. This increase was blocked by eliminating extracellular free Ca2+ or adding Co2+, nifedipine, or verapamil, suggesting mediation by voltage-sensitive calcium channels. 5. Neurons were sorted on the basis of high [Ca2+]i and placed into dissociated culture. After 24 h, neurons in culture retained indo-1 fluorescence, suggesting that populations of neurons can be collected on the basis of their levels of [Ca2+]i. 6. These results demonstrate that flow cytometric analysis allows the characterization of a variety of Ca(2+)-regulatory mechanisms in populations of freshly dissociated embryonic neurons. Although only a proportion of embryonic day 17 neurons exhibit voltage-sensitive calcium channels, all neurons have developed the ability to sequester and extrude Ca2+.


GABAA receptor antagonist bicuculline alters response properties of posteroventral cochlear nucleus neurons. 1. The role of GABAergic inhibitory inputs onto posteroventral cochlear nucleus (PVCN) neurons in the anesthetized chinchilla was investigated through iontophoretic application of the GABAA receptor agonist muscimol and the GABAA receptor antagonist bicuculline. The majority of the neurons studied displayed phasic temporal response patterns. 2. All the neurons were sensitive to bicuculline and displayed an increase in discharge rate, which was greatest during the post-onset portion of the response. Most of the tested neurons were also sensitive to muscimol, which appeared to mimic the putative effect of endogenous GABA. 3. Bicuculline reduced the average first-spike latency and the average variability of the first-spike latency. Muscimol had the opposite effect. 4. Bicuculline did not significantly alter the threshold but rather increased discharge rate at suprathreshold intensities. 5. The width of the excitatory response area was not significantly increased by application of bicuculline. The increase in discharge rate occurred within the units' excitatory response areas. 6. The shape of the rate-intensity functions was not altered by bicuculline application. 7. We conclude that GABAergic inhibitory inputs control the post-onset discharge rate of some PVCN neurons. They may suppress tonic activity, resulting in more phasic discharge patterns.


Long-term regulation of neuronal calcium currents by prolonged changes of membrane potential. Although rapid-onset, short-term regulation of neuronal Ca currents by neurotransmitters and second messengers is well documented, little is known about conditions that can cause longer-lasting changes in Ca channel function. We report here that persistent depolarization is accompanied by slowly developing long-term reduction of neuronal Ca currents. Rat myenteric neurons grown in cell culture for 1-7 d were studied with the tight-seal whole-cell recording technique. Macroscopic Ca-channel currents had decaying and sustained components at all days studied. When the neurons were grown in medium containing 25 mM KCl, which depolarized them to -40 mV and caused significant elevation of intracellular Ca, the densities of both components of Ca-channel current decreased by 40-80%. Several results suggest that different mechanisms underlie the downregulation of the two components. (1) The density of the decaying component decreased approximately four times faster than did that of the sustained component. (2) When neurons were returned to control medium, which contained 5 mM KCl, the density of the sustained component returned to control levels within 24 hr, while that of the decaying component did not recover significantly. (3) Inhibitors of RNA and protein synthesis reduced or prevented downregulation of the sustained but not of the decaying component. (4) The dihydropyridine antagonist nitrendipine, which prevented the sustained elevation of intracellular Ca in neurons grown in 25 mM KCl, prevented downregulation of the sustained component but had no effect on downregulation of the decaying component. We suggest that these forms of regulation of Ca current density could help neurons adapt to altered levels of electrical activity and may contribute to changes in synaptic strength that occur during periods of increased or decreased electrical activity.


Selective regulation of Gi alpha 1 expression and function in PC12 cells by cAMP. Hormone and neutrotransmitter receptor systems regulate both the activity and expression of GTP-binding proteins (G-proteins). However, relatively little is known about the mechanism by which this regulation occurs. One G-protein subtype, Gi alpha 1, is expressed primarily in neuronal cells. Here, we demonstrate the selective regulation of Gi alpha 1 mRNA and protein levels by cAMP. Treatment of PC12 cells with forskolin increases Gi alpha protein levels. Similarly, incubation of PC12 cells with agents that increase intracellular levels of cAMP, including forskolin, dibutyryl-cAMP, and 8-bromo-cAMP, induce a two- to fourfold increase in Gi alpha 1 mRNA levels. Furthermore, the effect of increased intracellular cAMP is specific for Gi alpha 1 mRNA expression; the levels of mRNA encoding other G-protein subtypes remain unaltered. cAMP-stimulated Gi alpha 1 expression occurs within hours of treatment and is sustained for days. Increasing intracellular cAMP by activation of cell surface adenosine receptors also increases Gi alpha 1 mRNA levels. Treatment of PC12 cells with phorbol esters, NGF, or depolarizing concentrations of KCl did not increase Gi alpha 1 mRNA expression, demonstrating that Gi alpha 1 expression is specifically regulated by cAMP. Guanine nucleotide-mediated inhibition of adenylate cyclase activity was measured in order to determine if the change in Gi alpha protein expression was accompanied by a change in G-protein function. Adenylate cyclase activity in PC12 cells treated with an adenosine analog and therefore expressing higher levels of Gi alpha protein is more sensitive to inhibition by guanine nucleotides than in nontreated PC12 cells.(ABSTRACT TRUNCATED AT 250 WORDS)


Clustering of muscle acetylcholine receptors requires motoneurons in live embryos, but not in cell culture. Previous culture studies have demonstrated that muscle cells autonomously express and cluster ACh receptors (AChRs) and that contact by neurites induces a reorganization of these clusters. We studied these phenomena in zebrafish embryos where the same cells could be examined in vivo and in vitro, and where contacts between cells could be viewed repeatedly. Receptor clusters first appeared when the pioneer growth cones emerged from the spinal cord, were always associated with labeled branches, and developed normally in the presence of neuromuscular transmission blockers. When motoneurons were removed, the muscles failed to cluster receptors. In contrast, muscle cells grown in cell culture uncontacted by nerves clustered AChRs. Our results suggest that clustering of AChRs in living embryos is induced by the presence of neurites and does not occur in the absence of neuronal contact. We suggest that conditions in cell culture, which differ from those in the intact embryo, induce clusters on isolated muscle cells. Moreover, our results demonstrate that receptors cluster without binding transmitter and in the absence of neuronal activity.


Systemic and local immune responses to enhanced-potency inactivated poliovirus vaccine in premature and term infants. Serum neutralizing, nasopharyngeal neutralizing, and IgA antibodies to polioviruses 1, 2, and 3 were detected in preterm and term infants who had received three doses of an enhanced-potency inactivated poliovirus vaccine at 2, 4, and 12 months of age. After the third dose of this vaccine, 95% or more of the infants tested had detectable serum neutralizing antibodies to polioviruses types 1, 2, and 3. Nasopharyngeal neutralizing and IgA antibodies were detected in 43% to 91% of the infants. The peak geometric mean titers of serum and nasopharyngeal antibodies against polioviruses types 1, 2, and 3 were similar for both groups. These preliminary data indicate that preterm infants are capable of mounting systemic and local immune responses to enhanced-potency inactivated poliovirus vaccine that are comparable to those made by term infants.


Electrodiagnosis reliability in the diagnosis of infant botulism. Infant botulism is confirmed by isolation of Clostridium botulinum from stool culture or by toxin assay. Although electrodiagnosis has been described as a diagnostic tool in infant botulism, our 11-year review of toxin-confirmed cases suggests that electrodiagnosis is not a reliable tool. In the case report presented, results of electrodiagnosis were negative but enema effluent contained adequate concentrations of organism and toxin to confirm the diagnosis.


An MMPI typology of cocaine abusers.

An MMPI typology of cocaine abusers. Three types were found in a cluster analysis of scores on the Minnesota Multiphasic Personality Inventory (MMPI) of 100 men voluntarily presenting for treatment of cocaine abuse. Type 1 (n = 59) had a mean MMPI profile consistent with the hypothesized grouping of persons who self-medicate with cocaine as a means of overcoming depression. The relatively normal mean MMPI profile obtained by Type 2 (n = 37) suggested little indication of significant psychopathology. Type 3's (n = 4) mean MMPI profile suggested either severe disturbance or profile invalidity. Types were found to differ in the nature of drug use, reactions to cocaine, education level, and social class, but not on the scales of the Adjective Check List.


New measures for assessing alcohol and drug abuse with the MMPI-2: The APS and AAS. We introduce two new scales for assessing substance abuse problems with the Minnesota Multiphasic Personality Inventory-2 (MMPI-2): the Addiction Potential Scale (APS), a 39-item empirically derived scale, developed by contrasting the responses of a large residential substance abuse sample with responses from both normative and psychiatric control groups; and the Addiction Acknowledgement Scale (AAS), a 13-item face-valid scale, constructed rationally and with attention to internal consistency. Both new scales are shown to discriminate well between groups and substantially better than other selected substance abuse scales. Covariation between the scales and joint effectiveness are examined. Finally, limitations for their practical utility are expressed, and considerations for future research are identified.


Adenosine 5'-monophosphate enhances the dark and isoproterenol-induced rise in rat pineal N-acetyltransferase activity. Exposure of rats to light during darkness or blockade of the pineal beta-adrenoceptor in stimulated pineal glands results in a rapid fall in pineal N-acetyltransferase (NAT) activity. Maintenance of a high level of NAT activity requires continuous stimulation of the pineal beta-adrenoceptors. It is not known what factors in the pinealocyte are responsible for this rapid inactivation of NAT activity. In the present study we have attempted to investigate a possible regulatory role of 5'-AMP on pineal NAT activity. The results show that 5'-AMP further enhances dark-induced as well as isoproterenol-induced NAT activity by approximately 3-fold but does not alter unstimulated daytime NAT activity. Theophylline, an inhibitor of 5'-AMP synthesis, when administered early in the dark phase, caused a rise in pineal cAMP with a concomitant fall in pineal NAT activity. These findings indicate that 5'-AMP could play a role in the activation of pineal NAT. The possibility that the rapid inactivation of NAT is due to a rapid removal of 5'-AMP by, for example, phosphorylation, remains to be investigated.


