The 3,3'-bis-pyridinium mono-oximes as antidotes against organophosphorous intoxication.

In an attempt to develop effective antidotes against organophosphorous intoxication, three new structurally related bispyridinium mono-oximes with a 2-oxopropane bridge were synthesized. The compounds were evaluated for in-vivo therapeutic protection and cholinesterase reactivation in blood and various brain regions. In neuromuscular function studies against diisopropyl-fluorophosphate and isopropyl methylphosphonofluoridate poisoning, the oximes produced significant protection. The compounds produced marked peripheral reactivation and beneficial effects on neuromuscular transmission. The reactivation of cholinesterase in cerebral cortex by the oximes, in spite of their being quaternary salts is a notable feature.


Some features of Cannabis plants grown in the United Kingdom from seeds of known origin.

The cannabinoid content of UK-grown plants (up to the 6th generation) from Moroccan, Sri Lankan and Zambian seedstock was determined by TLC, GLC and HPLC. All plants from the 5th and 6th series resembled their parents, and UK-grown plants were always much greener than those grown overseas. Cannabinoid content remained broadly typical of the source countries. However, tetrahydrocannabinolic acid (THCA) consistently predominated over tetrahydrocannabinol (THC) to a far greater extent than in the original plants; the THCA/THC ratio was 17 in UK-grown plants compared with 2.0 in the plants from the original areas. Two types of plant emerged from the Moroccan seedstock, one tending to increased cannabidiol (CBD), the other tending to zero levels of this component. The first generation Sri Lankan plants revealed one type of plant with an increased CBD/THC ratio (1.7 compared with 0.11) but this returned to the original value in the succeeding generations. Other Sri Lankan plants had low or undetectable levels of CBD. Moroccan and Sri Lankan CBD-rich plants did not contain cannabichromene, although this cannabinoid was found in THC-rich plants. Zambian plants did not appear to show such a pattern. Zambian seedstock plants had total tetrahydrocannabivarin (diol and acid) levels greater than THC but the ratio was progressively reversed in succeeding generations. The study concludes that the ratios of particular cannabinoids is greatly influenced by the environment.


Transfer of heparin across the human perfused placental lobule.

A system of dual perfusion of an isolated lobule of term human placenta was used as a model to study the transfer of heparin from maternal to foetal circulation. The metabolic viability of the system was assessed by measuring beta-HCG and alkaline phosphatase levels in both maternal and foetal perfusates. Creatinine and antipyrine were used as markers to determine juxtaposition of the maternal and foetal circulations. Results of this study indicate that following administration of a single bolus dose of heparin into the maternal circulation, its concentration declined slowly from 99.01 +/- 2.98 at 15 min to 97.23 +/- 4.12% and transfer of heparin in the foetal circulation was linear and increased from 0.10% +/- 0.05% at 15 min to 0.46 +/- 0.19% over a period of 120 min. The maternal (MAUC) and foetal (FAUC) concentration-time integrals were found to be 70160 +/- 1332 and 340 +/- 30 int. units min mL-1, respectively. Placental permeability of heparin and creatinine, calculated as the ratio of foetal concentration to the integral maternal-foetal concentration difference, was 8.65 x 10(-5) +/- 0.80 x 10(-5) and 0.033 +/- 0.006 mL min-1 g-1 of perfused placental weight, respectively. These data suggest that heparin was transferred from the maternal to the foetal circulation in small quantities.


The effects of various peptides on human isolated gut muscle.

The effects of eleven peptides of gastrointestinal origin have been studied on the contraction, relaxation and spontaneous activity of circular and longitudinal muscle strips from different regions of the human gastrointestinal tract. The effects varied with the peptides and sometimes with the region and muscle layer. There was either contraction, no effect, or relaxation and/or inhibition of an acetylcholine-induced contraction. Responses to some peptides are consistent with the possibility that they may contribute directly to the control of motility: galanin, neurotensin and substance P might be involved in contraction, and vasoactive intestinal peptide, peptide histidine isoleucine and peptide histidine methionine might be inhibitory transmitters.


Transport characteristics of cephalosporin antibiotics across intestinal brush-border membrane in man, rat and rabbit.

The uptake of orally active cephalosporins, ceftibuten and cephradine, by intestinal brush-border membrane vesicles isolated from man, rat and rabbit was studied. In the presence of an inward H+ gradient, ceftibuten but not cephradine was taken up into intestinal brush-border membrane vesicles of man and rat against the concentration gradient (overshoot phenomenon). In rabbit jejunal brush-border membrane vesicles, the uptake of both cephalosporins in the presence of an inward H+ gradient exhibited the overshoot phenomenon. In human and rat vesicles, the initial uptake of ceftibuten was strongly inhibited by compound V, an analogue of ceftibuten, but the uptake of cephradine was not affected by any of the cephalosporins tested, whereas in the rabbit brush-border membrane vesicles, initial uptake of both ceftibuten and cephradine were markedly inhibited by all cephalosporins and dipeptides used. These results suggest that the transport characteristics of human and rat intestinal brush-border membrane for cephalosporins are comparable, and that rabbit is an inadequate animal for investigating the transport characteristics of beta-lactam antibiotics.


Metabolic activation of sennoside C in mice: synergistic action of anthrones.

Sennosides A and C directly injected into the caecum of mice showed equal purgative activity. Intracaecal administration reduced time to onset of diarrhoea induced by sennoside C from about 3 h after oral administration to about 24 min. At 2.3 h after oral administration of sennoside C, nearly equimolar amounts of aloe-emodin anthrone and rhein anthrone were detected in the large intestine of mice. The purgative effect of oral sennoside C could be reduced by pretreating mice with chloramphenicol. This was observed as a decreased formation of total anthrones in the large intestine. Both anthrones and an equimolar mixture of both anthrones directly injected into the caecum exerted a purgative effect, although the activity was lower for aloe-emodin anthrone. The intracaecal ED50 values were 54.5 (24.1-89.6), 11.4 (5.0-15.7) and 11.2 (6.1-14.6) mumol kg-1 for aloe-emodin anthrone, rhein anthrone and an equimolar mixture of both anthrones, respectively. We concluded that aloe-emodin anthrone and rhein anthrone, formed mainly by intraluminal bacterial action, are the true active metabolites of sennoside C in mice and that both anthrones synergistically exert their purgative effects on mice.


Transport of paraquat and mexiletine from the blood into the rat intestinal lumen and peritoneal cavity.

Transport of paraquat and mexiletine from the blood into the intestinal lumen and the peritoneal cavity was examined after their intravenous administration (paraquat: 20 mg kg-1, mexiletine: 10 mg kg-1) to rats. The average amounts of paraquat transferred into the intestinal lumen and the peritoneal cavity were 1.39 and 22.8% of the dose in 120 min, respectively. The average amounts of mexiletine transferred into the intestinal lumen and the peritoneal cavity were 6.1 and 2.5% of the dose in 120 min, respectively. The transfer rate of 3H2O into the peritoneal cavity after intravenous administration (1.85 MBq) was greater than that into the intestinal lumen. In view of the hydrophilic nature of paraquat cation, a solvent drag effect due to movement of water might contribute to transport of paraquat from the blood to the peritoneal cavity. Differences in transport behaviour across the two membranes could be due to differences in the geometrical factors such as the surface area and the distribution of blood vessels. Differences might also be due to differences in physicochemistry and pharmacological effects of both substances.


Semicarbazide-sensitive amine oxidase from the smooth muscles of dog aorta and trachea: activation by the MAO-A inhibitor clorgyline.

Semicarbazide-sensitive amine oxidase (SSAO) has been identified in the dog trachea and aorta smooth muscles. The dog SSAO is blocked by hydrazine inhibitors. SSAOs from several different vascular smooth muscle sources, such as the rat and bovine aorta, and human umbilical artery, as well as the bovine plasma, are insensitive to the MAO-A inhibitor clorgyline; the dog SSAO on the other hand is significantly activated by clorgyline. Two methods, i.e. radioenzymatic and fluorometric methods, have been applied to substantiate this clorgyline-induced activation. The activation was detected with respect to the deamination of different substrates, such as benzylamine, beta-phenylethylamine and longer carbon chain aliphatic amines, but not with respect to methylamine. The clorgyline effect is reversible, non-competitive and time-independent; it depends on electrostatic and hydrophobic interactions between clorgyline and hydrophobic regions of the dog SSAO enzyme.


Contractile responses of chicken rectum to stimulation of Remak's nerve or purinoceptors: effect of suramin.

Stimulation of Remak's nerve produced a rapid contraction of the rectal muscle which was resistant to blockade by hyoscine, followed by a slow contractile response which was cholinergic. With hyoscine (1 microM) in the Tyrode solution, the non-cholinergic contractile response to nerve stimulation was compared with the responses to adenosine triphosphate (ATP) and alpha,beta-methylene adenosine triphosphate (alpha,beta-Me ATP). In the presence of suramin (300 microM), the contractile response to ATP was increased by about 100%, whereas that to nerve stimulation was inhibited by approximately 17%. Suramin (60 microM) also discriminated between the contractile responses to ATP and nerve stimulation, only the former being potentiated by the drug. It seems probable that suramin potentiated the action of ATP in the rectum through inhibition of ectonucleotidase activity. If so, the potentiation should not extend to alpha,beta-Me ATP, as this analogue of ATP is resistant to inactivation by the enzyme. Suramin inhibited the contractile response to alpha,beta-Me ATP. Thus the neurotransmitter which mediated the hyoscine-resistant contractile response to stimulation of Remak's nerve was dissimilar to ATP, in that it was not potentiated by suramin and presumably was not inactivated by ectonucleotidase activity.


L-dopa-like regulatory actions of L-threo-3,4-dihydroxyphenylserine on the release of endogenous noradrenaline via presynaptic receptors in rat hypothalamic slices.

Effects of L-threo-3,4-dihydroxyphenylserine (L-threo-DOPS) on the spontaneous release and the stimulus(2 Hz)-evoked release of endogenous noradrenaline were studied in rat hypothalamic slices with functioning L-aromatic amino acid decarboxylase (AADC) and with AADC inhibition. In non-inhibited slices, spontaneous release was not modified by L-threo-DOPS at 1 pM-100 nM, tended to increase at 1-10 microM and increased at 100 microM. Noradrenaline tissue content slightly increased at 100 microM. Stimulated release was concentration-dependently facilitated at 1-1000 pM and tended to decrease gradually from a maximum at 10 nM-10 microM. Under AADC inhibition, spontaneous release concentration-dependently increased at 10-100 microM by 60% of the increase seen in slices without AADC inhibition. Increase in noradrenaline tissue content was abolished. L-threo-DOPS produced a triphasic pattern on stimulated release; concentration-dependent facilitation at 1-1000 pM similar to that seen in slices with functional AADC, no facilitation at 10-1000 nM, and a concentration-dependent increment at 10-100 microM. The facilitation at 1 nM was stereoselective and was antagonized by (-)-propranolol 10 nM, and no facilitation at 100 nM was restored to the maximum by yohimbine 10 nM, DG-5128 10 nM or S-sulpiride 1 nM. Furthermore, L-threo-DOPS (1-1000 pM)-induced facilitation was competitively antagonized by L-dopa methyl ester, a competitive antagonist for L-dopa, with a pA2 value of 13.6, whereas it was noncompetitively antagonized by (-)-propranolol.


