The treatment of depression: prescribing practices of primary care physicians and psychiatrists. Depression is one of the most common mental disorders treated by primary care physicians. Concern has been expressed that primary care physicians underutilize antidepressants and overutilize anxiolytics in their management of depressive disorders. Data from the 1980, 1985, and 1989 National Ambulatory Medical Care Surveys were used to examine the pharmacologic treatment provided by primary care physicians and psychiatrists during office visits with patients diagnosed as depressed. The number and proportion of these visits that included an antidepressant prescription or an anxiolytic prescription were determined. Primary care physicians and psychiatrists both prescribed antidepressants more commonly than other classes of psychotropic medications during visits that included a depression diagnosis. Compared with psychiatrists, primary care physicians more commonly prescribed antidepressants for depressive disorders (1980, 55% vs 33%; 1985, 59% vs 41%; 1989, 57% vs 45%). In 1989, benzodiazepines were prescribed in 16% of the primary care visits for depression. More than half of these visits (56%) also resulted in an antidepressant being prescribed. Primary care visits for depression tended to be slightly longer than other primary care visits, but only about half as long as patient visits with psychiatrists. The pharmacologic treatment of depression by primary care physicians may be better focused than previously assumed. Future research should examine the informal psychological treatment routinely provided by primary care physicians to patients with depressive disorders.


Opioid properties of some derivatives of pethidine based on tropane. The preparation of some tropane analogues of pethidine and its reversed ester, chiefly with preferred 3 alpha-m-hydroxyphenyl chair conformations, is described. The former were secured from tropan-3-one in a sequence of reactions involving cyanide attack, hydrolysis, Grignard attack and then rearrangements. The reversed ester was obtained by treating tropan-3-one with lithium phenyl, followed by acylation. Configurational and conformational assignments follow from NMR analysis. The antinociceptive potencies of these compounds in mice are reported, and discussed in relation to non-phenolic congeners and the 4-arylpiperidine moiety of morphine.


Analogues of triprolidine: structural influences upon antihistamine activity. The synthesis of some geometrical isomers related to triprolidine is reported. Previous configurational assignments, by UV and proton NMR, are validated by high field nuclear Overhauser enhancement methods and the isomeric purity of tested E- and Z-isomers was greater than 99.5% as assessed by an HPLC method developed for these compounds. Affinity constants for triprolidine (E and Z) in guinea-pig ileum showed a potency ratio of approximately 600 whereas at cerebellar sites this ratio was only approximately 100, suggesting that the H1 receptor in these two tissues may not be identical. In-vivo tests using a lethal dose of compound 48/80 (a potent histamine-releasing agent) demonstrated that triprolidine itself was the most active compound to protect the animal among all the isomeric compounds tested: in all isomeric pairs the E-configuration possessed superior activity over Z. The disposition of the aryl groups in these geometrically constrained compounds mimics that seen in the structurally related chiral pheniramines which are sp3 hybridized and whose absolute stereochemistry is known.


The plasma disposition and renal elimination of digoxin-specific Fab fragments and digoxin in the rabbit. Administering digoxin-specific antibody fragments (DSFab, 1.9 mg kg-1, i.v.) to rabbits 1 h after digoxin (15 micrograms kg-1 or 12.5 microCi kg-1, i.v.) produced a redistribution of digoxin associated with a 5-fold elevation in total plasma concentration and 36-86% reductions in elimination half-life, apparent volume of distribution at steady-state and total body clearance (CLT). Renal clearance (CLR) was also reduced (54%), but urinary digoxin excretion was increased by one-third (35% vs 25%). This apparent anomaly is due to the large rise in total plasma digoxin concentration with a consequent increase in the area under the plasma concentration curve (AUC). The AUC, which is the denominator term in calculating CLR (and CLT), was increased to a greater extent than urinary digoxin excretion (numerator term in calculating CLR) so that an overall reduction in CLR occurred. The initial presence of digoxin appeared to alter the distribution of DSFab, since their plasma concentrations were markedly higher when the antibody was given after the hapten. The digoxin also reduced (from 3 to 1%) the amount of detectable DSFab in the urine.