Modulation of the discriminative stimulus effects of cocaine by mu and kappa opioids. The effects of cocaine alone and after pretreatment with selective mu and kappa opioids were determined in squirrel monkeys trained to discriminate i.m. injections of cocaine from vehicle in a two-lever discrimination procedure. Lever pressing was maintained under a fixed ratio schedule of food presentation. When administered alone, cocaine engendered dose-related increases in the proportion of cocaine-appropriate responding with an average ED50 of 0.19 mg/kg. Pretreatment with the mu agonists morphine (0.3 and 1.0 mg/kg), levorphanol (0.03 and 0.1 mg/kg) and methadone (0.1 and 0.3 mg/kg), as well as the mu partial agonist buprenorphine (3.0 and 5.6 micrograms/kg), potentiated the discriminative stimulus effects of cocaine such that the cocaine dose-effect functions were shifted to the left and the average ED50 for cocaine was reduced maximally by about one order of magnitude. None of the mu agonists consistently substituted for cocaine when administered alone, indicating that the observed interactions were not simply the result of additive discriminative stimulus effects. A similar potentiation by mu agonists was observed for the effects of cocaine on fixed ratio response rate. In contrast to the mu agonists, pretreatment with the kappa agonists N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4,5]dec-8-yll-4- benzofuranacetamide (CI 977; 3.0 and 5.6 micrograms/kg) and benzeneacetamide methane sulfonate (U 50,488; 0.3 and 1.0 mg/kg) attenuated the discriminative stimulus effects of cocaine in most monkeys, resulting in a modest (2- to 3-fold) increase in the average ED50 for cocaine.(ABSTRACT TRUNCATED AT 250 WORDS)


Effect of lithium and amiloride on collecting tubule transport enzymes. In humans and animals, the administration of Li or amiloride results in a defect in urinary acidification. Both agents are thought to cause this by a voltage-dependent mechanism in the distal nephron. This study was designed to determine the effects of chronic Li and amiloride administration on the two main transport enzymes in rat nephron collecting tubule, the Na-K-adenosine triphosphatase (ATPase) and the H(+)-ATPase. We also examined the effects of both agents on these enzymes in vitro. Amiloride administration resulted in a decrease in Na-K-ATPase and H(+)-ATPase activities in cortical collecting tubule and medullary collecting tubule. Therapeutic concentrations of amiloride in vitro inhibited Na-K-ATPase activity, but only in cortical collecting tubule. The effects of Li administration were different; it decreased Na-K-ATPase and H(+)-ATPase in both cortical collecting tubule and medullary collecting tubule. In cortical collecting tubule, the inhibitory effect on H(+)-ATPase activity was seen in vitro at a Li concentration similar to that found in urine. In contrast to the effect of Li on the H(+)-ATPase, in vitro Li stimulated Na-K-ATPase activity. These results suggest that the mechanism of action whereby these two agents result in distal renal tubular acidosis in humans and animals are different. In the collecting tubule, amiloride appears to act solely through a voltage-dependent mechanism by inhibiting cortical collecting tubule Na-K-ATPase. Li, by contrast, appears to have an additional effect in the cortical collecting tubule to inhibit the H(+)-ATPase. The biochemical differences seen with these drugs may explain the more severe acidemia universally found in animals after chronic Li administration.


Opioid and nicotinic analgesic and hyperalgesic loci in the rat brain stem. The effects of (-)-nicotine and ethylketazocine (EKC) on the latency of a low-intensity thermally evoked tail avoidance response were evaluated at different midline mesencephalic, pontomedullary and medullary sites of conscious intact rats. Guide cannulae were implanted surgically at six anterior-posterior stereotaxic locations (AP, -4.4 to +2.8) and drugs were microinjected (0.5 microliter) at different depths in each region. The analgesic effects of naltrexone and mecamylamine were evaluated at those sites exhibiting sensitivity to the hyperalgesic actions of (-)-nicotine and EKC. Additional experiments evaluated the validity and reproducibility of the low-intensity thermally evoked tail avoidance response and a low intensity hot plate response in detecting hyperalgesia. The chemostimulation studies suggest that there are opioid and nicotinic hyperalgesic processes distributed throughout the dorsal regions of the posterior mesencephalic and pontomedullary brain stem. The relative hyperalgesic potency of (-)-nicotine appears to exhibit a gradient from the dorsal posterior mesencephalic tegmentum to the midmedullary region, whereas only analgesia was produced more rostrally, in the central gray, or caudally within the posterior medulla. Within regions intermediate between the dorsal posterior, mesencephalic tegmentum and posterior medulla, (-)-nicotine produced biphasic dose-response and time action curves. The effects of EKC were similar to those of (-)-nicotine at most sites although (-)-nicotine was 55 times more potent than EKC when administered in the most active hyperalgesic regions.2+ may differ from region to region.


Blocking action of terodiline on calcium channels in single smooth muscle cells of the guinea pig urinary bladder. The blocking action of terodiline, a nonspecific organic Ca++ antagonist, on smooth muscle Ca++ channels of the guinea pig urinary bladder was investigated. Inward Ca++ currents were recorded from smooth muscle cells isolated from the urinary bladder using the whole-cell patch-clamp technique. In the absence of terodiline, a use-dependent reduction in the amplitude of inward Ca++ current was observed at a stimulus frequency of 0.2 Hz. When terodiline (1-10 microM) was applied, the use-dependent reduction was accelerated markedly, depending on the stimulus frequency. The blocking action of terodiline was also dose-dependent; the Kd value as measured at the end of 20 times repetitive stimulation at 0.2 Hz was 1.7 microM. In addition to such a use-dependent block, terodiline produced a hyperpolarizing shift in the steady-state inactivation curve. The results suggest that terodiline preferentially binds to the Ca++ channel in the open state and also in the inactivated state.


AHR-9294: a novel inhibitor of H,K-ATPase antagonizes gastric HCl secretion in vivo. 8-Methoxy-4-[(2-isopropylphenyl)amino]-3-quinolinecarboxylate ethyl ester (AHR-9294) inhibited acid secretion stimulated by histamine, pentagastrin or carbachol in rats, and by histamine or feeding in dogs. AHR-9294 was about half as potent as omeprazole and exhibited a shorter duration of action. Based on its inhibition of acid secretion induced by different secretagogues and its lack of effect on histamine-stimulated adenylate cyclase activity, AHR-9294 does not appear to operate at the histamine receptor or adenylate cyclase. Rather, studies on enriched oxyntic microsomal preparations showed AHR-9294 to be an effective inhibitor of the H+ pump enzyme, H,K-ATPase, suggesting this might be the site of antisecretory activity. Kinetic studies revealed that inhibition of both K(+)-activated ATPase and p-nitrophenylphosphatase by AHR-9294 was purely competitive with K+ and its congeners, indicating that AHR-9294 and its analogs belong to the class of compounds known as "K+)-site" inhibitors. On the other hand, inhibition by AHR-9294 was noncompetitive with both ATP and p-nitrophenylphosphatase on their respective rates of hydrolysis (i.e., both Vmax and the apparent Km were reduced, but Vmax/Km was unchanged). Studies on partial reactions of the H,K-ATPase showed that the rate of ATP/ADP exchange was unaffected by AHR-9294 and the steady-state level of phosphoenzyme was only partially reduced (thus ATP/enzyme interaction was not affected); however, the rate of K(+)-catalyzed dephosphorylation of phosphoenzyme was markedly decreased.(ABSTRACT TRUNCATED AT 250 WORDS)


Artificial nerve graft using glycolide trimethylene carbonate as a nerve conduit filled with collagen compared to sutured autograft in a rat model. A study was conducted to compare the regeneration of rat peroneal nerves across 0.5 cm gaps repaired with artificial nerve grafts (ANG) versus sutured autografts (SAG). The ANG model is composed of a synthetic biodegradable passive conduit made of glycolide trimethylene carbonate (GTMC) filled with a collagen matrix (predominantly Type I collagen, derived from calf skin, and with the telopeptide ends left intact). Axonal regeneration was studied in 11 long-term animals (two at 6 months and nine at 9 months). The nerves were studied by qualitative and quantitative histological, electrophysiological, and functional assays. Axonal regeneration with the ANG was equal to SAGs as measured by axonal diameters, physiological, and functional methods, although the SAG demonstrated statistically higher axonal counts.


Phenylmorphans and analogues: opioid receptor subtype selectivity and effect of conformation on activity. The morphine-like (+)-phenylmorphan, the atypical (-)-enantiomer, and some analogues have been tested in receptor binding assays selective for opioid mu 1, mu 2, delta, kappa 1, and kappa 3 receptors. The affinities of all of the compounds except one, including the atypical (-)-phenylmorphan, were greatest for mu 1 and mu 2 receptors. The only exception was the (+)-9 alpha-methyl analogue which had slightly greater affinity for the kappa 1 receptor. The selective receptor binding assays provide evidence that opioids in which the phenyl ring is constrained to be equatorial on the piperidine ring can have considerable affinity for mu receptors. In addition, dose-response curves were determined for (+)- and (-)-phenylmorphan using the mouse tail-flick assay with the (+)-enantiomer found to be about 7 times more potent. Pretreatment with the selective opioid antagonists beta-FNA (mu 1 and mu 2), naloxonazine (mu 1), nor-BNI (kappa 1), and naltrindole (delta) suggests that the antinociceptive activity of both enantiomers is mediated through mu receptors. The pretreatment with naloxonazine, which attenuated the antinociceptive effect, shows that both (+)- and (-)-phenylmorphan are mu 1 agonists while intrathecal administration shows that both are mu 2 agonists. Conformational energy calculations on the compounds were also performed using the MM2-87 program. Consistent with previous conformational results for the phenylmorphans (J. Med. Chem. 1984, 27, 1234-1237), the most potent antinociceptive compounds preferred a particular orientation of the phenyl ring.