Antiarrhythmic, electrophysiological and haemodynamic effects of prolonged oral dosing with Org 7797 in the anaesthetized rat.

The antiarrhythmic, electrophysiological and haemodynamic effects of chronic oral administration of Org 7797 ((16 alpha,17 beta)-17-methylamino-oestra-1,3,5(10)-triene-3, 16-diol-(Z)-2-butonedioate) were studied in rats. During dosing (10 mg kg-1 twice a day for 10 days) no effects on the electrocardiogram, monitored in conscious animals, were observed despite modest reductions (15-18%) in the maximum rate of depolarization of papillary muscle excised 1 or 6 h after completion of the dosing regime. Following anaesthesia, Org 7797 reduced the severity of arrhythmias induced by coronary artery occlusion and prevented the accompanying decrease in the ventricular fibrillation threshold (VFT) at 1 h after completion of dosing. By 6 h the effect on VFT had waned but protection against ischaemia-induced arrhythmias was retained despite a substantial decrease in Org 7797 plasma levels. Drug treatment did not modify arterial blood pressure, heart rate or stroke volume. We conclude that Org 7797 given chronically via the oral route exerts antiarrhythmic actions which may, at least in part, be due to sodium-channel block. In addition, our results suggest the presence of an active metabolite. The protective effects of Org 7797 were seen in the absence of electrocardiographic or haemodynamic changes suggesting that multiple oral doses of Org 7797 do not compromise normal cardiac function.


[Synthesis of metabolites of mosapramine. II. Synthesis of phenolic metabolites of mosapramine and their pharmacological activities].

Four phenolic metabolites of (+-)-3-chloro-5-[3-(2-oxo-1,2,3,5,6,7,8,8a-octahydroimidazo[1,2- a]pyridine-3-spiro-4'-piperidino)propyl]-10,11-dihydro-5H-dibenz [b,f]azepine (mosapramine), a new antipsychotic drug, were synthesized in order to determine their chemical structures. Pharmacological activities of the three main metabolites were compared with those of mosapramine. The activities of the metabolites were far less potent than those of mosapramine.


Inhibition of adenylate cyclase in the locus coeruleus mediates the hypnotic response to an alpha 2 agonist in the rat.

Recently, we determined that the transduction mechanism for the hypnotic response to dexmedetomidine, a highly selective alpha 2 agonist, resides in the locus coeruleus (LC) of the rat. Candidates for the effector mechanism of this alpha 2 adrenoceptor-mediated hypnotic response include inhibition of adenylate cyclase, which has been shown to be pivotal to the cellular response of alpha 2 agonists in some, but not in all, cases. The LC of rats were stereotaxically cannulated with an indwelling catheter, and after the 2nd day, the hypnotic response to 7 micrograms of dexmedetomidine into the LC (an effective hypnotic dose for 95% of animals) was tested. Other groups of rats were pretreated with the permeable nonhydrolyzable cyclic AMP (cAMP) analog, dibutyryl cAMP (dB cAMP), at a dose of 0.2 to 1.2 ng into the LC, or 2.75 to 275 micrograms.kg-1 i.p. rolipram, a cAMP-specific phosphodiesterase inhibitor, and the hypnotic response to 7 micrograms of dexmedetomidine into the LC was tested. Both dB cAMP and rolipram reversed the hypnotic response to dexmedetomidine. To test for the specificity of these hypnotic-reversing perturbations, rats were pretreated with Rp-adenosine-3',5'-cyclic phosphorothioate, a cAMP-dependent protein kinase inhibitor, and the experiments were repeated. The hypnotic-reversing property of either dB cAMP or rolipram could be prevented by blocking cAMP-dependent protein kinase ("A" kinase) activity with Rp-adenosine-3',5'-cyclic phosphorothioate.(ABSTRACT TRUNCATED AT 250 WORDS)


The positive chronotropic effect induced by BRL 37344 and CGP 12177, two beta-3 adrenergic agonists, does not involve cardiac beta adrenoceptors but baroreflex mechanisms.

The presence of a beta-3 adrenoceptor (in addition to the classical beta-1 and beta-2 adrenoceptors) and its involvement in the control of heart rate was investigated in the dog. Experiments were carried out in conscious normal and sinoaortic denervated dogs (i.e. animals deprived of baroreceptor pathways). In normal dogs, infusion of isoproterenol, BRL 37344 (4-[-[(2-hydroxy-(3-chlorophenyl) ethyl)-amino]propyl]phenoxyacetate) (a beta-3 adrenergic agonist) or CGP 12177 (4-[3-t-butylamino-2-hydroxypropoxy]benzimidazol-2- one) (a beta-1 beta-2 adrenergic antagonist reported to act as an agonist for the beta-3 adrenergic receptor) increased heart rate with an order of potency: BRL 37344 > isoproterenol >> CGP 12177. [125I]Cyanopindolol binding (2-2000 pM) was saturable and Scatchard analysis indicated the presence of an homogenous population of binding sites. KD was 12.8 +/- 18.5 pM and maximum binding was 94.2 +/- 12.5 fmol/mg of protein. Competition binding studies on dog heart membranes using 150 pM [125I] cyanopindolol indicated an order of potency (CGP 12177 > isoproterenol > BRL 37344) different from that observed in cardiovascular studies. Isoproterenol stimulated adenylate cyclase activity in heart membranes from normal dogs, whereas CGP 12177 and BRL 37344 were without any stimulating action. The positive chronotropic effects of isoproterenol, BRL 37344 and CGP 12177 were accompanied with a reduction in arterial blood pressure. In sinoaortic denervated animals, isoproterenol infusion provoked tachycardia and hypotension. BRL 37344 and CGP 12177 were without any significant effect on heart rate but induced a rapid and dramatic hypotension.(ABSTRACT TRUNCATED AT 250 WORDS)


Pharmacological characterization of PD 118717, a putative piperazinyl benzopyranone dopamine autoreceptor agonist.

PD 118717 (7-[3-[4-(2-pyrimidinyl)-1-piperazinyl]-propoxy]-2H-1- benzopyran-2-one sulfate) proved to be a dopamine (DA) D-2 autoreceptor agonist in biochemical and electrophysiological studies in rats and to exhibit an antipsychotic-like profile in behavioral tests in rodents and monkeys. In vitro binding studies indicated that PD 118717 bound selectively to DA D-2 vs. D-1 receptors and exhibited agonist binding properties (biphasic inhibitory curves and GTP shift) similar to DA. It also had significant affinity for serotonin-(5-HT)1A but not 5-HT1B and 5-HT2 receptors. PD 118717 was active in antagonizing the tau-butyrolactone-induced accumulation of dopa in rat striatum and mesolimbic regions. PD 118717 also depressed the firing of DA neurons in substantia nigra pars compacta of rats. In both of the latter tests the effects of PD 118717 were reversed by haloperidol. PD 118717 decreased brain DA metabolism, decreased DA utilization, decreased accumulation of dopa after inhibition of L-aromatic amino acid decarboxylase, stimulated serum corticosterone and inhibited stimulated serum prolactin levels. PD 118717 did not alter striatal acetylcholine levels; nor did it induce locomotor stimulation or stereotypy in normal animals, suggesting a lack of postsynaptic DA stimulation of normosensitive DA receptors. In tests designed to reveal even weak postsynaptic DA agonist effects, PD 118717 stimulated locomotor activity in 6-hydroxydopamine-lesioned animals and relatively higher doses induced a low degree of stereotyped behavior when combined with the DA D-1 agonist SKF 38393. PD 118717 decreased the accumulation of 5-hydroxytryptophan in brain, an effect probably due to an agonist action at 5-HT1A receptors. PD 118717 decreased spontaneous locomotor activity in rodents, antagonized amphetamine-stimulated hyperactivity in mice and inhibited Sidman avoidance in monkeys, effects seen with antipsychotic agents. Unlike DA antagonist antipsychotics, PD 118717 did not induce extrapyramidal dysfunction in monkeys. PD 118717 displayed behavioral activity after p.o. dosing and its effects did not show tolerance on repeated dosing. In conclusion, PD 118717 has the profile of a DA autoreceptor agonist in neurochemical and neurophysiological tests and produces effects suggestive of antipsychotic efficacy without neurological side effect liability in preclinical behavioral tests.


DuP 734 [1-(cyclopropylmethyl)-4-(2'(4''-fluorophenyl)-2'- oxoethyl)piperidine HBr], a potential antipsychotic agent: preclinical behavioral effects.

It has been suggested that sigma receptors may be involved in the etiology of psychosis and that 5-hydroxytryptamine2 (5-HT2) antagonists may have utility in treating the negative symptoms of psychosis as well as reducing the side effects associated with the typical antipsychotic haloperidol. We have evaluated the potential antipsychotic effects of 1-(cyclopropylmethyl)-4-(2'(4''-fluorophenyl)-2'-oxoethyl)piper i din e HBr (DuP 734), a selective and potent sigma and 5-HT2 receptor ligand with weak affinity for D2 receptors, in behavioral animal models that are not necessarily dependent on dopamine antagonism. DuP 734 potently blocked mescaline-induced scratching (ED50 = 0.35 mg/kg, p.o.) and aggressive activity (ED50 = 1.9 mg/kg, p.o.) and was relatively much weaker as an apomorphine antagonist (ED50 = 12 mg/kg, p.o.). This was in contrast to the typical antipsychotic agents such as haloperidol and chlorpromazine, which were very potent in all three tests. In rats, DuP 734 did not antagonize avoidance behavior or induce catalepsy, and, therefore, differed from the potent dopamine receptor antagonist antipsychotics. It did, however, reduce lever response rates in a random interval 60-sec food reward schedule of reinforcement (ED50 = 6.0 mg/kg, p.o.) in rats. The results suggest that DuP 734 may have antipsychotic activity without the liability of motor side effects typical of neuroleptics. Although DuP 734 itself did not antagonize avoidance activity, it significantly enhanced the potency of haloperidol in blocking avoidance behavior by 3-fold (by shifting the ED50 from 0.94 to 0.36 mg/kg, p.o.), whereas the ED50 of haloperidol for blockade of escape behavior and induction of catalepsy was not affected.(ABSTRACT TRUNCATED AT 250 WORDS)


DuP 734 [1-(cyclopropylmethyl)-4-(2'(4''-fluorophenyl)-2'-oxoethyl)- piperidine HBr], a sigma and 5-hydroxytryptamine2 receptor antagonist: receptor-binding, electrophysiological and neuropharmacological profiles.