Binding potency of paroxetine analogues for the 5-hydroxytryptamine uptake complex. The in-vitro inhibition constants (Ki) of 14 structural analogues of the potent 5-hydroxytryptamine (5-HT)-uptake inhibitor paroxetine were determined to assess the structure-affinity relationship of these derivatives. A goal of these studies was to determine those positions on paroxetine which could be derivatized without significantly decreasing the affinity of the drug for the binding site, so that radiolabels such as [18F]fluoroalkyl groups might be appended for future in-vivo imaging studies of the 5-HT uptake system. Using the methyl moiety as a steric probe for these studies, it was found that the rank order of potency of various methyl-substituted paroxetine analogues for inhibiting the binding of [3H]paroxetine to the 5-HT re-uptake site was: 4'-approximately equal to 3'-approximately equal to 2''- > 2'-approximately equal to 1- > 5''- > 6''-methyl. The in-vitro equipotent molar ratios (EPMR, Ki(analogue)/Ki(paroxetine)) of the analogues were determined, and the EPMRs of the 4'-, 3'-, and 2''-methyl derivatives were 1.9, 2.2 and 2.2, respectively. The 4'- and 2''-fluoromethyl and -fluoroethyl analogues were synthesized, and the EPMRs of the 4'- and 2''-fluoromethyl derivatives were determined to be 2.0 and 3.5, and those of the 4'- and 2''-fluoroethyl analogues were 5.2 and 6.2, respectively. The 2''-fluoromethyl analogue was unstable in aqueous solutions, and it is not a promising ligand for in-vivo studies.(ABSTRACT TRUNCATED AT 250 WORDS)


Plasma protein binding of quinine: binding to human serum albumin, alpha 1-acid glycoprotein and plasma from patients with malaria. The binding of quinine to human serum albumin (HSA), alpha 1-acid glycoprotein (AAG) and plasma obtained from healthy subjects (10 caucasians and 15 Thais) and from Thai patients with falciparum malaria (n = 20) has been investigated. In healthy volunteers, plasma protein binding expressed as the percentage of unbound quinine was 7.9-31.0% (69-92.1% bound). The mean percentage of unbound quinine found with essentially fatty acid-free HSA (40 g L-1) was 65.4 +/- 1.5% (mean +/- s.d.) and was comparable with the value (66.3 +/- 3.8%, mean +/- s.d.) for Fraction V HSA (40 g L-1). This suggests that fatty acids do not influence the plasma protein binding of quinine. Binding of quinine to 0.7 g L-1 AAG was high (mean unbound 61.0 +/- 5.0%), indicating that quinine is bound primarily to AAG and albumin, although other plasma proteins such as lipoproteins may be involved. The mean percentage of unbound quinine was slightly less in caucasians (14.8 +/- 6.7% unbound), compared with healthy Thai subjects (17.0 +/- 6.7% unbound). The higher binding of quinine in caucasian subjects was associated with a higher plasma AAG concentration observed in caucasians. Mean percentage of unbound quinine was significantly lower in Thai patients with malaria (10.9 +/- 4.0%) than in the healthy Thai subjects. The increase in the extent of quinine binding corresponded with the increase in the acute-phase reactant protein, AAG in the patients with malaria. Overall, when the data were combined there was a significant correlation (r = 0.846, P < 0.005) between the binding ratio (bound/unbound) of quinine and the plasma AAG concentration.(ABSTRACT TRUNCATED AT 250 WORDS)


A rapid filtration assay for the glycine binding site on the NMDA receptor in rat cortical membranes using [3H]dichlorokynurenic acid. A filtration binding assay using [3H]dichlorokynurenic acid to label the glycine binding site on the N-methyl-D-aspartic acid receptor has been evaluated on rat cortical membranes. This ligand binds to a single population of binding sites following mass action kinetics with a KD of 29 nM and a capacity of 5.73 pmol (mg protein)-1. The pharmacological specificity of the binding site is identical to that previously reported for this binding site using [3H]glycine as a radioligand. Agonists showed lower affinity and antagonists higher affinity when [3H]dichlorokynurenic acid was used compared with [3H]glycine. The higher affinity of [3H]dichlorokynurenic acid compared with [3H]glycine make it the more suitable compound with which to label the glycine site.


Comparison of the effects of cromakalim in trachea isolated from normal and albumin-sensitive guinea-pigs. The effects of the K(+)-channel activator, cromakalim, on spontaneous tone and constrictor responses to vagal stimulation or acetylcholine were compared in trachea isolated from groups of guinea-pigs that were: untreated; sensitized and chronically exposed to inhaled albumin; or sham sensitized. Responses were assessed as changes in intraluminal pressure in the isolated, Krebs-filled trachea, increases and decreases in intraluminal pressure directly reflecting constriction and dilatation, respectively. Cromakalim reduced resting intraluminal pressure in normal trachea but in sensitized trachea mixed effects occurred, many preparations exhibiting increases in intraluminal pressure, particularly at lower concentrations of cromakalim. Cromakalim attenuated the frequency-dependent increases in intraluminal pressure evoked by stimulation of the vagus nerve in a concentration-dependent manner and to a similar degree in trachea from each of the three groups tested. The degree of attenuation was similar in the absence and presence of the cyclo-oxygenase inhibitor flurbiprofen. In untreated trachea, responses to a range of concentrations of applied acetylcholine were attenuated by cromakalim. In sensitized trachea the response to the lowest concentration of applied acetylcholine was attenuated by cromakalim but responses to higher concentrations of were unaffected. The results indicate that the direct relaxant effect of cromakalim is altered in sensitized trachea, which may indicate abnormal K(+)-channel behaviour in the smooth muscle cell membrane. Attenuation by cromakalim of vagal responses occurs in both normal and sensitized trachea, due chiefly to a pre-junctional effect on cholinergic neurotransmission which is independent of the generation of cyclo-oxygenase products.