(Aminoalkoxy)chromones. Selective sigma receptor ligands. A series of (aminoalkoxy)chromones has been prepared, members of which bind potently (16-100 nM) at the sigma binding site and bind weakly (greater than 1000 nM) at the dopamine D2 receptor and 33 other receptors, second messenger systems, and ion channels. At the sigma receptor, the preferred position of attachment for the aminoalkoxy side chain to the chromone ring followed the rank order: 7-position greater than 5-position greater than 6-position. Chromones that contained a 2-substituent that was not coplanar with the chromone ring system showed improved binding over compounds with coplanar substituents. The most potent compound at the sigma site, 7-[[7-(4-hydroxypiperidyl)heptyl]oxy]-2-phenylchromone (74), had receptor affinities (IC50) of 16 nM at the [3H]DTG site, 19 nM at the [3H]-(+)-3-PPP site, and 4000 nM (Ki) at the dopamine D2 receptor. The most selective compound examined, 6-[[6-(4-hydroxypiperidyl)hexyl]-oxy]-2-cyclopentylchromone (58), exhibited IC50s of 51 nM at the [3H]DTG site, 55 nM at the [3H]-(+)-3-PPP site, and 21,000 nM (Ki) at the dopamine D2 receptor. Compound 44 (6-[[6-(4-hydroxypiperidyl)hexyl]oxy]-3-methylflavone, NPC 16377) was systemically effective (ip and po) in two behavioral models predictive of antipsychotic compounds and systemically active in animal models of ischemia.


Substitution on the Phe3 aromatic ring in cyclic delta opioid receptor-selective dermorphin/deltorphin tetrapeptide analogues: electronic and lipophilic requirements for receptor affinity. In an effort to explore structural features affecting receptor recognition in a series of conformationally restricted tetrapeptides related to the cyclic, delta opioid receptor-selective analogue, [formula: see text] electronic, lipophilic, and steric effects at the Phe3 residue were assessed by substitution at different positions of the side-chain aromatic ring by halogens, alkyl, hydroxyl, and nitro groups. Effects on opioid receptor binding affinity and selectivity were determined. The results, which are generally consistent with reports of analogous modifications in linear and cyclic pentapeptide enkephalins, indicate that steric, lipophilic, and electronic properties are all important determinants of delta opioid receptor recognition. Specifically, modifications which increase lipophilicity or exert electron-withdrawing effects on the aromatic ring enhance binding affinity, while hydrophilic, bulky, or electron-releasing modifications are detrimental. These observations are in excellent agreement with quantitative structure-activity relationship (QSAR) results reported for Phe4 modifications in linear opioid pentapeptide enkephalin analogues, suggesting that the Phe3 tetrapeptide side chain and the Phe4 pentapeptide side chain interact with the same delta receptor binding subsite.


Synthesis and biological activity of 8a-phenyldecahydroquinolines as probes of PCP's binding conformation. A new PCP-like compound with increased in vivo potency. The synthesis and chemical resolution of cis- and trans-fused 8a-phenyldecahydroquinolines 3 and 4 are described together with the affinity of the four optically pure compounds for the PCP recognition site of the NMDA receptor complex. These compounds were also evaluated for their antagonistic effects on cGMP levels in male Swiss Webster mice, and (-)-4 was found to exhibit in vivo potency comparable to that of MK-801. The results of the binding studies are interpreted in terms of a preferred orientation of PCP's N-H bond in binding to its NMDA receptor-associated recognition site.


Molecular recognition of amiloride analogs: a molecular electrostatic potential analysis. 1. Pyrazine ring modifications. Ab initio molecular electrostatic potential (MEP) patterns are used to determine the electrostatic requirements for the formation of a stable blocking complex between amiloride analogs and the epithelial sodium channel of Rana ridibunda. MEP maps calculated in the 3-21G(*) and STO-3G basis sets for amiloride and analogs with pyrazine ring modifications are used to interpret differences in the microscopic rate constants for analog-channel binding determined by Li et al. MEP maps of the protonated analogs are correlated to differences in the value of kon, the microscopic association constant. Those analogs with kon values similar to amiloride are found to have a MEP maximum that is localized over the side chain, as well as strong, distinguishing minima in the MEP pattern off the carbonyl oxygen and positions 3, 4, and 5 of the pyrazine ring. MEP maps of a model-encounter complex (protonated analog and formic acid anion) are correlated to differences in koff, the microscopic dissociation constant. The major conclusions of this work are that (1) a stable blocking complex is formed with analogs which have a deep, localized minimum off the 6 position of the pyrazine ring, (2) the stability of the blocking complex is directly related to the depth of that minimum, (3) substitution at position 5 affects not only the depth but also the location and size of the minimum off position 6, and (4) steric factors may influence the optimal binding of the 6-position ligand to the ion channel. The MEP analysis also suggests that the distance between the proton donors of the chelating guanidinium moiety and the deep, localized minimum off position 6 of the pyrazine ring may define an important spatial requirement for all those analogs which form a stable blocking complex with the channel.


Expression of platelet-derived growth factor (PDGF) and PDGF receptor genes in the developing rat brain. Steady-state levels of rat central nervous system (CNS) platelet-derived growth factor (PDGF) A- and B-chain mRNAs were measured by a polymerase chain reaction method employing a synthetic gene internal standard, and the rates of transcription of PDGF A- and B-chain genes in CNS were estimated by a nuclear runoff assay. The abundance of PDGF B-chain mRNA was an order of magnitude below that of PDGF A-chain mRNA, while the rate of PDGF B-chain transcription was only slightly below that for the PDGF A-chain gene, indicating that the half-life of PDGF B-chain in CNS is shorter than that of PDGF A-chain mRNA. No developmental alterations in expression of the PDGF A- and B-chain genes were detected. By contrast, Northern blots showed that steady-state levels of mRNAs encoding the two PDGF receptor proteins, alpha and beta, were markedly higher in embryonic day 15 and postnatal day 6 rat brains than in later life. These results suggest that the actions of PDGF on the brain in vivo are regulated not at the level of PDGF A and B-chain gene expression, but rather by changes in the level of expression of PDGF alpha- and beta-receptor genes.


Calcium-activated neutral proteinase (calpain) activity in C6 cell line: compartmentation of mu and m calpain. Calcium-activated neutral proteinase (calpain) activity was determined, including in cytosol and membrane fractions, in rat glioma C6 cell line. The mu and m forms of calpain were separated by DEAE and phenylsepharose column chromatography and with removal of the endogenous inhibitor calpastatin. C6 cells contained more mcalpain than the mu isoform. More than 70% of mcalpain activity was membrane-associated and 20% was cytosolic. Isolated plasma membrane also contained 69% of the mcalpain activity. In contrast, approximately 80% of mucalpain activity was cytosolic and 16% was membranous. Half-maximal activity for mu and mcalpain was obtained at 1 microM and 0.2 mM CaCl2, respectively. Trypsin dissociation of cells reduced activity. Triton X-100 stimulated mcalpain activity of the whole homogenate and the membrane pellet but not of the cytosol. Activity of the myelin marker enzyme adenosine 2'3'-cyclic nucleotide 3'-phosphohydrolase (CNPase), was also found in C6 cells. The identification of calpain and CNPase in C6 cells is in keeping with an interpretation that C6 differentiation resembles, at least in part, that of the myelin-forming oligodendroglial cells.


The significance of immunosuppression and hormonal alteration in neurotraumatology. Thirty head trauma patients receiving no steroid treatment were investigated for a period of 20 days. The patients were divided into three groups according to their Glasgow Coma Scale (GCS) scores: GCS = 4-8 and GCS = 9-13. We studied their immunological and hormonal status with special reference to the relationship among severity of trauma, immunosuppression and hormonal alteration. The immunity tests included lymphocyte transformation, lymphocyte phenotyping and delayed hypersensitivity (DH) skin tests. The hormone study consisted of serum cortisol and ACTH level determinations. The following results were obtained: (1) The entire group suffered from significant suppression in lymphocyte responses to mitogens PHA and Con A on day 1 and day 10 after injury, but later they improved; (2) There was a significant fall (P less than 0.05) in OKT3 OKT4 and active T-cell on day 10, especially in patients with GCS less than or equal to 8; (3) A significant increase in OKT8 and decrease in OKT4 was concurrently noted causing an inversion in the ratio of OKT4 to OKT8 on the 10th day (patients 0.96:1, normal 1.6:1, P less than 0.01); (4) No significant change was observed in the number of B-cells, suggesting that cell-mediated immunity may be affected earlier and more severely than antibody producing capacity; (5) Delayed hypersensitivity skin test showed a high incidence of anergy in the severe (GCS less than or equal to 8) and fetal cases. A high mortality in anergic cases was also noted; (6) Elevated ACTH and cortisol levels detected upon arrival rapidly declined to normal value at day 5. In addition, we found no correlation between the curve of serum ACTH and cortisol alteration and the biphasic suppression curve of lymphocyte transformation in later study period. These findings suggest that the severity of natural resistance impairment seems to be related to the gravity of clinical manifestations and to the serum hormonal alteration only in the initial stages. Also, we find that the development of anergy may be an index of poor prognosis.