It has been suggested that sigma receptor antagonists may be useful as antipsychotic drugs and that 5-hydroxytryptamine (5-HT2) receptor antagonists produce improvements of the negative symptoms of schizophrenia. [1-(Cyclopropylmethyl)-4-(2'-(4''-fluorophenyl)-2'- oxoethyl)-piperidine HBr] (DuP 734) is a novel compound with high affinity for the sigma (Ki = 10 nM) and 5-HT2 (Ki = 15 nM) receptors, but low affinity for dopamine receptors (Ki > 1000 nM) as well as 33 other receptors, ion channels and second messenger systems in vitro. DuP 734 did not inhibit the synaptosomal uptake of dopamine, 5-HT or norepinephrine. Oral administration of DuP 734 potently blocked 5-hydroxy-L-trytophan (5-HTP)-induced head twitch in the rat (ED50 = 6.5 mumol/kg), indicating 5-HT2 antagonist activity. Extracellular single-unit recording studies demonstrated that DuP 734 antagonized the effect of the selective sigma ligand (+)-3-(3-hydroxyphenyl-N-(1-propyl) piperidine [(+)-3-PPP] on dopamine neuronal activity in the substantia nigra of the rat with an ED90 of 3.6 mumol/kg i.v. The sigma receptor agonists (+)-SKF 10,047 and phencyclidine both elicited rotational behavior in rats with unilateral lesion of the substantia nigra. The rotational behavior induced by either (+)-SKF 10,047 or phencyclidine was dose-dependently antagonized by DuP 734 with oral ED50 of 8.7 and 19.6 mumol/kg, respectively. The 5-HT2 receptor antagonist ICI 169,369, even at high doses (up to 33 mumol/kg, s.c.), did not antagonize the rotational behavior induced by (+)-SKF 10,047.(ABSTRACT TRUNCATED AT 250 WORDS)


[3H]DuP 734 [1-(cyclopropylmethyl)-4-(2'-(4''-fluorophenyl)-2'- oxoethyl)-piperidine HBr]: a receptor binding profile of a high-affinity novel sigma receptor ligand in guinea pig brain.

The in vitro binding properties of 1-(cyclopropylmethyl)-4-(2'-(4''-fluorophenyl)-2'-oxoethyl)pipe ridi ne HBr, [3H]DuP 734, a novel sigma receptor ligand, were examined in homogenates of guinea pig brain. Specific [3H]DuP 734 binding (10 microM haloperidol-displaceable) in cerebellum was dependent on pH, temperature and membrane protein concentration, reversible, saturable and of high affinity (KD = 228 +/- 34 pM; Bmax = 3856 +/- 340 fmol/mg protein). [3H]DuP 734 binding was substantially reduced by treating the membrane with proteases and completely abolished by heat denaturation. [3H]DuP 734 binding was unaffected by the presence of ions or guanine nucleotides. The pharmacological characteristics of [3H]DuP 734 binding in cerebellum displayed the same rank order and stereospecificity as previously reported for sigma receptors in brain. [3H]DuP 734-labeled sigma receptors were heterogeneously distributed throughout the central nervous systems with highest densities present in pons/medulla, hypothalamus, spinal cord and cerebellum. In addition to labeling sigma receptors, a second, lower affinity, haloperidol-insensitive [3H] DuP 734 binding site was observed in the cerebral cortex. This second site could not be positively identified as a neuronal receptor because a series of standard compounds were unable to displace [3H]DuP 734 from the haloperidol-insensitive site; only structural analogs of DuP 734 proved effective in displacing [3H]DuP 734 from the haloperidol-insensitive site. In summary, [3H]DuP 734 is a novel ligand that binds with high affinity to sigma receptors in brain.


Pharmacology of gamma-aminobutyric acidA receptor complex after the in vivo administration of the anxioselective and anticonvulsant beta-carboline derivative abecarnil.

In rodents, the effect of the beta-carboline derivative isopropyl-6- benzyloxy-4-methoxymethyl-beta-carboline-3-carboxylate (abecarrnil), a new ligand for benzodiazepine receptors possessing anxiolytic and anticonvulsant properties, was evaluated on the function of central gamma-aminobutyric acid (GABA)A receptor complex, both in vitro and in vivo. Added in vitro to rat cortical membrane preparation, abecarnil increased [3H]GABA binding, enhanced muscimol-stimulated 36Cl- uptake and reduced the binding of t-[35S]butylbicyclophosphorothionate ([35S]TBPS). These effects were similar to those induced by diazepam, whereas the partial agonist Ro 16-6028 (tert-butyl-(S)-8-bromo-11,12,13,13a-tetrahydro-9-oxo-9H- imidazo[1,5-a]-pyrrolo-[2,1-c][1,4]benzodiazepine-1-carboxylate) showed very weak efficacy in these biochemical tests. After i.p. injection to rats, abecarnil and diazepam decreased in a time-dependent and dose-related (0.25-20 mg/kg i.p.) manner [35S]TBPS binding measured ex vivo in the cerebral cortex. Moreover, both drugs at the dose of 0.5 mg/kg antagonized completely the convulsant activity and the increase of [35S]TBPS binding induced by isoniazide (350 mg/kg s.c.) as well as the increase of [35S]TBPS binding induced by foot-shock stress. To better correlate the biochemical and the pharmacological effects, we studied the action of abecarnil on [35S]TBPS binding, exploratory motility and on isoniazid-induced biochemical and pharmacological effects in mice. In these animals, abecarnil produced a paralleled dose-dependent (0.05-1 mg/kg i.p.) reduction of both motor behavior and cortical [35S]TBPS binding. Moreover, 0.05 mg/kg of this beta-carboline reduced markedly the increase of [35S]TBPS binding and the convulsions induced by isoniazid (200 mg/kg s.c.).(ABSTRACT TRUNCATED AT 250 WORDS)


Characterization of 2,4,5-trihydroxyphenylalanine neurotoxicity in vitro and protective effects of ganglioside GM1: implications for Parkinson's disease.

The neurotoxic properties of 2,4,5-trihydroxyphenylalanine (TOPA; the 6-hydroxylated derivative of dopa) was investigated in cultures of central neurons. Application of solutions of TOPA to cerebellar granule cells resulted in a concentration- and time-dependent neuronal death, with prolonged (24 hr) exposure producing a clear left-handed shift in the dose-response relationship from the one observed with a 60-min exposure (LD50: 4 and 29 microM, respectively). This toxicity was largely blocked by the non-N-methyl-D-aspartate antagonist 6-cyano-7-nitroquinoxaline-2,3-dione. Solutions of TOPA were also toxic to mesencephalic neurons after acute or chronic exposure, displaying the same leftward shift in LD50. This latter preparation contained a minor population of dopaminergic, tyrosine hydroxylase immunopositive cells which were likewise sensitive to the excitotoxic effects of TOPA. Neurotoxic activity of TOPA appeared to depend upon its oxidation in solution, as judged using chemical analysis and reducing agents. The monosialoganglioside GM1 was effective in protecting against neurodegeneration induced by brief or prolonged exposure to solutions of TOPA. These results suggest that an abnormal production or accumulation of TOPA or its oxidation product(s) might be involved in excitotoxicity directed to areas of the brain with dopaminergic innervation, and in other brain areas in Parkinson's disease patients on long-term dopa therapy. The selective action of gangliosides in disrupting the pathological consequences of glutamate receptor activation proposes their use as chemoprophylactic agents for preventing or arresting the neuronal losses accompanying such situations.


Augmented sensitivity of D1-dopamine receptors in lateral but not medial striatum after 6-hydroxydopamine-induced lesions in the neonatal rat.

Lesioning of neonatal rats with the neurotoxin 6-hydroxydopamine (6-OHDA) reduced striatal dopamine (DA) levels to 3% of control levels and produced marked increases in the behavioral effects of the selective D1-DA receptor agonist SKF-38393 in these animals when tested as adults. However, no differences were observed, either in basal or D1-DA-stimulated striatal cAMP formation or in forskolin-stimulated or GTP-stimulated cAMP production, between control and lesioned animals. C-fos-like immunoreactivity after SKF-38393 was significantly greater in dorsolateral vs. ventromedial aspects of the striatum in lesioned animals. Like the c-fos response, augmented electrophysiological responsiveness to SKF-38393 occurred in lesioned rats in lateral, but not medial, portions of the striatum. No differences were found in nucleus accumbens in sensitivity to SKF-38393 between control and lesioned rats. Although autoradiographic determination of D1-DA receptor binding throughout the striatum and nucleus accumbens revealed no differences between unlesioned and lesioned rats, tyrosine hydroxylase-like immunoreactivity was reduced with a regional distribution inversely related to c-fos-like immunohistochemical expression. These findings demonstrate that regionally enhanced electrophysiological sensitivity of striatal neurons to D1-DA receptor agonists after neonatal 6-OHDA-induced lesions is associated with regional changes in c-fos-like immunoreactivity and tyrosine hydroxylase-like immunohistochemistry, but not with changes in D1-DA receptor autoradiography or D1-DA-stimulated adenylyl cyclase activity. Such regional consequences of 6-OHDA-induced lesions in neonates may contribute to the unique behavioral patterns observed when these rats are challenged with L-dopa or D1-DA agonists as adults.


Effects of intrahippocampal injections of N-methyl-D-aspartate receptor antagonists and scopolamine on working and reference memory assessed in rats by a three-panel runway task.

In order to elucidate the roles of hippocampal N-methyl-D-aspartate-type excitatory amino acid receptors in working and reference memory in rats, the effects of intrahippocampal injections of selective and competitive N-methyl-D-aspartate receptor antagonists such as CGS 19755 (cis-4-phosphonomethyl-2-piperidine carboxylic acid), 3-[(+-)-2-carboxypiperazin-4-yl]propyl-1-phosphonic acid and 2-amino-5-phosphonovaleric acid on this behavior were examined with a three-panel runway task. The results were compared with the effect of the muscarinic receptor antagonist scopolamine. In the working memory task, CGS 19755 and 3-[(+-)-2-carboxypiperazin-4-yl]propyl-1-phosphonic acid at 10 and 32 ng/side, injected bilaterally into the dorsal hippocampus before testing, produced a significant increase in the number of errors (attempts to pass through two incorrect panels of the three panel-gates at four choice points). This also occurred after the rats were given systemic injection of these drugs at 3.2 and 10 mg/kg. In the reference memory task, neither CGS 19755 nor 3-[(+-)-2-carboxypiperazin-4-yl]propyl-1-phosphonic acid affected the number of errors, whether given at doses up to 32 ng/side intrahippocampally or up to 10 mg/kg systemically. Working memory errors also increased significantly after intrahippocampal injections of d-2-amino-5-phosphonovaleric acid at 100 and 320 ng/side, but were not affected by I-2-amino-5-phosphonovaleric acid at doses up to 1 microgram/side. On the other hand, intrahippocampal scopolamine at 1.0 and 3.2 micrograms/side increased significantly working memory errors, without affecting reference memory errors.(ABSTRACT TRUNCATED AT 250 WORDS)


Synthesis and biological characterization of alpha-(4-fluorophenyl)-4-(5-fluoro-2-pyrimidinyl)-1-piperazinebutanol and analogues as potential atypical antipsychotic agents.