Receptors involved in the modulation of 5-hydroxytryptamine release in bovine cerebral arteries. The uptake of [3H]5-hydroxytryptamine (5-HT) in bovine cerebral arteries was reduced by cocaine (1 microM), ouabain (100 microM), pretreatment with 6-hydroxydopamine (6-OHDA) (1.46 mM, 10 min) and metitepine (1 microM). Electrically-stimulated tritium release was decreased by tetrodotoxin (0.8 microM), Ca-free medium, denervation with 6-OHDA (1.46 mM, 10 min), 5-HT (10 microM), noradrenaline (1 microM) and the agonist of alpha 2-adrenoceptors B-HT 920 (0.1 and 1 microM), enhanced by metitepine (1 microM, antagonists of presynaptic 5-HT1 receptors) and rauwolscine (1 microM, antagonist at alpha 2-adrenoceptors, and also of 5-HT1D receptors) and not affected by ketanserin (1 microM, antagonist of 5-HT2 receptors), methysergide (0.1 microM, antagonist of 5-HT1 and 5-HT2 receptors) and phentolamine (1 and 3 microM antagonist of alpha-adrenoceptors and less potent of 5-HT1 receptors). The inhibitory action of 10 microM 5-HT was partially reversed by phentolamine (3 microM) and cocaine (1 microM) and completely reversed by both metitepine (1 microM) and rauwolscine (1 microM). Ketanserin (1 microM), methysergide (0.1 microM) or phentolamine (1 microM) had no effect. Rauwolscine (1 microM) antagonized the inhibition induced by both noradrenaline (1 microM) and B-HT 920 (0.1 and 1 microM). 5-HT induced tritium release which was inhibited by cocaine (an antagonist of 5-HT3 receptors) and denervation with 6-OHDA.(ABSTRACT TRUNCATED AT 250 WORDS)


Cardiovascular effects of LAS 30538, a new vascular selective Ca(2+)-channel blocker. A new compound, 1-[2-(2,6-dimethylphenoxy)ethyl]-alpha,alpha-bis-(p-fluorphenyl)-4 -piperidine methanol (LAS 30538), was found to have potent vasodilator effects. Its vasorelaxant activity was demonstrated in rat perfused hindlimbs contracted with 80 mM K+, having an IC50 value of 40 nM. In conscious spontaneously hypertensive rats, LAS 30538 administered orally, caused dose-dependent sustained falls in systolic blood pressure with an ED30 value of 11 mg kg-1. In pithed rats, LAS 30538, strongly inhibited vasoconstriction induced by the alpha 2-adrenoceptor agonist B-HT 933 and the calcium agonist compound Bay K8644 with ED50 values of 4 mg kg-1 p.o. and 1.3 mg kg-1 i.v., respectively. Results from electrophysiological studies carried out using guinea-pig papillary muscles partially depolarized by 22 mM K+ are consistent with LAS 30538 acting as a Ca(2+)-channel blocker. When compared with verapamil, in guinea-pig and rabbit isolated heart preparations, LAS 30538 caused less cardiodepression and bradycardia. The results suggest that LAS 30538 may have some advantages over other Ca(2+)-channel blockers such as verapamil in causing less myocardial depression for a given level of vasodilatation.