Protective effects of a PGI2 analogue OP-2507 on hemorrhagic shock in rats--with an evaluation of the metabolic recovery using near-infrared optical monitoring. The protective effects of the PGI2 analogue, OP-2507 against hypoxic tissue injury were investigated in a 60-70 min acute hemorrhagic shock model in 29 rats. To assess the metabolic recovery of mitochondria after tissue injury induced by hemorrhagic hypotension with a mean arterial pressure of 30 mmHg, we have non-invasively monitored changes in the brain tissue parameters of cyt. aa3 redox state, blood oxygenation and relative blood volume by spectrophotometry through the closed skull and intact skin. Pretreatment with 0.1 mg/Kg s.c. of OP-2507 at 30-40 min before induction of shock was performed on 14 rats (OP-treated group). The remaining 15 rats were used as a control (control group). There was a consistent prolongation of survival time and a significant improvement in survival rate after reinfusion of the shed blood in the OP-treated group. In this group there was a rapid and complete reoxidation of cyt. aa3 with a mean overshoot of 9 +/- 5.5% above the baseline value after reinfusion. On the other hand, in the control group the extent of reoxidation was significantly lower, with a minimal 11 +/- 3.2% below the base line. In order to evaluate the mechanisms involved 10 mg of NaCN i.v. was administered to the living rats at 60-70 min after reinfusion of the blood in both groups. In the OP-treated group, brain Hb saturation increased up to 20% above the pre-cyanide infusion level. However in the control group there was a non-significant increase in the Hb oxygenation level. These observation indicate that in the OP-treated group oxygen consumption by mitochondria is significantly higher than that in the control group. Thus, enhanced oxygen utilization could lead to the active restoration of injured tissue by promoting oxidative phosphorylation. Under these experimental conditions the oxidative response of cyt. aa3 is concluded to correlate closely with the prognosis of shock animals in both groups. These results indicate the potential usefulness of OP-2507 in protecting the brain and other organs from oxygen insufficiency as a result of tissue ischemia and anoxia.


The beta-adrenergic receptor/adenylate cyclase system in the cardiac ventricles of a hypertrophic cardiomyopathy rat model. The WKY/NCrj rat strain is considered to be a good animal model for hypertrophic cardiomyopathy (HCM). The purpose of this study was to examine the beta-adrenergic receptor/adenylate cyclase system in the cardiac ventricles of these rats. beta-adrenergic receptor density (Bmax) in the right ventricle (RV) was higher in WKY/NCrj than in Wistar rats. In contrast, Bmax in the interventricular septum (IVS) was lower in WKY/NCrj than in Wistar rats. Isoproterenol-stimulated adenylate cyclase activity in cardiac ventricular slices showed that changes corresponded to the changes of Bmax in every type of studied cardiac ventricle. The intracellular adenylate cyclase pathway (GTP gamma S-, NaF- and forskolin-stimulated adenylate cyclase activity in cardiac ventricular particulate fraction) did not differ between WKY/NCrj and Wistar rats in any region of the cardiac ventricles. Catecholamine levels tended to be low in the RV and to increase in the IVS of WKY/NCrj rats. Our results suggest that the activity of the beta-adrenergic receptor/adenylate cyclase system varies in different cardiac ventricles of the WKY/NCrj rat model for HCM.


[Effects of prostaglandin E1 and dibutyryl cyclic AMP on hemodynamics after cardiopulmonary bypass in valve replacement surgery]. In view of vasodilating action of prostaglandin E1 (PGE1) and dibutyryl cyclic AMP (DBcAMP) we investigated the effect of each agent on hemodynamics after weaning from cardiopulmonary bypass (CPB) comparing with the effect in control group. PGE1 and DBcAMP were administered to patients who underwent valve replacement surgery with continuous low dose infusion at an average rate of 0.026 micrograms.kg-1.min-1 and 7.25 micrograms.kg-1.min-1 respectively. Following result was obtained. In PGE1 administered group, a significant reduction in pulmonary vascular resistance (PVR) and a significant decrease in mean arterial pressure (MAP) were observed during CBP, while there were no significant differences in other parameters, such as platelet counts, differences between core and peripheral temperature (delta T), urine output, systemic vascular resistance (SVR), cardiac index (CI), right-to-left shunt (Qs/Qt), oxygen delivery (DO2) and oxygen consumption (VO2). However, CI and platelet counts tended to increase but delta T and SVR tended to decrease. In DBcAMP administered group, there were no significant differences in all parameters compared with those of control group, showing a tendency of less improvement in hemodynamics than in PGE1 group. We have shown that the use of PGE1 rather than DBcAMP as vasodilator agent seems advantageous during open-heart surgery in patients especially with severe pulmonary hypertension, but it tends to cause severe hypotension during CPB.


[ESR study of free radical formation during ischemia-reperfusion injury in the rat brain and the protective effect of a new antioxidant]. By using the spin-trapping technique and electron spin resonance spectroscopy (ESR), we detected directly oxygen-derived free radicals in the brain exposed to ischemia and reperfusion. Forebrain ischemia was produced in the rat by bilateral occlusion of the common carotid arteries combined with hemorrhagic hypotension. The whole cerebral cortex was homogenized in the presence of the spin trap agent, N-tert-butyl-alpha-phenylnitrone, followed by a Folch extract. Spin-adducts were detected using ESR. As the index of tissue injury, the lipid peroxidation was estimated from both the amount of thiobarbituric acid reactive substance and the formation of conjugated diene. After 10 or 20 min of ischemia, reperfusion induced a burst of spin adduct formation which peaked at 5 min reperfusion time. The peak value increased with the ischemia time. The degree of lipid peroxidation, which was measured after 20 min of reperfusion, also increased with the ischemia time. When the oligomeric derivative was administered (9 mg . kg-1, i.p.) 30 min before ischemic insult, both spin adduct formation and lipid peroxidation were reduced. The results support the current view that free radicals produced upon reperfusion may be the direct cause of the subsequent lipid peroxidation.


[Angiotensin-converting-enzyme (ACE) in Crohn's disease]. Serum concentration of angiotensin-converting enzyme (ACE) were measured in 34 patients with Crohn's disease. ACE was decreased in Crohn's disease compared with healthy controls. Significant negative regression between ACE and CDAI was observed. It suggested intestinal ACE may play a regulatory role in intestinal inflammation of Crohn's disease. Among the patients, ACE did not show the significant change by use of prednisolone nor by location of disease. The mechanism of ACE decrease in Crohn's disease was not known, but this phenomena may be one of the reason of persistent inflammation of Crohn's disease.


[Juvenile optic neuropathy caused by Km variants of biotinidase]. A patient with a newly recognised variant of biotinidase deficiency presented with acute bilateral visual loss at the age of 10 years. A progressive optic neuropathy, a predominantly motor type neuropathy and spastic paraparesis developed over the following 5 years. Metabolic investigations revealed biotin depletion causing multiple biotin dependent carboxylase deficiency. The basic defect was a biotin recycling disorder due to a biotinidase Km variant with residual colorimetric activity of 4.4% of normal. Further investigations on plasma biotinidase showed biphasic kinetics with two different reduced Vmax values and two Km-values, one being almost normal and the other highly elevated. After a period of 2 months of oral substitution with biotin 10 mg per day the visual field defects improved as well as the distal spastic parapareses and motor neuropathy. We conclude that the differential diagnosis of unexplained bilateral optic neuropathy of juvenile onset, particularly when associated with upper and lower motor neuron disease should include biotinidase deficiency.


[Familial adenomatous polyposis from the ophthalmologic viewpoint]. Familial adenomatous polyposis is dominant autosomal heritable and pre-cancerous. Characteristic changes of ocular fundus, multiple and bilateral retinal dysplasias, permit diagnosis in presymptomatic stage, significant especially not at least for sporadic cases of polyposis recti and coli, which are 40 per-cent of all. Contrary to often wellknown risk-families this group can't be realized epidemiologically, but complications are the same.


Agonist and antagonist activities of ligands derived from naltrexone and oxymorphone. The pharmacological profile of naltrindole (NTI) and three of its analogues, N-methyl-NTI (N-Me-NTI), oxymorphindole (OMI) and naltriben (NTB) were studied in antinociceptive assays. The compounds were found to have agonist activities that appear to be mediated mainly by kappa opioid receptors because norbinaltorphimine (nor-BNI), the selective kappa opioid receptor antagonist inhibited their effects significantly. All of the compounds, behaved as antagonists at doses that were lower than those that produced agonist effects and they possessed a profile that was very selective for inhibiting the antinociceptive activities of delta opioid receptor agonists. Differential antagonism by NTB of the activities of DSLET and DPDPE was demonstrated.


Antinociceptive interactions of opioid delta receptor agonists with morphine in mice: supra- and sub-additivity. In this study, the antinociceptive interactions of fixed ratio combinations of intracerebroventricularly (i.c.v.) given morphine and subantinociceptive doses of the delta agonists, [D-Pen2, D-Pen5]enkephalin (DPDPE), [D-Ala2, Glu4]deltorphin (DELT) or [Met5]enkephalin (MET) were examined using the mouse warm water tail flick test. When morphine was coadministered with DPDPE or DELT in a 4:1 and 9:1 mixture, respectively, a synergistic antinociceptive effect was observed. In contrast, when morphine was coadministered with MET in a 1:2 fixed ratio mixture, a subadditive interaction occurred. These results demonstrate both positive and negative modulatory interactions of delta agonists with morphine in an antinociceptive endpoint and that these interactions can be either supra- or subadditive. The data support the concept of a functional interaction between opioid mu and delta receptors and a potential regulatory role for the endogenous ligands of the opioid delta receptor.


Spectroscopic and biological studies of spin-labeled tetracycline. A new nitroxyl labeled tetracycline is synthesized. Proton NMR experiments of tetracycline, spin-labeled tetracycline, and the diamagnetic reduced form in DMSO-d6 are reported. The signals observed in the NMR spectra are all assigned. The NMR data revealed that the spin label is attached to the C-2 amide group on ring A of tetracycline. The spin-labeled tetracycline is also tested in vitro for antitumor activity and is found to be active against leukemia P338/ADR cell line and in melanoma LOX cell line.


5-hydroxytryptamine4 receptor agonists facilitate cholinergic transmission in the circular muscle of guinea pig ileum: antagonism by tropisetron and DAU 6285. The effect of 5-hydroxytryptamine (5-HT), BIMU 8 (endo-N-(8-methyl-8-azabicyclo [3.2.1.] oct-3-yl)-2,3-dihydro-3-(1-methyl)ethyl-2-oxo-1H-benzimidazole-1- carboxamide hydrochloride) and cisapride was studied on the electrically-induced neurogenic cholinergic twitch contractions in the guinea pig ileum circular muscle. These compounds caused a concentration-dependent increase in the amplitude of submaximal twitch contractions with the following rank order of potency: 5-HT greater than BIMU 8 = cisapride. The effect of 5-HT was competitively antagonized by tropisetron (ICS 205-930) (apparent pA2 value: 6.4), suggesting an interaction at 5-hydroxytryptamine4 (5-HT4) receptors. The novel benzimidazolone derivative DAU 6285 (endo-6-methoxy-8-methyl-8-azabicyclo [3.2.1.] oct-3-yl-2,3-dihydro-2-oxo-1H-benzimidazole-1-carboxylate hydrochloride), antagonized the effect of 5-HT, BIMU 8 and cisapride with apparent pA2 values in the range 7.1-7.3. Our findings demonstrate that cholinergic neurones innervating the circular coat are endowed with excitatory 5-HT4 receptors. DAU 6285 is approximately 5-9-fold more potent than tropisetron as antagonist at these receptors.