A series of 1-(pyrimidin-2-yl)piperazine derivatives were prepared and evaluated in receptor binding assays and in in vivo behavioral paradigms as potential atypical antipsychotic agents. Compound 16 (BMS 181100 (formerly BMY 14802)) emerged as the lead compound from within the series on the basis of its good activity and duration of action in the inhibition of both conditioned avoidance responding and apomorphine-induced stereotopy in the rat. Compound 16 not only failed to induce catalepsy in the rat but was quite effective in reversing the cataleptic effect of neuroleptic agents, thus indicating a low propensity for causing extrapyramidal side effects. In comparison to reference antipsychotic agents, 16 appeared to be less sedating and was relatively weaker in causing muscle incoordination. The compound was essentially inactive in binding to dopamine D2 receptors and its chronic administration to rats did not result in dopamine receptor supersensitivity. It exhibited modest to weak affinity for 5-HT1A and alpha 1 receptors but was found to be a fairly potent ligand for sigma binding sites (IC50 vs (+)-[3H]-3-PPP = 112 nM). Although the resolved enantiomers of racemic 16 did not show dramatic differences from racemate or from each other in most tests, the R(+) enantiomer was up to 11-fold more potent than its antipode in binding to sigma sites. Several studies have indicated that 16 may be a limbic-selective agent which may modulate dopaminergic activity by an indirect mechanism. The compound has been selected for clinical evaluation in the treatment of psychosis.


DL-tetrazol-5-ylglycine, a highly potent NMDA agonist: its synthesis and NMDA receptor efficacy.

At physiological pH, the spatial arrangement of the three charges of DL-tetrazol-5-ylglycine (5) could be viewed as similar to those found in certain conformations of the two excitatory amino acids (EAAs)--aspartic and glutamic acids. Given significant binding to one or more EAA receptors, 5 would offer unique modeling and perhaps biological opportunities. We have previously shown it to be the most potent NMDA agonist known, with a unique and marked in vitro neutrotoxicity at depolarizing concentrations. Now we report the details required for its synthesis, together with its potency and efficacy in two assays of functional activation of the NMDA receptor, namely agonist-influenced [3H]MK801 binding and agonist-induced release of the neurotransmitter [3H]-norepinephrine from brain slices. In both these assays DL-tetrazol-5-ylglycine proved to be more potent and efficacious than NMDA and cis-methanoglutamate. It was more potent than, and equally efficacious to, L-glutamate in [3H]MK801 binding. The structural features of 5 may well reflect optimal agonist interaction at the NMDA receptor site. (We considered the possibility that some decarboxylation of DL-tetrazol-5-ylglycine may have occurred during testing. This would give 5-(aminomethyl)tetrazole (13), the tetrazole acid analog of glycine; and glycine is involved in NMDA receptor activation. Compound 13 does not affect [3H]glycine binding at the strychnine-insensitive glycine binding site, and [3H]MK801 binding studies showed that the (aminomethyl)-tetrazole, even if is formed, would probably have no effect on the activity of tetrazol-5-ylglycine at the NMDA receptor.


Computer-aided mapping of the beta-adrenoceptor. 1. Explanation for effect of para substitution on blocking activity at the beta-1-adrenoceptor.

Anomalously low affinities for the beta-1-adrenoceptor are seen for members of a series of para-substituted N-isopropylphenoxypropanolamines in which the substituent is able to conjugate with the aromatic ring. The energy of conjugation was calculated using the AM1 semiempirical molecular orbital method and appears to correlate with the loss of binding energy, and hence affinity for the receptor. This suggests that binding is associated with movement of the substituent out of the plane of the aromatic ring due to steric interference with the receptor. A previously unrecognized binding site for aromatic groups off the para position is also identified.


Bioisosteric replacement of the alpha-amino carboxylic acid functionality in 2-amino-5-phosphonopentanoic acid yields unique 3,4-diamino-3-cyclobutene-1,2-dione containing NMDA antagonists.

In this report, a novel bioisostere of the alpha-amino acid, 3,4-diamino-3-cyclobutene-1,2-dione, has been incorporated into a series of compounds which are NMDA antagonists. These compounds, which are achiral and easily prepared, demonstrated good affinity at the NMDA receptor by their ability to displace [3H]CPP binding in vitro. In particular, the phosphonic acid 24 provided protection against NMDA-induced lethality in mice equivalent to 2-amino-7-phosphonoheptanoic acid (5). This was considered an encouraging result in lieu of the fact that 24, like 5, lacks the conformational rigidity of the more potent NMDA antagonists. In addition, analogs that incorporate the 1,2,4-oxadiazolidine-3,5-dione heterocycle of quisqualic acid and the unsaturation of kainic acid were prepared to explore selectivity at the non-NMDA receptor subtypes.


Helical structure of P pili from Escherichia coli. Evidence from X-ray fiber diffraction and scanning transmission electron microscopy.

The structure of the P pili from Escherichia coli has been studied using X-ray fiber diffraction and scanning transmission electron microscopy (STEM). Analysis of the fiber diffraction data indicates that the pili are constituted largely of structural subunits arranged helically with approximately 33 subunits in 10 turns in an axial repeat of 244.5 +/- 1.8 A. Radial electron density distributions calculated from equatorial diffraction data and STEM data indicate that the pili are about 65 A in diameter with a small central cavity roughly 15 A across. The principal protein component of the pili is PapA, which has a molecular weight of 16.5 kDa. Assuming that each subunit consists of a single PapA molecule, the mass-per-unit-length of the pili predicted from the X-ray data is 2.23 kDa/A. Measurements of mass-per-unit-length were also made through the analysis of STEM images. These measurements indicate a value of 2.13 +/- 0.14 kDa/A. STEM images demonstrated the presence of thin, thread-like structures emerging from the ends of pili and spanning breaks in the pili structure. These structures, which have been observed under other conditions, have been termed fibrillae. In the STEM images the fibrillae appear about 20 A in diameter. The mass-per-unit-length of the fibrillae was estimated using the STEM data to be 0.4 kDa/A. These data are consistent with the fibrillae representing an unwound or unraveled form of the pili proteins overstretched to about five times the length they would have in the intact pili.


3H-D-aspartate release from cerebellar granule neurons is differentially regulated by glutamate- and K(+)-stimulation.

Neurotransmitter release in response to either 55 mM K+ or 25 microM glutamate as well as its dependency on Ca2+ from different sources was compared in cultured glutamatergic cerebellar granule cells from rat brain. The intracellular Ca2+ concentration was monitored at the single cell level in neurites as well as cell bodies employing the fluorescent Ca2+ indicator fura-2. Transmitter release was assayed using 3H-D-aspartate to label the exogenously accessible glutamate pools, which in these neurons is believed to also include the transmitter pool. In an attempt to distinguish whether transmitter release was dependent on an intact cytoskeleton or not, the colchicine-like drug Nocodazole, which also blocks transport of vesicles, was used. K(+)-stimulated transmitter release consisted for the major part (around 70%) of a Ca(2+)-dependent, Nocodazole sensitive release component and this K(+)-induced release appeared to be almost exclusively dependent on N-type Ca2+ channels. In contrast, 50% of the glutamate-induced Ca(2+)-dependent release was triggered by Ca2+ from a Dantrolene sensitive intracellular Ca2+ pool. Since these neurons undergo a pronounced maturational change in which neurotransmitter vesicles become increasingly prominent, the Ca2+ responses and transmitter release evoked by the two different stimuli were investigated as a function of the culture period. K+ and glutamate were found to increase intracellular [Ca2+] differentially. In 1-day-old cultures K+ elicited a small albeit significant increase in [Ca2+]i while glutamate was completely without effect. In 7-day-old neurons both agents induced a large increase in [Ca2+].(ABSTRACT TRUNCATED AT 250 WORDS)


Prevalence of RAS oncogene mutation in head and neck carcinomas.

RAS genes encode for a protein (p21) known to play an important role in the regulation of normal signal transduction and cell growth. Activation of RAS genes have been strongly implicated in the pathogenesis of cancer in cell line studies, animal models and in human tumors. RAS genes have been shown to be mutated in 10 to 15% of human solid tumors but the frequency of mutation varies widely depending on the tumor type. The prevalence of RAS mutation has not been well-established in head and neck squamous cell carcinomas (SCC). The purpose of our study was to screen a relatively large number (50) SCC tumors using a gene amplification technique, the polymerase chain reaction (PCR). H-RAS gene mutation is identified by diagnostic restriction length polymorphism, created by introducing specific mismatched primers in the PCR. The first 20 tumors were also amplified and directly sequenced for K-RAS codon 12 and 13. Four of the 50 screened tumors were positive for H-RAS codon 12 mutation. All tumor DNA screened normal at codon 61 and the first 20 tumors were also normal at K-RAS codon 12 and 13. The prevalence of RAS mutations appears to be low in head and neck squamous cell carcinomas. Tumors positive for point mutation in the H-RAS gene revealed some unusual clinical characteristics.


Measurement of proliferative index in squamous cell carcinoma by flow cytometry.

Squamous cell carcinoma of the upper aerodigestive tract presents significant problems in determining appropriate treatment regimens. Staging aided by sophisticated investigations allows for planning of treatment, but there is a definite need for a specific and reproducible marker to quantify biological aggressiveness. For some classes of tumors the DNA ploidy of cells, as determined by flow cytometry, has shown good correlation to pathologic grading and prognosis. Using a mathematical model, it is possible to calculate the S-phase fraction, which is indicative of proliferative activity and may reflect tumor aggressiveness. However, this parameter is often difficult to determine reliably in squamous cell carcinoma. An optimal marker would be a measureable protein related to proliferation. An attempt was made to use flow cytometry to measure the nuclear enzyme topoisomerase II to assess proliferation in cultured cell lines. Although the antibody was specific to the extracted protein, constituents of the rabbit serum bound non-specifically throughout fixed cells. Further purification of this antibody preparation could realize its diagnostic potential. Antibody against proliferating cell nuclear antigen was more specific, resulting in good correlation flow cytometrically with the S-phase fraction of the cultured cell lines. The results obtained with these protein markers deems further investigation into their use as prognostic indicators. The protocol has been established to apply this technology to tumor samples and establish a meaningful parameter of biological behavior of tumors, upon which treatment regimens can be confidently based.


Aberrant crypts in human colonic mucosa: putative preneoplastic lesions.

Aberrant crypts are recognized in methylene blue-stained, unsectioned, colonic mucosa by their increased size, elliptical lumenal opening, thicker epithelial layer, and increased pericryptal region. Aberrant crypt foci in rodents are observed as early as 2 weeks and for at least 9 months after a single dose of carcinogen, have a distribution that parallels that of tumors, and have an increased number of aberrant crypts per focus with time after the carcinogen dose. The ability to quantify these lesions in the entire colon of rodents in less than an hour suggests that aberrant crypts may provide a highly efficient in vivo bioassay for colon carcinogens. Since aberrant crypt foci appear to be the earliest identifiable putative precursors of colon cancer, they represent lesions that can be characterized further for the earliest genetic and biochemical alterations. In F344 rats, we have demonstrated that aberrant crypts have multiple histochemically-detectable enzyme alterations. Using similar techniques, we were the first to demonstrate aberrant crypts in unsectioned human mucosa. After embedding and sectioning, these microscopic aberrant crypts resemble rare lesions described earlier in the literature after extensive serial sectioning. In rats and humans, aberrant crypts may be histologically normal or display varying degrees of dysplasia and histochemically-detectable altered enzyme activities. These putative, preneoplastic lesions should reveal early changes that precede colon cancer and ways to alter their progression.