The interaction between noradrenaline and ATP upon polyphosphoinositide metabolism and contraction in tail arteries from normo- and hypertensive rats. The effects of, and interaction between, noradrenaline and alpha,beta-methylene ATP upon polyphosphoinositide (PPI) breakdown, investigated by measuring the accumulation of inositol phosphates, and contraction, were studied in tail arteries from normo- (WKY) and spontaneously-hypertensive (SHR) rats. Noradrenaline (10(-7)-10(-3) M) evoked a prazosin (10(-6) M)-sensitive, concentration-dependent increase in total inositol phosphate accumulation in both WKY and SHR rats. No significant differences were observed in either the maximal response or in the concentration range over which noradrenaline evoked this response, between these two populations. Noradrenaline (5 x 10(-7)-5 x 10(-5) M) evoked a concentration-dependent contraction of arteries from both SHR and WKY rats. The responses to noradrenaline were about 2-fold greater at all effective concentrations of noradrenaline in SHR compared with WKY rats. alpha,beta-Methylene ATP (10(-6) M) did not alter noradrenaline-stimulated total inositol phosphate accumulation, in arteries from either SHR or WKY rats, measured either as the maximal response or as the EC50. alpha,beta-Methylene ATP (5 x 10(-6) M), by itself, evoked a contractile response, which was quantitatively similar in SHR and WKY rats, and was additive with the contractile responses to noradrenaline (5 x 10(-7)-5 x 10(-5) M). The maximum response produced by a combination of noradrenaline and alpha,beta-Methylene ATP was quantitatively similar to that produced by noradrenaline alone. No evidence of synergism between alpha,beta-Methylene ATP and noradrenaline upon contraction was observed.(ABSTRACT TRUNCATED AT 250 WORDS)


In-vivo pharmacological studies of 2-N-carboxamidinonormianserin, a histamine and 5-hydroxytryptamine antagonist lacking central effects. The in-vivo pharmacological properties have been examined of FCC5 (2-N-carboxamidino-1,2,-3, 4, 10, 14b-hexahydrodibenzo (c.f.) pyrazino (1, 2-alpha)azepine hydrochloride), a guanidino analogue of mianserin. FCC5 (30-100 micrograms kg-1, i.v.) caused long-lasting (> 1 h) attenuation of histamine- and 5-hydroxytryptamine (5-HT)-induced bronchoconstriction in the anaesthetized guinea-pig. FCC5 (< or = 1 mg kg-1, i.v.) had no effect on submaximal bronchoconstrictor responses caused by i.v. acetylcholine or the thromboxane A2-mimetic U46619 ((15S)-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5Z,13E-dienoic acid). The pressor effects of 5-HT in anaesthetized and pithed rats were inhibited by FCC5 (0.3-1.0 mg kg-1, i.v.). Higher doses of FCC5 (3 mg kg-1, i.v.) reduced bradycardia and depressor responses to 5-HT in anaesthetized rats. In anaesthetized cats and rats and also pithed rats, FCC5 (0.1-1.0 mg kg-1, i.v.) caused sympathomimetic effects as demonstrated by pressor responses and tachycardia. FCC5 (0.1-0.3 mg kg-1, i.v.) inhibited pressor responses to tyramine whereas those to noradrenaline and sympathetic nerve stimulation were potentiated. Oedema in the rat paw caused by intraplantar 5-HT was inhibited by FCC5 (ID50 0.76 mg kg-1, i.p.; and 2.7 mg kg-1, p.o.). In decerebrate rats which had been spinalized at T6-8, fenfluramine-induced facilitation of the flexor reflex of the anterior tibialis muscle was inhibited by mianserin (ID50 0.36 mg kg-1, i.p.) but not by FCC5 (< or = 3 mg kg-1, i.p.).(ABSTRACT TRUNCATED AT 250 WORDS)


The effect of serum Ca2+ compensation on the efficacy of chlorpropamide in alloxan-diabetic rabbits. A decrease in serum Ca2+ concentration was observed in alloxan-diabetic rabbits, with recovery of serum Ca2+ levels achieved following insulin therapy. This suggested that the diabetic condition directly relates to the decrease in serum Ca2+ levels. The efficacy of chlorpropamide in alloxan-diabetic rabbits after intravenous injection (150 mumol kg-1) was less than that in normal rabbits as measured by the serum insulin levels, and the efficacy did not change when the dose was increased. However, compensation of serum Ca2+ levels in alloxan-diabetic rabbits caused an increase in the efficacy of chlorpropamide observed as an increase in serum insulin and a decrease in serum glucose levels.


The calcium channel blocker LAS 30538, unlike nifedipine, verapamil, diltiazem or flunarizine, potently inhibits insulin secretion in-vivo in rats and dogs. The effects of a novel calcium channel blocker, LAS 30538 (1-[2-(2,6-dimethylphenoxy)ethyl]-alpha,alpha-bis-(p-fluorophenyl)-4- piperidine methanol), were studied on glucose tolerance and insulin secretion in rats and dogs in-vitro and in-vivo. Some comparisons were made with nifedipine, verapamil, diltiazem, flunarizine, diazoxide, cromakalim and minoxidil. LAS 30538, like a number of calcium channel blockers, was found to inhibit insulin secretion in-vitro, but was 1000-fold more potent than verapamil or diltiazem in this respect. LAS 30538 differed from the other calcium channel blockers studied in that it also potently inhibited insulin secretion and impaired glucose tolerance in-vivo. The evidence that LAS 30538 is more potent than diazoxide as a hyperglycaemic agent in-vivo suggests that this could be a useful drug for the treatment of hyperinsulinaemia in man.