Antipyretic effect of central arginine8-vasopressin treatment: V1 receptors specifically involved? Intracerebroventricular (i.c.v.) administration of the neurohypophyseal neuropeptide arginine8-vasopressin (AVP) results in a dose-dependent attenuation of endotoxin-induced fever (EIF) in rats. Specific antagonists of the neuropeptided(CH2)5[Tyr(Me)2]AVP for V1 receptors, d(CH2)5[dlle2lle4]AVP for the V2 receptors and Des-Gly,NH2d(CH2)5[Tyr)Me2)Thr4Orn8]vasotocin, an antagonist of the oxytocin receptors (AOXT), failed to modify EIF when administered i.c.v. Relatively high doses (100 ng) of all three peptide antagonists effectively blocked the antipyretic effect of AVP. Administered in smaller doses (10 or 30 ng), however, a more specific interaction was observed, i.e. the V1 antagonist being the only effective compound in preventing the effect of AVP. Although the data indicate that peptide-antagonist interactions should be interpreted carefully, the present experiments confirm previous observations on the involvement of V1-type receptors in the antipyretic action of AVP and suggest additional interactions with V2 vasopressinergic and oxytocinergic receptors.


Levels of thiobarbituric acid reactive substances and the cytocidal potential of gammalinolenic and docosahexaenoic acids on ZR-75-1 and CV-1 cells. To clarify the mechanism by which gammalinolenic acid (GLA) is more tumoricidal than docosahexaenoic acid (DHA), we have compared the incorporation of the respective exogenously added ethyl esters GLAe and DHAe into the phospholipids of tumorigenic ZR-75-1 and non-tumorigenic CV-1 cells relative to the ability of the cells to survive and to accumulate thiobarbituric acid reactive substances (TBARS). GLA and DHA were incorporated in the phospholipids to the same extent, but GLA disappeared more rapidly than DHA in both cell lines. GLAe induced about twice as much intracellular TBARS as DHAe in both cell lines, but killed ZR-75-1 cells four times more effectively than DHAe. DHAe induced 11-15 fmoles malondialdehyde-equivalents (MDA-eq)/cell in both ZR-75-1 and CV-1 cells, whereas GLAe induced 5-6 times more TBARS in ZR-75-1 cells (26-30 fmoles MDA-eq/cell) than in CV-1 cells (5-6 fmoles MDA-eq/cell). The results show that there is no difference in GLA and DHA incorporation into phospholipids, but that their metabolism differs in the two cell types. The data also suggest that the cytocidal potential is related to TBARS levels in a nonlinear fashion. The relationship between excess prostaglandin production and excessive cell death due to GLA is discussed.


Phospholipid composition of cultured human endothelial cells. Detailed analyses of the phospholipid compositions of cultured human endothelial cells are reported here. No significant differences were found between the phospholipid compositions of cells from human artery, saphenous and umbilical vein. However, due to the small sample sizes, relatively large standard deviations for some of the phospholipid classes were observed. A representative composition of endothelial cells is: phosphatidylcholine 36.6%, choline plasmalogen 3.7%, phosphatidylethanolamine 10.2%, ethanolamine plasmalogen 7.6%, sphingomyelin 10.8%, phosphatidylserine 7.1%, lysophosphatidylcholine 7.5%, phosphatidylinositol 3.1%, lysophosphatidylethanolamine 3.6%, phosphatidylinositol 4,5-bisphosphate 1.8%, phosphatidic acid 1.9%, phosphatidylinositol 4-phosphate 1.5%, and cardiolipin 1.9%. The cells possess high choline plasmalogen and lysophosphatidylethanolamine contents. The other phospholipids are within the normal biological ranges expected. Phospholipids were separated by high-performance liquid chromatography and quantified by lipid phosphorus assay.


Expression of IGF-1 mRNA in the murine subventricular zone during postnatal development. Insulin-like growth factor-1 (IGF-1) stimulates the proliferation and maturation of neuroglia in vitro. To further investigate its role in gliogenesis, in situ hybridization was utilized to determine whether IGF-1 mRNA was expressed in the subventricular zone (SVZ) of the postnatal mouse forebrain. The SVZ is a transient germinal zone and in the neonate is the principle source of oligodendroglia for myelinating fiber tracts of the forebrain. Strong hybridization signal was detected over cells in the SVZ at postnatal day (PND) 4, the earliest time point examined. Positive signal persisted in the SVZ at PND 8, however, the number of IGF-1-labeled cells declined rapidly during the second postnatal week. IGF-1 mRNA was not uniformly distributed throughout the SVZ and the majority of labeled cells were located within its so-called 'border' region. In contrast to the SVZ, IGF-1 mRNA-expressing cells were only rarely found in forebrain fiber tracts. IGF-1 transcripts were not detected in ependymal lining or choroid plexus of the lateral ventricle. In light of its known gliotrophic activity, the localization of IGF-1 mRNA in the SVZ suggests that locally produced IGF-1 may act as a mitogen or differentiation-inducing agent during gliogenesis.


Region-specific expression of GABAA receptor alpha 3 and alpha 4 subunits mRNAs in the rat brain. The expression of mRNAs encoding the alpha 3 and alpha 4 subunits of the gamma-aminobutyric acid A (GABAA) receptor in the rat brain was investigated by in situ hybridization histochemistry. Both subunits showed a wide but uneven distribution, which did not coincide with the distribution of any other subunit so far reported. The cerebral cortex, anterior olfactory nucleus, lateral septum, subiculum, lateral and medial nuclei of the amygdaloid complex, anterior nuclei of the thalamus, pars compacta of the substantia nigra, trigeminal sensory nuclei, and cochlear nucleus were some of the areas where strong expression of mRNA for both the alpha 3 and alpha 4 subunits was detected. In the mitral cell layer of the olfactory bulb, the preoptic area and locus coeruleus, strong expression of only the alpha 3 subunit was detected. In the granular cell layer of the olfactory bulb, caudate-putamen, tenia tecta, pyramidal cell layer of the CA region and granular cell layer of the dentate gyrus in the hippocampal formation, dorsomedial and ventrolateral nuclei of the thalamus, dorsal part of the lateral geniculate body, preolivary nuclei and pontine nuclei, only the alpha 4 subunit showed strong expression. The diverse distribution of these two subunits is considered to indicate that each has a different role in the central nervous system.


Co-expression of glycine receptor beta subunit and GABAA receptor gamma subunit mRNA in the rat dorsal root ganglion cells. We examined the expression of the beta subunit mRNA of the glycine receptor and the gamma subunit mRNA of the GABAA receptor in the rat dorsal root ganglion (DRG) using in situ hybridization histochemistry with oligonucleotide probes. About 44% and 37% of the all DRG neurons were labeled by the probes for glycine receptor beta subunit and GABAA receptor gamma subunit mRNAs. Labeled neurons were mostly large cells that simultaneously expressed both glycine receptor beta subunit and GABAA receptor gamma subunit mRNA as demonstrated using consecutive sections. Thus, we suggest the possibility that both GABA and glycine presynaptically regulate the activity of neurons involved in low-threshold mechanoreception at axo-axonic synapses in the spinal cord.


Survival and migration of transplanted male glia in adult female mouse brains monitored by a Y-chromosome-specific probe. A Y-chromosome-specific probe and in situ hybridization technology have been used to monitor the survival and migration of neonatal male glia isografted to the left cerebral hemisphere of adult female mice. More than 95% of the cultured donor glia were glial fibrillary acidic protein (GFAP)-positive astrocytes. By 4 weeks, large numbers of transplanted glia were found in both cerebral hemispheres; the extent of glial migration was greatest in white matter tracts. This method provides a new way of identifying all surviving donor cells within the brains of immunologically compatible hosts.


Reserpine increases striatal neurotensin mRNA levels. Reserpine administration has been shown to increase striatal tissue levels of neurotensin with a time course similar to that for striatal dopamine depletion. To determine whether reserpine treatment may increase striatal neurotensin synthesis we have examined striatal neurotensin mRNA levels using in situ hybridization histochemistry. The number of striatal cells expressing neurotensin mRNA was increased 6 h, but not 1 h, after reserpine administration. Thus, the increase in striatal tissue levels of neurotensin after reserpine may be due in part to an increase in peptide synthesis.


Distribution and cellular localization of preproenkephalin mRNA in the ovine brain and pituitary. In this study in situ hybridization histochemistry was used to determine the regional and cellular localization of preproenkephalin (PPE) mRNA in the sheep brain and pituitary. Coronal brain sections were hybridized with an 35S-labelled synthetic 45-mer deoxyribonucleotide probe complementary to a portion of the bovine PPE gene. The specificity of the probe was confirmed by Northern blot analysis. The highest density of labelled cell bodies was found in the nucleus accumbens, caudate-putamen, olfactory tubercle, the central nucleus of the amygdala, the paraventricular nucleus of the hypothalamus, the suprachiasmatic nucleus and in the gigantocellular division of the medullary reticular formation. Labelled cells were also found in the olfactory bulb, prefrontal cortex, piriform cortex and cerebral cortex and in the vicinity of the locus coeruleus, parabrachial nucleus and the nucleus of the solitary tract. In the pituitary a dense PPE mRNA signal was observed in the intermediate lobe; cells in the anterior or neural lobe did not express PPE mRNA. The widespread distribution of cells containing PPE mRNA transcripts within the ovine brain agrees with a similar distribution in the rat. The data suggest that PPE neurons may be involved in diverse physiological functions including the processing of sensory and nociceptive information and in the regulation of endocrine and motor responses.