Effect of psychotropic drugs on histamine H2-receptors in rat isolated uterus.

The effects of the monoamine oxidase inhibitors iproniazid, nialamide and phenelzine, the neuroleptics haloperidol and thioridazine, and the benzodiazepines diazepam and clonazepam on histamine H2-receptors were assessed on rat isolated uterus. The monoamine oxidase inhibitors showed a slight non-competitive antihistamine H2 activity, while diazepam and clonazepam were devoid of any action. Haloperidol and thioridazine inhibited in a dose-dependent manner the tonic component of KCl induced contraction, while thioridazine under the same conditions exhibited a slight antihistamine H2 activity. These data show that the drugs tested are devoid of or elicited only a slight antihistamine H2 activity at high non-therapeutic concentrations.


Comparison of oral-steroid sparing by high-dose and low-dose inhaled steroid in maintenance treatment of severe asthma.

It is not clear whether high doses of inhaled steroids have a greater sparing effect than low doses on the requirement for systemic steroids. In a randomised, double-blind, multicentre study, we compared the effects of high-dose (1500 micrograms/day) and low-dose (300 micrograms/day) inhaled beclomethasone dipropionate (BDP) in patients with severe asthma requiring a daily oral prednisolone dose of 10-40 mg. During a 3-month run-in period, we tried to achieve optimum asthma control by means of oral steroid and inhaled BDP 300 micrograms/day. The patients were then allocated to high-dose (n = 71) or low-dose (n = 72) treatment by an independent observer who took into account various prognostic factors. BDP was administered by means of an aerosol inhaler with a spacer device. The dose of systemic steroid was reduced as much as possible during the 6-month study period while keeping the peak expiratory flow (PEF) constant and asthma clinically stable. There was no difference between the low-dose and high-dose treatment groups in the mean reduction in oral prednisolone dose achieved by the end of the study (5.2[ SD 7.9] vs 5.0 [9.4] mg/day). The maximum response to inhaled steroid was seen, however, only after several months' therapy in both groups. There were no differences between the groups in use of on-demand beta-agonist inhalations or in asthma symptoms, and PEF values were constant throughout the study. Both doses of BDP were well tolerated. High doses of inhaled steroid offer no further benefit over low doses in the maintenance treatment of severe steroid-dependent asthma when the inhaled steroid is administered with a spacer device.


Bone turnover in malnourished children.

Pyridinoline (PYD) and deoxypyridinoline (DPD) are cross-linking aminoacids of collagen that are located mainly in bone and cartilage. When bone matrix is resorbed these cross-links are quantitatively excreted in the urine and therefore represent specific markers. We have measured the urinary excretion rate of PYD and DPD in 46 severely malnourished boys to assess their skeletal turnover and to relate this to their subsequent rate of growth. The children were aged 13 months (SD 6), and height-for-age was -3.6 (1.6) Z-score, and weight-for-height was -2.4 (0.8) Z-score. PYD excretion when malnourished and after "recovery" was 11.2 (4.6) nmol h-1m-2 and 32.2 (10.8) nmol h-1m-2 and DPD excretion was 2.6 (1.3) nmol h-1m-2 and 7.5 (3.0) nmol h-1m-2, respectively. The ratio of the two cross-links did not change with recovery. These data show that cartilage and bone turnover is much lower in the malnourished than in the recovered child. There was no difference in the degree of depression of turnover between the children with marasmus, marasmic-kwashiorkor, or kwashiorkor. The rate of height gain during recovery was significantly related to cross-link excretion, age, and weight-for-height on admission. These three factors accounted for 44% of the variance in the height velocity of the children. PYD and DPD excretion rate could be used to assess therapeutic interventions designed to alleviate stunting.


Comparison of saliva and serum for HIV surveillance in developing countries.

Saliva has been proposed as a non-invasive alternative to serum for HIV antibody testing. In a field study in Myanmar (formerly Burma), we evaluated such an alternative to identify the frequency of HIV infection in a surveillance programme of high-risk and low-risk sentinel groups. Duplicate vials of saliva and serum were collected from 479 high-risk and 1039 low-risk subjects. One vial of each pair was analysed blind in two laboratories, one in the USA and the other in Myanmar. The US laboratory followed WHO confirmatory strategy III with three different enzyme-linked immunosorbent assays (ELISAs), while the laboratory in Myanmar followed strategy I with one ELISA. Serum testing in the US was the gold standard. The Cambridge ELISA with saliva was a more effective surveillance tool (sensitivity 90.5%, specificity 99.5-100%) for describing the frequency of subjects with HIV antibodies than the serum ELISA supplied to Myanmar by WHO (95.9% and 98.3%, respectively). Saliva is recommended as a safe and effective alternative to serum for HIV antibody testing with ELISA in surveillance programmes in developing countries.


Lipoprotein(a) and accelerated coronary artery disease in cardiac transplant recipients.

High concentrations of serum lipoprotein(a) (Lp(a)) are associated with an increased risk of atherosclerotic vascular disease in the nontransplanted population. However, its relation with accelerated coronary artery disease (CAD) in cardiac transplant recipients has not been reported. We measured serum Lp(a) in 130 cardiac transplant recipients undergoing routine follow-up, which included annual coronary angiography. The median Lp(a) concentration in 33 patients with CAD was 71 mg/dl, which was significantly higher than the corresponding value of 22 mg/dL in the 97 patients without CAD (p = 0.0006). Multivariant analysis showed the serum Lp(a) value to be a higher significant risk factor for CAD irrespective of the other factors included in the regression analysis. Thus a high concentration of serum Lp(a) is an important, independent risk factor for the development of accelerated CAD in transplant recipients.


Fine-needle extrathoracic lymph-node aspiration in HIV-associated sputum-negative tuberculosis.

HIV-associated tuberculosis is increasingly seen in Zimbabwe and other developing countries. The clinical and radiological features are often atypical, and diagnostic confusion may arise when sputum smears are negative. Patients with suspected intrathoracic tuberculosis frequently have palpable extrathoracic lymph nodes. In this study, Ziehl-Neelsen staining of aspirates from extrathoracic lymph nodes revealed acid-fast bacilli in 20 (71%) of 28 patients with suspected tuberculosis, and 20 (87%) of 23 patients in whom a final diagnosis of tuberculosis was made Aspiration is simple, rapid, and cheap and may be of value in the diagnosis of tuberculosis, especially in developing countries with limited diagnostic and therapeutic resources.


Recognition of pneumonia by primary health care workers in Swaziland with a simple clinical algorithm.

In developing countries primary health care workers are being trained to manage and treat acute respiratory infections with a protocol developed by the WHO. We tested the ability of nurses and nursing assistants in Swaziland to recognise the signs and symptoms of pneumonia; with the results of a paediatrician's examination as "gold standard", sensitivities and specificities were calculated. Danger signs of stridor and abnormal sleepiness were poorly recognised (sensitivity 0-50%) by the health care workers, as was audible wheeze. Severe undernutrition, tachypnoea, and chest wall indrawing were well recognised. Overall, the recognition of pneumonia was good (sensitivity 71-83%, specificity 84-85%). These findings highlight topics for emphasis in training.


Oral and gastrointestinal manifestations of epidermolysis bullosa.

The mouth, oesophagus, and anus are often involved in dystrophic and junctional epidermolysis bullosa, but the frequency is unknown. Among 246 patients with epidermolysis bullosa, dysphagia developed in 76% of those with recessive dystrophic, in 20% of those with dominant dystrophic, in 15% of those with junctional, and in 2% of those with simplex forms. Lingual adhesions or microstomia occurred in dystrophic epidermolysis bullosa only, but were eight times more common in recessive than in dominant subtypes. These lesions are provoked by the trauma of eating and further reduce food intake, which exacerbates constipation caused by anal blisters and results in malnutrition. Management requires specialised multidisciplinary care.


Red blood cell transfusion in warm-type autoimmune haemolytic anaemia.

Blood transfusions are regarded as hazardous in patients with warm-type autoimmune haemolytic anaemia (AIHA) because of potential intensification of haemolysis and a presumed high incidence of alloimmunisation. We have retrospectively analysed data of 79 multitransfused patients (74 adults, 5 children) with detectable warm autoantibodies and transitory or persisting haemolytic anaemia. All patients had received blood transfusions on at least two occasions. Patients were reexamined at least twice within the first 6 months of transfusion (duration of follow-up 6 months-12 years). 53 patients had received blood transfusions because of decompensated AIHA, all of whom presented with detectable autoantibodies against red blood cells. None of these patients had transfusion-related alloimmunisation or a definite increase in haemolysis, even when the transfused red cells were serologically incompatible because of free serum autoantibodies. The other 26 patients had no signs of AIHA at presentation (negative direct and indirect antiglobulin test), but received blood transfusions for anaemia due to various other causes. 23 of these 26 patients went on to develop alloantibodies as well as autoantibodies upon transfusion, and 3 patients developed autoantibodies alone. Our findings do not support the generally accepted notion that transfusion therapy should be avoided in AIHA patients. Rather, they indicate that the incidence of alloimmunisation as well as adverse haemolytic transfusion reactions are less common in AIHA patients than in other multitransfused patients.


The extent of man from Vitruvius to Marfan.

It is frequently stated that patients with Marfan's syndrome have an arm span greater than height. This implies a characteristic different from the proportions in normal adult man, in whom span and height are often thought to be equal. Such equality of span and height, which allows man to be portrayed within a square, has been a widely held concept, immortalised by Leonardo da Vinci, that dates from the Roman Vitruvius. However, in the past two hundred years, anthropometry has shown that span exceeds height in 59-78% of normal adult white men. Thus not only is the classic concept of equality of span and height generally incorrect, but also a span greater than height cannot be considered characteristic of Marfan's syndrome. Paradoxically, in some affected individuals, Vitruvian equality of height and span may occur.


1993 revised classification system for HIV infection and expanded surveillance case definition for AIDS among adolescents and adults.

CDC has revised the classification system for HIV infection to emphasize the clinical importance of the CD4+ T-lymphocyte count in the categorization of HIV-related clinical conditions. This classification system replaces the system published by CDC in 1986 (1) and is primarily intended for use in public health practice. Consistent with the 1993 revised classification system, CDC has also expanded the AIDS surveillance case definition to include all HIV-infected persons who have < 200 CD4+ T-lymphocytes/microL, or a CD4+ T-lymphocyte percentage of total lymphocytes of < 14. This expansion includes the addition of three clinical conditions--pulmonary tuberculosis, recurrent pneumonia, and invasive cervical cancer--and retains the 23 clinical conditions in the AIDS surveillance case definition published in 1987 (2); it is to be used by all states for AIDS case reporting effective January 1, 1993.


[Comparison of long-term results of surgical and nonsurgical therapy in acute aortic dissection].