Negative regulatory regions are present upstream in the three mouse neurofilament genes. We have cloned and examined the 5' flanking regions of the heavy (NF-H), light (NF-L) and mid-sized (NF-M) mouse neurofilament (NF) genes in order to begin to characterize the regions of each gene that regulate NF transcription. Chimeric plasmids bearing the CAT reporter gene and deletion mutants of the upstream NF genes were transiently transfected into neuronal (PC12 and Neuro 2A) and non-neuronal (HeLa) cell lines. Constructs bearing upstream regions to -4000 in NF-H, to -5600 in NF-L and to -4500 in NF-M were expressed at low levels in neuronal and in non-neuronal cells. Progressive deletion of 5' flanking sequence to -385 in NF-H, to -325 in NF-L and to -505 in NF-M caused a several-fold increase of transcription from the transfected plasmids. Increases of transcription by deletion mutants followed a similar pattern in neuronal and in non-neuronal cell lines. Negative upstream regions are located between -1314 and -385 in NF-H, between -936 and -325 in NF-L and between -874 and -505 in NF-M. Additional negative regions are present further upstream in NF-L and in NF-H. The negative regions of NF-H and of NF-L suppress transcription when placed in either orientation in front of the SV40 or a heterologous NF promoter. These studies demonstrate that the three mouse NF genes possess similar functional features, namely, that of a relatively strong and promiscuous promoter with negative upstream elements. The role of the negative elements in regulating NF expression remains unclear.


A novel neuroendocrine gene product: selective VGF8a gene expression and immuno-localisation of the VGF protein in endocrine and neuronal populations. The VGF8a gene was recognised on the basis of its inducibility by NGF in rat pheochromocytoma (PC12) cells. Using immunocytochemistry, we have localised the corresponding VGF protein product in various neuronal groups, including primary sensory and enteric neurons, and in endocrine cells of the adrenal medulla, adenohypophysis and gut. VGF8a gene expression, as detected by RNAse protection analysis, largely correlated with such distribution.


Rat pituitary cells selectively express mRNA encoding the short isoform of the y2 GABAA receptor subunit. The GABAA receptor belongs to the ligand-gated ion channel receptor superfamily and appears to be composed of from 4 to 5 subunits which interact with each other. Molecular cloning of cDNAs encoding the different subunits of the GABAA receptor has revealed an unexpected heterogeneity which includes at least 4 homologous classes of subunits: these classes, designated as alpha, beta, gamma, and delta contain multiple variants. We have measured the steady-state levels of mRNAs encoding different (alpha 1, alpha 4, beta 1, beta 2, beta 3, gamma 2 long, gamma 2 short and delta) GABAA receptor subunits in the rat anterior pituitary and cerebellum using a polymerase chain reaction (PCR)-derived method. We found that pituitary cells express mRNAs encoding the alpha 1, beta 1 and beta 3 GABAA receptor subunits whereas the transcripts for alpha 4, beta 2 and delta were undetectable. We also found that pituitary cells selectively express the short isoform of the gamma 2 subunit mRNA. These data indicate that the expression of the various GABAA receptor subunits is cell-specific and support the concept that the diversity in function and pharmacology of the GABAA receptor is based on the ability of cells to specify the expression of selective GABAA receptor subunits.


Amphetamine and haloperidol modulate preprotachykinin A mRNA expression in rat nucleus accumbens and caudate-putamen. In situ hybridization was used to measure the effect of repeated amphetamine (1.5 mg/kg) and haloperidol (0.5 mg/kg) treatment for 7 days on the expression of preprotachykinin A (PPT-A) mRNA in rat nucleus accumbens (Acb) and caudate-putamen (CPu). Amphetamine elevated the level of PPT-A mRNA in Acb, but not in CPu. Haloperidol decreased the levels in Acb shell and CPu, but not in Acb core. Haloperidol injected together with amphetamine, prevented the amphetamine-induced increase in PPT-A mRNA expression in both Acb core and shell.


Novel G protein-coupled receptors: a gene family of putative human olfactory receptor sequences. We have taken advantage of the sequence conservation in the G protein-coupled receptor superfamily to isolate a fragment of a novel G protein-coupled receptor sequence using polymerase chain reaction (PCR) amplification of human genomic DNA. Screening of human genomic and hippocampal cDNA libraries with this amplified receptor fragment revealed a number of related sequences. Sequence analysis of four genomic clones and one cDNA clone clearly identifies these as related members of the G protein-coupled receptor family, as the deduced amino acid sequence reveals putative transmembrane domains and conserved amino acid residues. Southern blot analysis of restriction digests of human genomic DNA indicates that these receptor subtypes are likely to belong to a family of related genes. One of the proposed receptor sequences indicates the presence of pseudogenes in this family. Based on the homology of these sequences to a family of recently described receptors expressed exclusively in rat olfactory epithelium, it is suggested that these receptors represent a family of human odorant receptors.


Direct visualization of serotonin1D receptors in the human brain using a new iodinated radioligand. The development of the new ligand serotonin-5-O-carboxymethyl-glycyl [125I]tyrosinamide (abbreviated [125I]GTI) allows for the direct visualization of serotonin1B and serotonin1D (5-HT1B/1D) sites. Autoradiographic techniques were used to demonstrate the selective binding of this ligand to 5-HT1D sites in human post-mortem brain materials. The distribution of [125I]GTI binding sites was compared to [3H]5-HT sites in the presence of different displacers. The results show the selective binding of [125I]GTI to sites in the basal ganglia and substantia nigra which corresponds to 5-HT1D receptors.


Induction of constitutive heat shock protein 73 mRNA in the dentate gyrus by seizures. We examined the effects of generalized seizures on heat shock protein (hsp) mRNA induction in the rat brain using in situ hybridization. Seizures induced by electroconvulsive shock, electrical or cocaine kindling caused a selective induction of the constitutive hsp 73 gene in the dentate gyrus. In these seizure paradigms, not thought to induce widespread tissue damage, neither the heat-inducible hsp 72 gene nor a member of the hsp 90 family (hsp 84) were induced. Hsp 73 may play a role in the adaptation and/or in the maintenance of dentate granule cell integrity following seizures.


Limbic seizures induce a differential regulation of the expression of nerve growth factor, brain-derived neurotrophic factor and neurotrophin-3, in the rat hippocampus. Small unilateral electrolytic lesions placed in the hilus of the dentate gyrus produce limbic seizures. We have investigated the effects of these hilar lesions on the levels of the mRNAs encoding for 3 neurotrophic factors (NTF): nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT3). 'In situ' hybridization histochemistry with synthetic oligonucleotides was used to analyze their mRNA distribution and levels. In agreement with previously published data (Science, 245 (1989) 758-761), NGF mRNA was found bilaterally, quickly and transiently increased in granule cells of the dentate gyrus. Only 2 h after the onset of limbic seizures, mRNA levels for BDNF were also found to be dramatically elevated in both sides of the hippocampus, reaching a maximum 30-fold increase in the granule cell layer of the dentate gyrus 5 h after the lesion. Moreover, increased levels of this mRNA were also been found in the pyramidal layer of the CA3 (5-fold) and CA1 (15-fold) hippocampal fields. In contrast, NT3 mRNA was found to be clearly and bilaterally decreased in dentate gyrus granule cells, reaching 5- to 6-fold decreased levels at 12 h after lesion. Taken together, these results clearly show a different regulation of neurotrophic factors genes (NGF, BDNF and NT3) expression in the different hippocampal fields, as a consequence of seizure-producing hilar lesions.


D2 dopaminergic regulation of striatal preproenkephalin mRNA levels is mediated at least in part through cholinergic interneurons. The effect of administration of the muscarinic antagonist scopolamine on the increase in striatal preproenkephalin (PPE) mRNA following a 6-hydroxydopamine (6-OHDA) lesion or chronic D2 dopamine (DA) antagonist treatment was examined by dot-blot hybridization. Administration of scopolamine dose-dependently attenuated the 6-OHDA lesion-induced increase in striatal PPE mRNA. Administration of the D2 DA antagonist eticlopride to naive rats increased striatal PPE mRNA in a dose- and time-dependent fashion. Chronic coadministration of scopolamine attenuated the eticlopride-induced increase in striatal PPE mRNA. Chronic administration of scopolamine alone did not alter striatal PPE mRNA levels. In contrast, chronic administration of eticlopride, scopolamine or the two combined decreased striatal preprotachykinin (PPT) mRNA to the same extent, suggesting that there was no direct interaction between D2 dopaminergic and cholinergic mechanisms in the regulation of striatal PPT mRNA. These data indicate that DA differentially regulates striatal PPE and PPT mRNA and suggest that dopaminergic regulation of striatal PPE mRNA is mediated in part through D2 DA effects on striatal cholinergic neurons.


The role of target muscles in the expression of calcitonin gene-related peptide mRNA in the spinal nucleus of the bulbocavernosus. Previous studies have shown that calcitonin gene-related peptide (CGRP) mRNA steady-state levels and CGRP immunoreactivity in motoneurons of the spinal nucleus of the bulbocavernosus (SNB) are inversely related to the gonadal steroid environment in male rats. Since both the SNB motoneurons and their target muscles, the bulbocavernosus and the levator ani, are steroid sensitive, gonadal steroids may act at either site to regulate CGRP expression. In the present study, we tested the hypothesis that gonadal steroids influence CGRP expression in SNB motoneurons through their effects on the bulbocavernosus and levator ani muscles. We determined the levels of alpha-CGRP mRNA and immunoreactive CGRP in SNB motoneurons of adult male rats following injection of the bulbocavernosus with muscle extracts from bulbocavernosus/levator ani of castrated rats, paralysis of the bulbocavernosus or pudendal nerve cuts. Following injection of the bulbocavernosus/levator ani with extracts from castrated rats, the level of CGRP expression and the number of SNB motoneurons with alpha-CGRP message were increased. These studies suggest that the bulbocavernosus/levator ani muscles from castrated rats produce a factor that increases levels of CGRP. Injections of extract prepared from the bulbocavernosus and levator ani muscles of gonadally intact rats did not change the expression of alpha-CGRP mRNA in the SNB. Paralysis of the bulbocavernosus/levator ani with a local anesthetic increased the number of SNB motoneurons expressing alpha-CGRP mRNA and CGRP immunoreactive neurons. To determine whether nerve damage accounted for the observed effects following injection of anesthetic, the pudendal nerves were cut bilaterally.(ABSTRACT TRUNCATED AT 250 WORDS)


Molecular cloning of calmodulin mRNA species which are preferentially expressed in neurons in the rat brain. A cDNA clone designated NGB, which was isolated from a rat brain expression library, detected two mRNA species of 1.8 and 4.0 kb which are highly enriched in brain tissue. cDNAs NGB1 and NGB2 corresponding to these two mRNAs have been isolated and characterized. Sequence data showed that both mRNA species contain the same open reading frames but differ in their 3' untranslated regions. The open reading frame encodes a calmodulin protein of 148 amino acids. Both mRNA species are derived from the rat CaMI gene by utilization of different polyadenylation addition sites. Analysis of the 3' untranslated sequence which is unique to the larger mRNA species revealed a putative AU-rich 'destabilizer' sequence which is thought to be involved in mechanisms of selective mRNA breakdown. In situ hybridization studies revealed that the two calmodulin mRNAs are expressed strongly in neuronal cells in the adult rat brain. Levels of the two mRNA species increased during early postnatal development.


Differential activation of NGF receptor and early response genes in neural crest-derived cells. Nerve growth factor (NGF) binds to a specific cell surface receptor (NGFR) that exists in high affinity (now called trk) and low affinity (now called p75NGFR) forms. NGF-responsive neurons express both forms of the receptor, while Schwann cells, during early development and after nerve injury, express only low affinity p75NGFR. In an attempt to determine whether NGF alters patterns of gene expression in p75NGFR-bearing Schwann cells, we examined the regulation of three early response genes (NGFI-A, NGFI-B, and c-fos) in JS1 rat schwannoma cells. Although these genes are markedly activated by NGF in PC12 (rat pheochromocytoma) cells, NGF has no effect on their transcription in JS1 cells. In contrast to PC12 cells, NGFI-A and NGFI-B are constitutively expressed in JS1 cells, whereas the c-fos gene is not expressed. Treating JS1 cells with cycloheximide (CHX), an inhibitor of protein synthesis that commonly potentiates induction of early response genes by presumably inhibiting synthesis of transcriptional repressors, markedly induces the transcription of NGFI-A and c-fos as well as p75NGFR genes. These data suggest that transcriptional repression plays a major role in the regulation of these genes and that the markedly different regulation of NGFI-A, NGFI-B, and c-fos, all of which encode transcriptional regulators, may be important in guiding the differentiation of these cell types.


Hypoxia-ischemia induces transforming growth factor beta 1 mRNA in the infant rat brain. Transforming growth factor beta 1 (TGF beta 1) mRNA expression was examined after hypoxia-ischemia in rat brains using in situ hybridization. Twenty-one-day-old Wistar rats had unilateral ligation of the right carotid artery followed by either 15 or 90 min inhalational hypoxia. Fifteen min of hypoxia resulted in moderate damage with selective neuronal loss in cortical layer 3 and in the hippocampus of the ligated hemisphere. Seventy-two hours after hypoxia TGF beta 1 expression was markedly increased above control levels in those sites. Levels were normal after 120 h. Ninety min of hypoxia led to an infarction of the lateral cerebral cortex and hippocampus of the ligated hemisphere. One hour after hypoxia TGF beta 1 mRNA was expressed in the hippocampus of the damaged side. Seventy-two and 120 h after hypoxia, expressing cells were found throughout the cerebral cortex, piriform cortex, striatum, thalamus and hippocampus of the infarcted side. These data show that TGF beta 1 mRNA expression is induced after a hypoxic-ischemic insult in the brain. TGF beta 1 may be involved in post-asphyxial repair mechanisms.


Reversible effect of calcium-binding protein regucalcin on the Ca(2+)-induced inhibition of deoxyuridine 5'-triphosphatase activity in rat liver cytosol. The effect of regucalcin, a calcium-binding protein isolated from rat liver cytosol, on deoxyuridine 5'-triphosphatase (dUTPase) in the cytosol of rat liver was investigated. Addition of Ca2+ up to 5.0 microM to the enzyme reaction mixture caused a significant decrease of dUTPase activity, while Zn2+, Cd2+, Co2+, Al3+, Mn2+ and Ni2+ (10 microM) did not have an appreciable effect. The Ca(2+)-induced decrease of dUTPase activity was reversed by the presence of regucalcin; the effect was complete at 1.0 microM of the protein. Regucalcin had no effect on the basal activity of the enzyme. Meanwhile, the reversible effect of regucalcin on the Ca2+ (10 microM)-induced decrease of dUTPase activity was not altered by the coexistence of Cd2+ or Zn2+ (10 microM). The present data suggest that liver cytosolic dUTPase is uniquely regulated by Ca2+ of various metals, and that the Ca2+ effect is reversed by regucalcin.


The nerve growth factor receptor: a multicomponent system that mediates the actions of the neurotrophin family of proteins. Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin 3 (NT-3) are members of a family of structurally related proteins termed neurotrophins that promote the growth and survival of neurons in the central and peripheral nervous systems. Each of these proteins bind to at least two membrane receptors. One is the low affinity nerve growth factor receptor (p75), which binds each member of the neurotrophin family. The other is one of a family of tyrosine kinase receptors--trkA binds only NGF, the related trkB receptor binds BDNF and NT-3, and trkC binds NT-3 alone. This article reviews kinetic and biochemical information on p75 and its relationship to the trk gene products.


Cyclic AMP in prokaryotes.

Cyclic AMP in prokaryotes. Cyclic AMP (cAMP) is found in a variety of prokaryotes including both eubacteria and archaebacteria. cAMP plays a role in regulating gene expression, not only for the classic inducible catabolic operons, but also for other categories. In the enteric coliforms, the effects of cAMP on gene expression are mediated through its interaction with and allosteric modification of a cAMP-binding protein (CRP). The CRP-cAMP complex subsequently binds specific DNA sequences and either activates or inhibits transcription depending upon the positioning of the complex relative to the promoter. Enteric coliforms have provided a model to explore the mechanisms involved in controlling adenylate cyclase activity, in regulating adenylate cyclase synthesis, and in performing detailed examinations of CRP-cAMP complex-regulated gene expression. This review summarizes recent work focused on elucidating the molecular mechanisms of CRP-cAMP complex-mediated processes. For other bacteria, less detail is known. cAMP has been implicated in regulating antibiotic production, phototrophic growth, and pathogenesis. A role for cAMP has been suggested in nitrogen fixation. Often the only data that support cAMP involvement in these processes includes cAMP measurement, detection of the enzymes involved in cAMP metabolism, or observed effects of high concentrations of the nucleotide on cell growth.


Target organ-specific inactivation of drug metabolizing enzymes in kidney of hamsters treated with estradiol. Chronic treatment of hamsters with estradiol for several months has previously been shown to decrease the specific content of cytochrome P450 in the kidney, a target of hormonal carcinogenesis, but not in liver. The reason for this decrease in metabolic enzyme activity is unknown and has been examined in this investigation. We now report that the decrease in specific content of renal cytochrome P450 by 73% in response to estradiol was not affected by co-treatment with tamoxifen for 1 month. The subcutaneous infusion of 250 micrograms/day estradiol for 7 days lowered renal cytochrome P450 by 71% from control values and was therefore used for further mechanistic studies. This treatment decreased renal activities of estradiol 2- or 4-hydroxylase by 77 to 80%, of 7-ethoxycoumarin-O-deethylase by 66% of control values, respectively, and completely eliminated aryl hydrocarbon hydroxylase activities, whereas liver enzymes remained unaffected. After 7 days of infusion of estradiol, fluorescent products of lipid peroxidation were more than doubled in hamster kidney but remained unchanged in liver. The possibility of enzyme destruction by binding of estradiol 2,3-quinone to metabolizing enzymes was investigated in vitro. In the presence of 2-hydroxyestradiol, cumene hydroperoxide, and microsomes, conditions known to favor the oxidation of the steroid to quinone, the binding of catechol estrogen metabolite to microsomal protein increased 60 fold over control values in the absence of cofactor. Purified rat liver cytochrome P450c also oxidized 2-hydroxyestradiol to 2,3-estradiol quinone. The rate of oxidation was linear for the first 2-3 min, but thereafter decreased with time.(ABSTRACT TRUNCATED AT 250 WORDS)


In vitro effects of reactive O2 species on the beta-receptor-adenylyl cyclase system. The irreversible loss of activity of the sarcolemma-localized beta-receptor-adenylyl cyclase system (beta-RAS) in myocardial ischemia is a well documented phenomenon. Alterations in the sarcolemma (SL) induced by reactive O2 species could be responsible for this loss. Therefore the influence of oxidation of SH-groups and lipid peroxidation induced by Fe2+/Vit. C on the beta-RAS activity was studied. During incubation of SL with Fe2+/Vit. C a transient enhancement followed by a continuous loss of the beta-RAS activity (isoprenaline-, NaF-, Gpp(NH)p-, forskolin-stimulated and basal activity) was observed. In contrast there occurred a continuous loss of SH-groups and lipid peroxidation, beginning immediately after the start of incubation. Loss of SH-groups and lipid peroxidation as well as changes in the beta-RAS did not take place in the presence of the antioxidant t-Butyl-4-hydroxyanisole (BHA) or the Fe(2+)-chelator EGTA. In view of the known ischemia-induced formation of reactive O2 species our results show that these powerful oxidants could contribute to the modulation of the beta-RAS during myocardial ischemia.