The early and long-term results of both surgical and nonsurgical therapy for 160 patients with acute aortic dissection in our institution were analyzed. Of the 83 patients with acute type A aortic dissection, 47 received surgery during acute stage, 10 received it in the chronic stage, and 26 received nonsurgical therapy. Of the 77 patients with acute type B aortic dissection, 27 received surgery during acute stage, 22 received it in the chronic stage, and 28 received nonsurgical therapy. Ten-year survival rates for patients receiving surgery during the acute stage were 62% for type A and 64% for type B dissection. This survival rate for type A patients was significantly higher than that for type A patients not receiving surgery, but the corresponding difference for type B patients was not significant. Present data indicate that immediate surgical intervention is indicated in patients with acute type A dissection, and elective operation in the subacute stage in patient with uncomplicated type B dissection following medical therapy in the acute stage.


[Problems in ulcer surgery after the introduction of H2-receptor antagonists].

Recently, antisecretory drugs such as H2-receptor antagonists (H2-RA) or proton pump inhibitor have been used for peptic ulcer patients widely in Japan. However, there are possibilities that long term administration of H2-RA might cause changes in intragastric environment. The present study was designed to clarify the changes of surgical treatment in Japan Surgical Society training hospitals, before and after introduction of H2-RA. Serum gastrin and antral G-cell number was measured after administration of H2-RA (1 mg/kg 14 days continuous infusion) in rat. Also, acid secretion and gastrin response stimulated by adrenalin (40 ng/kg.min) were measured in duodenal ulcer patients. 1) In the view of surgical treatment, elective operation highly decreased after the introduction of H2-receptor antagonists, and showed the increase of the rate of emergency operation up to 70%. 2) Hypergastrinemia and antral G cell hyperplasia were observed after administration of H2-RA in rats. 3) Acid secretion stimulated by adrenalin which is considered as antral G cell dependent, showed a higher response in H2-RA treated cases than in those untreated. 4) Antrectomy was carried out in 43.4% of the patients treated with H2-RA versus 18.9% to the patients untreated.


[Effect of gastric mucosal denervation on gastric carcinogenesis].

Numerous nerve fibers containing various neuropeptides are found in gastric mucosa. They play an important role not only in regulation of gastric secretion, motility and microcirculation but also in regeneration and differentiation of gastric mucosa. These nerve fibers are reduced in chronic atrophic gastritis which is considered a lesion closely related to carcinogenesis. We investigated the effect of gastric gastric mucosal denervation (vagotomy) on gastric carcinogenesis by using two experimental rat models in which chronic atrophic gastritis is induced by duodenogastric reflux. At first, following administration of MNNG, vagotomy with duodenogastric reflux enhanced gastric carcinogenesis compared to reflux only. At second, in the model of gastric remnant in which no carcinogenic agent was given, both B-I and B-II gastrectomy with vagotomy showed an increase of carcinoma and/or adenoma at the anastomotic site compared to those without vagotomy. Moreover, in vagotomized groups, there were an increase of labeling index of PCNA positive cells in gastric mucosa and a marked reduction of intramucosal neutral mucin in PAS-Alcian blue staining. These results indicate that the lack of gastric mucosal innervation not only induces the decrease of gastric mucosal cell function and cytoprotection but also enhances the increase of immature cell regeneration.


[Mutations of ras and p53 genes in human non-small cell lung cancer cell lines and their clinical significance].

We examined 77 non-small cell lung cancer (NSCLC) cell lines for mutations of 3 ras genes and p53 gene, and ras mutations were detected by designed RFLP assay generated by mismatched primers during the PCR step and p53 gene mutations were detected using SSCP analysis. The incidence of ras mutations were 27/77 (35%) while that of p53 gene mutations were 57/77 (74%). The incidence of ras mutations in cell lines with p53 mutations were not different from that without p53 mutations, suggesting that they occurred independently. Neither ras nor p53 mutations correlated with histologic subtype, disease extent, in vitro culture time nor prior therapy status. The patients whose cell lines had any ras mutations survived for shorter period of time not only among the patients who were treated with curative intent but among those treated with palliative treatment. The Cox proportional hazards model predicted the higher risk for patients with ras mutations but not those with p53 mutations. We conclude that ras and p53 mutations are frequent, apparently independent genetic alterations which play different roles in NSCLC and that this information should be utilized in surgical oncology in the near future.


[Problems in multidisciplinary treatment from the standpoint of clinical characteristics in human cancers].

We investigated the amplification and expression of oncogenes in human gastric and breast cancers. The biological malignancy of the cases with oncogene amplification/expression was higher than that of the cases without amplification/expression. Moreover, the case with amplification/expression of oncogene was found to be highly correlated with distant organ metastasis. In strongly suggests the necessity of postoperative adjuvant therapy. In addition, these data will have to be taken into consideration as deciding on the way of operation and therapy.


[Evaluation of immunoreactivity to erbB-2 protein as a marker of prognosis in bile duct carcinoma].

Recent studies of erbB-2 expression have shown that the erbB-2 oncoprotein correlated with poor prognosis of patients with breast cancer. Surgical treatment of the bile duct carcinoma is currently unsatisfactory. To evaluate erbB-2 oncoprotein as a marker of prognosis, we analyzed 68 bile duct carcinomas immunohistologically, using monoclonal antibody against erbB-2 oncoprotein, as well as clinicopathological data and outcome. High incidence of expression of erbB-2 oncoprotein was shown in bile duct carcinoma. Positive rates of erbB-2 oncoprotein correlated with stage of bile duct carcinoma. Survival of patients with erbB-2 expression cancer was shorter than those without erbB-2 expression cancer and erbB-2 expression has a prognostic value in bile duct carcinoma.


[Pathophysiology and management of patients with diabetes mellitus in gastrointestinal surgery, with special reference to hepatectomy and pancreaticoduodenectomy].

In the 235 patients with hepatectomy, 111 with pancreaticoduodenectomy (PD) and 547 with gastrectomy for the past 15 years and 5 months, we compared the incidence of postoperative complications between patients with diabetes mellitus (DM) and those without DM, and studied glucose metabolism and management of diabetic patients. DM was most frequently found at 27.7% in hepatectomy, especially 38.7% in cirrhotic patients, followed by 24.3% in PD and only 5.9% in gastrectomy. The incidence of postoperative complications was not different between DM and non-DM after gastrectomy and PD. It was significantly higher in DM than in non-DM after hepatectomy (38.5% vs. 11.8%), although there was no statistically significant difference in the cirrhotic patients with hepatectomy (34.9% vs. 23.5%). The studies on insulin metabolism preoperatively determined by oral glucose tolerance test and postoperative control of DM revealed that diabetic patients with hepatectomy, especially hepatogenous DM, had a significantly reduced insulin uptake in the liver and exogenous insulin resistance to glucose. After PD for diabetic patients, a long-term care of impaired pancreatic exocrine and endocrine functions was considered to be required, especially paying attention to lipid and zinc metabolism.


Characterization of soluble platelet guanylyl cyclase with peptide antibodies.

Soluble guanylyl cyclase partially purified from bovine and human platelets was characterized with antibodies raised against synthetic peptides corresponding to different sequences of the alpha 1- and beta 1-subunits of the bovine lung enzyme. On immunoblots, the platelet guanylyl cyclase was recognized by the four antisera used, with the exception of an antiserum against the C-terminus of the beta 1-subunit which did not react with the human platelet but with the bovine platelet beta 1-subunit. Furthermore the human platelet beta 1-subunit exhibited a slightly lower molecular mass than the bovine protein. The C-terminal antibodies precipitated native platelet and lung guanylyl cyclase activity. In contrast an antibody against a peptide out of the putative catalytic domain, which is highly conserved between all guanylyl cyclases sequenced so far, did not precipitate native guanylyl cyclase, although it recognized both subunits on immunoblots, suggesting that the respective amino acid sequence is located in an inner site of the protein.


[Pathophysiology and therapy of malignant neuroleptic syndrome].

Rigidity, hyperthermia, and elevated levels of creatine phosphokinase are the essential features of "neuroleptic malignant syndrome" (NMS), a clinical condition caused either by pharmacological treatment with dopamine receptor antagonists or by the withdrawal of dopamine receptor agonists. An acute dopaminergic transmission block in the basal ganglia and the hypothalamus is thought to be the pathophysiological mechanism of NMS. NMS, the "malignant dopamine depletion syndrome", and the akinetic Parkinsonian crisis may be considered identical conditions with regard to their presumptive pathogenesis. Centrally acting dopamine receptor agonists and the peripheral calcium antagonist, dantrolene, are the most common adjunctive pharmacological approaches to NMS, although their efficacy has been questioned. The motor symptoms and the autonomic nervous system disturbances of NMS may be related to a relative glutamatergic transmission excess, as a consequence of a dopaminergic block. Therefore, we recommend the application of N-methyl-D-aspartate (NMDA) receptor antagonists such as amantadine or memantine for the management of NMS. These drugs counteract excitatory amino acid neurotransmission and exhibit hypothermic and central muscle relaxant properties. However, identification and reversal of the causative event, such as neuroleptic drug therapy or change of anti-parkinsonian medication, remain the cornerstones for reducing the incidence and mortality of NMS.


Sulphasalazine-induced eosinophilic pneumonia.

Eosinophilic pneumonia and skin rash developed in a 21-yr-old man while taking sulphasalazine. After discontinuation of the drug and treatment with steroids the pulmonary infiltrates and rash resolved completely. A short review of the literature concerning sulphasalazine-induced lung disease is presented.


Gastroenterological Aspects of Cystic Fibrosis. Symposium proceedings. The Hague, The Netherlands, 10 April 1992.

Cystic fibrosis (CF) is the most common hereditary disease amongst Caucasians with a potentially lethal outcome. The basic defect is a dysregulation of the chloride channels leading to a relative dehydration of the luminal surface of the exocrine cells. The prognosis of CF patients is predominantly related to the progression of their pulmonary disease. This prognosis has improved dramatically during the last decennia and many patients live into adulthood. Gastrointestinal disturbances and, consequently, malabsorption of nutrients represent a major therapeutic problem in CF. A better understanding of the nutritional problems, including the treatment of pancreatic insufficiency, appears to contribute to an improved prognosis. However, the nutritional status of many patients is still poor and studies to optimise treatment are of paramount importance. In addition, with the increased life span, hepatobiliary complications will contribute to the morbidity of these patients and preventive treatment may be warranted. The aim of this Symposium was to improve our knowledge in this important field by considering the interesting and up-to-date contributions of many experts.


Protein kinase C and dopamine transport--1. Effects of amphetamine in vivo.

Rats, injected with small doses of amphetamine (0.03-0.1 mg/kg, i.p.), showed an increase in the soluble and a decrease in the activity of the particulate protein kinase C (PKC) in the striatum, while large doses of amphetamine (0.3-1.0 mg/kg) had the opposite effect of decreasing the soluble and increasing the particulate activity of PKC. These effects were manifested as a change in the Km for calcium, without an alteration in the Vmax. They were attenuated by pretreatment with benztropine, a dopamine (DA) uptake blocker and by alpha-methyl-p-tyrosine (alpha-MT), a DA synthesis inhibitor. The effects of 0.1 mg/kg amphetamine were insensitive to pretreatment with reserpine but were attenuated by the DA antagonists, SCH 23390 or sulpiride. These results suggest that the changes in activity of PKC induced by a small dose of amphetamine were mediated by an activation of DA autoreceptors, through an increase in the biophase concentration of DA at the synapse. In contrast, the effects of 1.0 mg/kg amphetamine on activity of PKC were attenuated by reserpine and by the DA agonists, LY 171555 or SKF 38393. They were, furthermore, potentiated by simultaneous treatment with sulpiride, which indicates that the two drugs act by different mechanisms. These results suggest that larger doses of amphetamine altered the activity of PKC at the DA transport site.


Protein kinase C and dopamine transport--2. Effects of amphetamine in vitro.

Synaptoneurosomes, incubated with amphetamine, showed a biphasic dose-response change in activity of PKC and release of DA. Activity of particulate PKC activity and release of DA were decreased at 0.01-10.00 nM, while activity of both was increased at 1-10 microM. The effects of 0.1 nM amphetamine were attenuated by pretreatment with N-ethoxy-carbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), a peptide-coupling agent which inactivated DA receptors. They persisted if the autoreceptors were protected with sulpiride or apomorphine (0.05 mg/kg), prior to the treatment with EEDQ. In these protected rats, the amphetamine-induced change in activity of PKC was attenuated by sulpiride. In contrast, the effects of the larger doses of amphetamine on the activity of PKC were not affected by pretreatment with EEDQ. Subcellular fractionation of tissues, incubated with 1 microM amphetamine, showed an increase in activity of particulate PKC, only in the synaptic plasma membrane fraction, while tissues incubated with reserpine showed a decrease in activity of particulate PKC, only in the vesicular fraction. Similar results were seen when synaptic plasma membrane or synaptic vesicles were incubated directly with amphetamine or reserpine. These findings suggest the presence of multiple metabolic pools of PKC: a reserpine-sensitive pool, present in synaptic vesicles and an amphetamine-sensitive pool, present in the synaptic plasma membrane. That the latter pool of PKC may be involved in the transport of DA was indicated by the good correlation between the ability of drugs to inhibit the activity of PKC and their ability to inhibit the amphetamine-induced release of DA.


Caroverine depresses the activity of cochlear glutamate receptors in guinea pigs: in vivo model for drug-induced neuroprotection?

With the aid of microiontophoretic techniques the action of caroverine, a quinoxaline-derivative, was tested on the receptor-linked depolarisation of the subsynaptic membrane of cochlear afferents. This membrane can be depolarised by the afferent transmitter agonist glutamate, mediated by NMDA and non-NMDA receptors and by acetylcholine, one of the different transmitter substances, released physiologically on axodendritic efferent synapses. Caroverine antagonized the membrane response to glutamate in an enduring but reversible manner. In contrast, the drug exhibited no effect on the depolarising action of acetylcholine. Therefore, the pharmacological profile of caroverine corresponded to the action of selective glutamate receptor antagonists. Since glutamate is likely to be the major mediator of neurotoxicity in the central nervous system, the selective glutamate-antagonism of caroverine is of particular interest, due to its putative neuroprotective competence. Caroverine is currently available clinically in some countries as a spasmolytic drug. Following these results it is proposed to test the drug for clinical efficacy in putatively glutamate-induced, excitotoxic disorders of the brain.


Nicotinic activity in the interpeduncular nucleus of the midbrain prolongs recovery from halothane anesthesia.

The influence of nicotinic transmission in the interpeduncular nucleus of the ventral midbrain on recovery from general anesthesia (3% halothane in oxygen) was assessed in rats. Immediately upon withdrawal of the anesthetic, nicotine (2 microliters, 10(-5) to 10(-1) M) was injected into the interpeduncular nucleus. Larger doses of nicotine (10(-2) and 10(-1) M) significantly (P < 0.05) prolonged the recovery of righting reflexes (to 371 +/- 55 and 362 +/- 67 sec, respectively, mean +/- SE), compared with injection of saline (187 +/- 19 sec). Prior intramuscular administration of the nicotinic antagonist, mecamylamine (2 mg/kg) significantly reduced the effect of 10(-2) M nicotine (to 211 +/- 43 sec). Injection of the nicotinic antagonist, hexamethonium (10(-1) M) led to a low mean recovery time (181 +/- 21 sec), not significantly different from control. Prolongation of recovery by 10(-2) M nicotine was not found to be significant when sites more dorsal to the interpeduncular nucleus were injected. An observed tendency for injection of nicotine to slow the post-anesthesia rate of breathing was not statistically significant and not correlated anatomically with the injection site in the midbrain. Increased release of acetylcholine has been shown previously to occur in the interpeduncular nucleus during anesthesia. The present results suggest that nicotinic activation of the interpeduncular nucleus facilitates or sums with the mechanisms in the brain that produce anesthesia under halothane.


Bethanechol-induced responses in mudpuppy parasympathetic neurons.

The effect of bethanechol on membrane potential and excitability was determined in mudpuppy parasympathetic postganglionic neurons. Bethanechol induced a large amplitude hyperpolarization, which was followed by a smaller amplitude depolarization, in 115 out of 135 cells tested. In approximately 20% of these cells, a brief depolarization preceded the hyperpolarization. During the bethanechol-induced hyperpolarization, the membrane input resistance decreased markedly, whereas the input resistance was increased during the subsequent depolarization. The hyperpolarization and depolarization were blocked by atropine and were unaffected by d-tubocurarine, thus, both appeared to be mediated by muscarinic receptors. The bethanechol-induced hyperpolarization was inhibited by the M2 muscarinic receptor antagonist AF-DX 116, whereas the bethanechol-induced depolarization was unaffected. Both a nonselective increase in membrane conductance and a decrease in membrane potassium conductance appeared to be involved in the generation of the bethanechol-induced depolarization. Evidence for the first mechanism was obtained in barium-treated cells in which bethanechol initiated a rapid onset depolarization, which was reversed at membrane potentials near 0 mV. Evidence for the second mechanism was obtained when the hyperpolarization was inhibited by AF-DX 116. In AF-DX 116-treated cells, the membrane input resistance was increased during most of the bethanechol-induced depolarization. Mudpuppy neurons initiate repetitive action potential activity in response to long depolarizing current pulses. Following application of bethanechol, with the hyperpolarization negated electrotonically, the number of action potentials produced by a depolarizing current pulse was greater than that produced prior to application of bethanechol. It is suggested that activation of muscarinic receptors on mudpuppy cardiac neurons influences multiple conductance systems and determines the excitability of these neurons.


The effects of sandostatin and somatostatin on nociceptive transmission in the dorsal horn of the rat spinal cord.

The role of somatostatin and a stable analogue, sandostatin (Octreotide), on the responses of spinal cord neurones in vivo was investigated in the rat. Electrical C-fibre stimulation was used as a model of acute nociception and the response to subcutaneous formalin was used as a model of longer term events. Intrathecal pre-treatment with sandostatin and somatostatin did not alter the C-fibre response, wind up or A beta responses of the cells. However, intrathecal pre-treatment with sandostatin and somatostatin inhibited both the first and second phases of the formalin response dose dependently. Thus, sandostatin (20 micrograms) and somatostatin (150 micrograms) inhibited the first phase (66 +/- 12% inhibition and 52 +/- 13% respectively) and second phase (91 +/- 2% inhibition and 39 +/- 16% inhibition respectively). The second phase of the formalin response was more sensitive to somatostatin and sandostatin than the first. Sandostatin was approximately 400 times more potent than somatostatin on the second phase of the response. Subcutaneous sandostatin (100 mg/kg) significantly inhibited both the first and second phase of the formalin response whereas the local peripheral administration of sandostatin (20 micrograms) only inhibited the second phase of the formalin response.


Cold weather injuries during peacetime military training.

Demographic data on 220 cold weather injuries seen over a 52-month period at the 67th Evacuation Hospital in Wuerzburg, Germany, was reviewed. Data were collected at the time of presentation and all diagnoses were made by a general/vascular surgeon. Statistics on age, gender, race, rank, unit, prior injury, use of tobacco products, classes on prevention, and activity at the time of injury were reviewed. Previously identified risk factors were confirmed except for tobacco use. There appeared to be no risk associated with gender or rank. Most injuries were sustained by soldiers performing low-risk activities for which no clear predisposing event could be ascribed. Prevention and early detection appear critical since injuries were not necessarily associated with specific actions or events.


Effect of induced molting on B cell and CT4 and CT8 T cell numbers in spleens and peripheral blood of White Leghorn hens.

Two trials were conducted to examine the possible effect of induced molting on splenic and peripheral blood T and B cells. Molting was achieved using shortened light exposure and a 14-day fast. Feed was removed on Day 0 and blood for analysis was removed on Days 3, 10, and 17 whereas spleens were removed on Days 4, 11, and 18. Fluorochrome-labeled anti-chicken CT4 and CT8 monoclonal antibodies were used to examine molting effects on chicken T cells and polyclonal anti-chicken immunoglobulin was used to detect chicken B cells. The labeled cell preparations were analyzed by flow cytometry. Molted hens had significantly decreased CT4+ peripheral blood T cells on Day 3 in both trials and on Day 10 in one trial. No effects on peripheral blood CT8+ T cells were observed. Splenic CT4+ T cells were decreased on Day 11 in one trial whereas splenic CT8+ T cells were significantly increased on Day 4 in two trials and on Day 11 in one trial. Peripheral blood and splenic B cells were largely unaffected in both trials. These results indicate that fasting to induce a molt does alter T lymphocyte subpopulations and these effects primarily occur early in the fasting procedure.


The turkey major histocompatibility complex: identification of class II genotypes by restriction fragment length polymorphism analysis of deoxyribonucleic acid.

Using a chicken Class II MHC clone in Northern blot analysis, tissue-specific expression of turkey Class II MHC genes was observed in the embryonic bursa of Fabricius as well as in the adult spleen. In contrast, there was no detectable expression in the embryonic liver, brain, or spleen. Southern blot analysis of BamHI-digested turkey DNA revealed two restriction fragment length polymorphism (RFLP) patterns that did not deviate significantly from single-gene Mendelian inheritance. Further analysis of PvuII-digested DNA from 325 turkeys showed four distinct RFLP patterns that segregated within the turkey lines studied. Because the chicken Class II MHC clone hybridized specifically to mRNA in immune-associated tissues, and because it identified polymorphisms among turkeys, the chicken clone is suggested to identify four turkey Class II MHC genotypes. The current study provides good evidence that RFLP analysis of DNA can be used as a means for molecular genotyping at the MHC in turkeys.


Distribution and amounts of taxol in different shoot parts of Taxus cuspidata.