Dideoxynucleoside triphosphates inhibit a late stage of SV40 DNA replication in vitro. The role of DNA polymerases in the replication of SV40 DNA was studied using a T-antigen-dependent assay supplemented with a human KB cell extract. Inhibition of DNA polymerase alpha by addition of aphidicolin or monoclonal antibodies prevented DNA synthesis, confirming the requirement for this enzyme in replication. The replication process was unaffected by ddTTP at a concentration (5 microM) inhibitory to DNA polymerases beta and gamma, however, higher concentrations of ddTTP (200 microM) caused an apparent accumulation of relaxed circular plasmid with a concomitant decrease in DNA synthesis. An analysis of this replication intermediate indicated that it was formed during the replication reaction and that the replicative cycle was nearly complete. A kinetic study of ddTTP inhibition strongly suggested DNA polymerase epsilon (PCNA-independent DNA polymerase delta) was the target of the inhibitor and that this enzyme functions during the final stages of DNA replication.


The tumor promoter pristane activates transcription by a cAMP dependent mechanism. Pristane is a naturally occurring isoprenoid which is believed to be derived from the phytyl moiety of chlorophyll. Thus it is not surprising that pristane is present in many common fruits or vegetables and furthermore can be detected in tissues of fish and mammals. Using the rat as an animal model, pristane can function as a potent tumor promoter. It is conceivable that pristane could play a role in the development of certain malignancies in higher mammals since it is commonly found in the diet. At the molecular level, pristane can induce changes in the plasma membrane, alter the conformation of chromatin, as well as selectively activate gene expression. This study was undertaken to identify specific transcriptional motifs which are responsive to pristane. A transcriptional promoter which contained a cAMP response element (CRE) was consistently stimulated by pristane in several mouse and primate cell lines. A promoter construct which contained a single copy of the TPA response element (TRE) was also activated by pristane but surprisingly a promoter which contained multiple copies of the TRE was not. Activation of the TRE required 10 fold higher concentrations of pristane relative to activation of the CRE. Within two hours after addition of pristane to monkey fibroblasts (CV-1) levels of cAMP were increased more than two fold relative to controls. These data indicated that pristane can increase the level of cAMP in CV-1 cells and consequently stimulate transcriptional promoters which contain a CRE.


Stimulatory guanine nucleotide-binding protein and adenylate cyclase activities in Bio 14.6 cardiomyopathic hamsters at the hypertrophic stage. The Bio 14.6 cardiomyopathic Syrian hamster is an animal model of human idiopathic cardiomyopathy. The pathogenesis of the disease in this animal has not yet been clearly elucidated. It is well known that alpha- and beta-adrenergic receptors are increased in the myocardium of this animal, but that isoprenaline does not produce an augmented response. We examined the activity of cardiac stimulatory GTP-binding protein (Gs), which couple with beta-adrenergic receptors to stimulate adenylate cyclase, in Bio 14.6 cardiomyopathic hamsters at 90 and 160 days of age. The cardiac norepinephrine concentration was significantly increased in Bio 14.6 hamsters compared with control hamsters (F1B) at 90 days of age (1,739 +/- 120 vs 1,470 +/- 161 ng/g wet tissue weight, p less than 0.05). Cardiac forskolin-stimulated adenylate cyclase activities at 90 and 160 days of age were lower in the cardiomyopathic hamsters than in the F1B controls (90 days old: 98 +/- 24 vs 122 +/- 29 pmol/min/mg protein, p less than 0.05; 160 days old: 74 +/- 13 vs 124 +/- 28 pmol/min/mg protein, p less than 0.01). Cardiac Gs activities at 90 and 160 days of age were significantly lower in Bio 14.6 hamsters than those in F1B hamsters (90 days old: 204 +/- 42 vs 259 +/- 49 pmol/min/mg protein, p less than 0.05; 160 days old: 156 +/- 39 vs 211 +/- 60 pmol/min/mg protein, p less than 0.05). We thus demonstrated functional defects in cardiac Gs protein and adenylate cyclase activity in the Bio 14.6 cardiomyopathic hamsters at 90 to 160 days of age (the hypertrophic stage of cardiomyopathy).(ABSTRACT TRUNCATED AT 250 WORDS)


Underdiagnosis of genital herpes by current clinical and viral-isolation procedures. The current clinical strategy for diagnosing genital herpes simplex virus (HSV) infection in women relies on clinical findings plus the selective use of viral culture. The effectiveness of this approach for identifying women with genital herpes is unknown. We performed physical examinations, colposcopy, Pap smears, viral cultures, and HSV type-specific serologic assays of 779 randomly selected women attending a sexually transmitted disease clinic. Evidence of HSV type 2 infection was detected in 363 women (47 percent), and 9 others (1 percent) had positive cultures indicative of urogenital or anal infection with HSV type 1. Of these 372 women, only 82 (22 percent) had symptoms. Fourteen women (4 percent) had viral shedding without symptoms, 60 (16 percent) had formerly had symptomatic episodes, and 216 (58 percent) had antibodies to HSV-2 with neither viral shedding nor a history of clinical episodes. Characteristic ulcerations of the external genitalia were present in only two thirds of the 66 women with positive HSV cultures; the others had atypical genital lesions or asymptomatic viral shedding. Isolation of HSV from a genitourinary tract specimen was the most sensitive (77 percent) test for confirming a first episode of infection. The detection of HSV-2-specific antibodies was the most sensitive (97 percent) way to confirm symptomatic reactivations of HSV-2 infection. HSV-2 serologic testing also identified the 290 women with asymptomatic HSV-2 infections (37 percent), including 14 (5 percent) who were shedding virus asymptomatically on the day of the examination. The current strategy for diagnosing genital HSV infection in women misses many cases. Newly developed type-specific serologic methods can identify women with recurrent genital HSV-2 infection, as well as those with unrecognized or subclinical infection.


Age-related ubiquitin deposits in dystrophic neurites: an immunoelectron microscopic study. Widespread neuritic dystrophy is a hallmark of Alzheimer's disease (AD) and, in a less severe form, of brain ageing in various mammalian species. By immunohistochemistry, diffuse dot-like staining for ubiquitin (Ubq), a polypeptide involved in the degradation of abnormal and short-lived proteins, has been associated with human brain ageing. The nature of the Ubq deposits was investigated by immunogold electron microscopy on autopsy samples from aged human and dog brains. Most of the dot-like staining was localized to the white matter and corresponded to myelinated dystrophic neurites filled by Ubq-labelled lysosomal dense bodies. They did not contain paired helical filaments or multilamellar bodies. A minority of Ubq deposits was represented by amorphous densities in focal enlargements of the myelin sheaths. Our findings show that the spectrum of Ubq changes in ageing brain is wider than formerly recognized, and support the hypothesis that a defective regulation of the lysosomal system might be involved in the pathogenesis of structural abnormalities both in the ageing brain and in Alzheimer's disease.


Recovery of intracellular pH in cortical brain slices following anoxia studied by nuclear magnetic resonance spectroscopy: role of lactate removal, extracellular sodium and sodium/hydrogen exchange. [31P]- and [1H]nuclear magnetic resonances recorded in an interleaved fashion were used in order to quantify high-energy phosphates, intracellular pH and lactate in cortical brain slices of the guinea-pig superfused in a CO2/HCO3(-)-buffered medium during and after anoxic insults. The volume-averaged intracellular pH and energy status of the preparation following anoxia were determined. In the presence of external Na+, intracellular pH normalized in 3 min and was significantly more alkaline from 10 to 12 min of recovery, but lactate remained elevated for 12 min of reoxygenation following anoxia. The amount of lactate removed was only 40% of the quantity of acid extruded showing operation of H+ neutralizing transmembrane mechanisms other than transport of lactic acid. Amiloride (1 or 2 mM) did not prevent the recovery of intracellular pH, but it blocked the "overshoot" of the alkalinization at 10-12 min of recovery. In a medium containing 70 mM K+, 60 mM Na+ and 0.1 mM Ca2+, the recovery of pH, but not lactate washout, was significantly delayed. Removal of external Na+ caused severe energetic failure, decreases both in oxygen uptake and in N-acetyl aspartate concentration, indicating loss of viable tissue. In Na(+)-free superfusion, lactic acidosis caused a more severe drop in intracellular pH than in the presence of Na+. Complexing of extracellular Ca2+ in the Na(+)-free medium inhibited the acidification by 0.38 pH units during anoxia which is as much as the acidification caused by lactate accumulation in the absence of Na+. In Na(+)-free medium intracellular pH recovered, however, from an anoxic level to a normoxic value in 6 min. Metabolic damage of the slice preparation induced by anoxia in the absence of Na+ was as profound in the presence as in the absence of Ca2+ showing that accumulation of Ca2+ is not the only reason for the damage. It is concluded that recovery of intracellular pH from lactic-acidosis can occur independently of energetic recovery and involves acid extrusion mechanism(s) that is(are) dependent on external Na+ and sensitive to high K+.


Metabolic inhibition and electrical properties of type-1-like cortical astrocytes. Type-1-like cortical mouse astrocytes were studied in homogeneous cultures. Membrane input resistance and membrane potential were measured during drug-induced inhibition of glycolysis (sodium fluoride), mitochondrial respiration (antimycin-a) and Na+/K+ pump activity (ouabain). It was found that the electrical properties of the astrocytes recovered after a 60 min period with inhibited glycolysis or mitochondrial respiration, exhibiting only small reversible depolarizations. A 60 min period of high K(+)-induced depolarization, of cell swelling or of Na+/K+ pump inhibition does not lead to irreversible changes. Total block of energy metabolism, however, causes (1) a large depolarization, which is mainly mediated by external calcium, and (2) a 10-fold increase in input resistance, suggestive of an uncoupling of gap junctions. After an exposure period ranging between 45 and 60 min these conditions lead to irreversible damage. This damage appears to be independent of extracellular calcium and the degree of depolarization and to be specifically mediated by events occurring after the 60-min period of inhibited cell metabolism, that is during the recovery period.