Different fresh shoot parts of male and female plants of Taxus cuspidata were extracted and analysed for taxol concentration by high performance liquid chromatography (HPLC). Extracted parts included: young needles (first 10 top needle pairs of 30 cm long branches), old needles (last 10 needle pairs of 30 cm long branches), green bark, dark bark (with intense secondary growth), young wood (originally surrounded by green bark), wood (originally surrounded by dark bark), young stems (surrounded by green bark and devoid of needles), and mature male cones. Dichloromethane extracts were analysed by HPLC and diode array spectroscopy. Taxol identification was done by retention time, U.V. spectra, and spiking with an authentic taxol standard; 1H-NMR analysis was done for needle extracts. All parts except male cones had measurable amounts of taxol; no effect of plant sex on taxol levels of the plant parts analysed was observed. Results indicated that the bark accounted for almost all the taxol present in stems devoid of needles. Needles showed the highest levels of taxol (overall average of 0.035 +/- 0.006% of the extracted dry weight), significantly higher than those displayed by dark bark samples (0.012 +/- 0.001% of the extracted dry weight). Different needle post-harvesting procedures were evaluated in relation to taxol yields, 96 h dark incubation at -12 degrees C and 96 h dark incubation at 25 degrees C under vacuum gave taxol yields equivalent to those of freshly extracted samples. However, results obtained for 96 h dark incubation at 60 degrees C indicated some extent of taxol degradation.


Contractile effects of prostaglandin E2 in rat rectum: sensitivity to the prostaglandin antagonists diphloretin phosphate and SC 19220.

Prostaglandin E2 (PGE2) applied cumulatively (1 nM-1 microM) induced concentration-dependent tonic contractions in the longitudinal muscle of isolated rat rectum. The PGE2 effects were not altered by guanethidine (50 microM), whereas atropine (3 microM), guanethidine plus atropine or tetrodotoxin (0.1 microM) reduced them to an almost equal extent and increased the EC50 values for PGE2. The after-contractions following electrical stimulation were enhanced by PGE2 (10 nM) and inhibited by atropine. Diphloretin phosphate (DPP, 100 microM) shifted the regression lines for PGE2 to the right in both untreated and tetrodotoxin-treated preparations, and thereby increased the EC50 values. Slopes of the concentration-effect lines for PGE2 before and after DPP differed in the presence of tetrodotoxin. The regression line for PGE2 with SC 19220 (100 microM) in tetrodotoxin-treated preparations was shifted to the right in a parallel fashion. It is concluded that PGE2 exerted both a neural (cholinergic) and a smooth muscle effect. There may be a competitive antagonism between SC 19220 and PGE2 but the block by DPP may be nonselective.


Effects of EP-receptor subtype specific agonists and other prostanoids on adenylate cyclase activity of duodenal epithelial cells.

Rank order of agonist potency for activation of adenylate cyclase by the naturally occurring prostanoids PGE2, PGF2 alpha, PGD2, the stable PGI2 analogue iloprost, and the TXA2 mimetic U 46619, provides evidence for the existence of a distinct PGE-receptor on guinea-pig duodenal enterocytes. The PGE-receptor is likely to be of the EP2-subtype since the specific EP2-agonist 11-deoxy-PGE1 stimulated adenylate cyclase activity with a 20-fold higher potency than the EP1-agonist 17-phenyltrinor-PGE2 and the EP3-agonists MB 28767 and GR 63799. In addition, sulprostone (acting on both EP1- and EP3-receptors) was ineffective. Since the specific EP1-antagonist SC 19220 did not inhibit PGE2-stimulated adenylate cyclase activity, the involvement of EP1-receptors could be further excluded. The synthetic prostaglandin E-analogues misoprostol and nocloprost stimulated adenylate cyclase almost identically, though they were about 10-fold less potent than the natural PGE2.


[The effect of acute poisoning of rats with carbaryl on the transamination reactions involving alpha-ketoglutarate. Part I. Activity of plasma aminotransferases].

White male Wistar rats were poisoned orally with a single carbaryl dose 474 mg/kg (1/2 LD50). In the plasma of control animals and 2, 4, 24 and 72 hours after administration of the insecticide the activity of aminotransferase catalysing the reactions between alpha-ketoglutarate and six amino acids (cysteine, lysine, phenylalanine, leucine, asparagine and valine) was determined. Aminotransferases activity was expressed as the amount of glutamic acid developing during incubation of 1 cm3 of plasma for 1 hour. Glutamic acid was determined spectrophotometrically after chromatographic separation on paper. The results of the investigations demonstrated that acute intoxication with carbaryl caused a statistically significant increase in the activity of two aminotransferases in the plasma of experimental animals. A rise in the activity of the enzyme catalysing the reaction between alpha-ketoglutarate and asparagine ketoglutarate and lysine was observed.


NO news is good news.

A startlingly simple molecule unites neuroscience, physiology, and immunology, and revises scientists' understanding of how cells communicate and defend themselves.


Impaired long-term potentiation, spatial learning, and hippocampal development in fyn mutant mice.

Mice with mutations in four nonreceptor tyrosine kinase genes, fyn, src, yes, and abl, were used to study the role of these kinases in long-term potentiation (LTP) and in the relation of LTP to spatial learning and memory. All four kinases were expressed in the hippocampus. Mutations in src, yes, and abl did not interfere with either the induction or the maintenance of LTP. However, in fyn mutants, LTP was blunted even though synaptic transmission and two short-term forms of synaptic plasticity, paired-pulse facilitation and post-tetanic potentiation, were normal. In parallel with the blunting of LTP, fyn mutants showed impaired spatial learning, consistent with a functional link between LTP and learning. Although fyn is expressed at mature synapses, its lack of expression during development resulted in an increased number of granule cells in the dentate gyrus and of pyramidal cells in the CA3 region. Thus, a common tyrosine kinase pathway may regulate the growth of neurons in the developing hippocampus and the strength of synaptic plasticity in the mature hippocampus.


HLA-DPB typing using co-digestion of amplified fragments allows efficient identification of heterozygous genotypes.

Twenty-four alleles have been defined for HLA-DPB based on their second exon sequences. This paper describes a novel method, co-digested amplified fragment length polymorphisms (CAFLP), for assigning these alleles to heterozygous patients, as well as to homozygous cell lines. The method depends on co-digestion of amplified DNA by restriction endonucleases and separation of the resultant fragments with polyacrylamide gel electrophoresis. Co-digestion by selected restriction enzymes produces a set of readily discernible fragments that are unique for a given haplotype because the selected restriction sites occur in cis. Consequently, this method provides haplotype information not available from independent digests and allows all known heterozygous genotypes to be identified. Analysis of 103 trios of mother, father, and child, plus 120 normal caucasians, demonstrates the reliability and simplicity of this procedure. This simple typing method results in unambiguous assignment of all current HLA-DPB genotypes in random samples with a high proportion of heterozygous individuals.


HLA-DP polymorphism in northern Italian celiac patients.

The contribution of HLA-DP genes to celiac disease susceptibility has been investigated in 95 Italian patients, 41 with childhood and 54 with adult disease onset. Polymerase chain reaction amplification, sequence-specific oligonucleotide probe hybridization and restriction fragment length polymorphism analyses have been carried out. All celiac patients and 56 out of 128 random healthy controls were DQw2-positive. The frequency of the DPB1*0101 allele was significantly increased (pc = 0.002, relative risk 5.21) in patients with celiac disease (23.2%) compared to the whole panel of controls (5.5%), but not to the 56 controls bearing DQw2 (10.7%). No significant difference in the frequency of DPB1*0101 was found between celiac patients with pediatric (24.4%) or adult (22.2%) onset. The DPB1*0101 allele was associated with both the DR3-DQw2 and DR7-DQw2 haplotypes. Moreover, our study has not confirmed the association with DPB1*0402 and DPB1*0301 previously reported in celiac children from southern Italy. The linkage of the DPB1*0101 allele with the DQ locus and the observation that the DP but not the DQ association appears to be ethnically dependent strongly support a secondary role of DP molecules in celiac disease susceptibility.


Distribution of HLA antigens in Spanish Gypsies: a comparative study.

We have studied the HLA-class I and class II antigen distribution in a sample of 75 Spanish Gypsies and 74 Spanish non-Gypsies by serology, restriction fragment length polymorphism, and protein chain reaction and hybridization with allele-specific oligonucleotide probes. When both population samples are compared, we find that Gypsies have a statistically significantly higher frequency of A1, A11, B61, Cw6, DQ5 and haplotypes DR16 DQ5 Dw21 and DR14 DQ5 Dw9 DR52b. Frequency of A3, A29, B44, DR4, DQ2, DQ8 and haplotypes DR1 DQ5 and DR7 DQ2 DB17 DR53 are significantly lower in this ethnic group. The analysis of the serological data in the two populations demonstrates that Cw6 can be split into long Cw6 (Cw6.1) and short Cw6 (Cw6.2). Haplotype A1-Cw6-B61-DR14-DQ5 is the most characteristic in Gypsies, with a frequency of 13%. Estimation of the genetic distances shows that Spanish Gypsies are closer to Indian Caucasoid populations than to the Spanish non-Gypsy population. HLA data support the proposed historical origin of this ethnic group.


The effects of 2,3,5-(triglutathion-S-yl)hydroquinone on renal mitochondrial respiratory function in vivo and in vitro: possible role in cytotoxicity.

Administration of 2,3,5-(triglutathion-S-yl)hydroquinone [2,3,5-(triGSyl)HQ] to rats causes severe renal proximal tubular necrosis. Although the cellular target(s) for 2,3,5-(triGSyl)HQ is not known, substantial evidence implicates mitochondria as the primary cellular target for aliphatic S-conjugates. To determine whether mitochondria are targets for 2,3,5-(triGSyl)HQ, the in vivo and in vitro effects of this conjugate on rat renal mitochondria (RRM) were investigated. In vitro exposure of RRM to 2,3,5-(triGSyl)HQ inhibited site I-supported respiration to a much greater extent than site II-supported respiration. Inhibition of mitochondrial function, as manifested by decreases in the respiratory control ratios, were a consequence of significant elevations in state 4 respiration. Inhibition of constitutive gamma-GT activity with AT-125 had no effect on the ability of 2,3,5-(triGSyl)HQ to decrease mitochondrial function. The effects of 2,3,5-(triGSyl)HQ on mitochondrial function in vivo were subsequently assessed. Shortly (0.5-2.0 hr) following administration of 2,3,5-(triGSyl)HQ (20 mumol/kg, iv) to rats, a significant elevation of state 4 respiration was observed. Thereafter (4-16 hr) state 4 respiration returned to control values and state 3 respiration became significantly depressed. A total collapse in RRM function occurred by 24 hr. The effects of 2,3,5-(triGSyl)HQ on state 4 respiration preceded significant elevations in blood urea nitrogen, which occurred at 8 hr. However, pretreatment of animals with probenecid, an inhibitor of organic anion transport, caused a significant decrease in the 2,3,5-(triGSyl)HQ-mediated elevations in state 4 respiration at 1 hr, without preventing the subsequent development of renal necrosis. In contrast, AT-125, which protected animals from 2,3,5-(triGSyl)HQ-mediated nephrotoxicity, had no effect on the early (1 hr) elevations in state 4 respiration but did prevent the later (8 hr) decreases in state 3 respiration. The data suggest that the early elevation in state 4 respiration observed in vivo is unlikely to contribute to 2,3,5-(triGSyl)HQ-mediated nephrotoxicity. The relationship between the decrease in state 3 respiration seen at later time points and the subsequent development of toxicity require further study before a cause and effect relationship can be determined.