Effect of mammalian lignans on fMLP-induced oxidative bursts in human polymorphonuclear leucocytes. We examined the effects of mammalian lignans, enterolactone, prestegane B and 2,3-dibenzylbutane-1,4-diol (DBB) on superoxide production and luminol-dependent chemiluminescence (LCL) response in human polymorphonuclear leucocytes (PMNs). The three lignans had no direct effect on the responses of human PMNs. DBB and prestegane B enhanced the superoxide production and LCL response induced by formylmethionyl-leucyl-phenylalanine (fMLP), but enterolactone inhibited fMLP-induced effects. The effects of DBB were stronger than those of prestegane B and the effects of DBB were inhibited by bromophenacyl bromide, mepacrine, N-(6-aminophenyl)-5-chloro-1-naphthalene, sulphonamide and trifluoroperazine, but not by gossypol, nordihydroguaretic acid, indomethacin, staurosporine, 1-(5-isoquinolinesulphonyl)-2-methylpiperazine dihydrochloride or (R,S)-2-methoxy-3-(octadecyl-carbamoyloxy)-propyl-2-(2-thiazoli o)-ethylphosphate. These results suggest that DBB primes the responses of human PMNs, and the priming effect is caused by the activation of phospholipase A2--and Ca(2+)-calmodulin-pathways, but not by the activation of lipoxygenase, cyclo-oxygenase and protein kinase C or by the release of platelet activating factor.


Effects of cupric chloride and tamrabhasma, a traditional Indian preparation of copper, on eicosanoid production by human gastric and colonic mucosa. Tamrabhasma is a traditional copper oxide-containing Indian preparation which has anti-ulcer activity. We have studied the effect of tamrabhasma and CuCl2.2H2O on prostaglandin formation by human gastric and colonic mucosa and submucosa, as prostaglandins have mucosal protective activity, and their release may contribute to the anti-ulcer effect. With the gastric mucosa, tamrabhasma 10 micrograms mL-1, but not 0.1 or 1 microgram mL-1, increased prostaglandin E (PGE) concentration by 38% (P < 0.05), with little or no effect on 6-keto-PGF1 alpha, thromboxane B2 or the leukotriene (LT) LTC4/LTD4. CuCl2.2H2O (10 micrograms mL-1) increased 6-keto-PGF1 alpha by 46% (P < 0.05), but 0.1, 1, 50 and 250 micrograms mL-1 did not significantly affect any of the prostanoids, and only the highest concentration reduced the amount of LTC4/LTD4. In the colon mucosa, tamrabhasma (0.1-10 micrograms mL-1) or CuCl2.2H2O (10-50 micrograms mL-1) increased all the prostanoids and this effect was greater than in the gastric mucosa but there was no significant change in LTC4/LTD4. CuCl2.2H2O showed a bell-shaped concentration-effect curve, with no significant effect at lower and higher amounts. Indomethacin (0.1-10 micrograms mL-1) caused a concentration-dependent reduction in the prostanoid amounts. The effect of tamrabhasma was probably not only due to the presence of Cu2+, as tamrabhasma was more effective than CuCl2.2H2O alone; in addition the solubility of CuO is very low. Increased prostanoid levels might explain, at least partly, the gastric mucosal protection by tamrabhasma. The results in the colon, however, raise the possibility that tamrabhasma should be examined for the treatment of inflammatory bowel disease.


Microsomal formation of N-benzyl-4-hydroxymethylaniline from N-benzyl-4-methylaniline. The in-vitro metabolism of N-benzyl-4-methylaniline was re-examined using male hamster and rabbit hepatic microsomes; both species generated N-benzyl-4-hydroxymethylaniline, confirmed by TLC and HPLC, comparison with authentic compound. Further confirmation of the formation of this metabolite was achieved by use of a rapid scan UV detector.


Digoxin-specific Fab fragments impair renal function in the rabbit. A 2 mg kg-1 intravenous bolus dose of digoxin-specific Fab fragments produced a 28% reduction in creatinine clearance in rabbits after 24 h. Urine output was reduced, while plasma and urinary creatinine concentrations were unaffected and increased, respectively. By 5 days the creatinine clearance had returned to normal. The fractional excretion of Na+ was nearly halved, indicating that the tubular reabsorption of Na+ increased to compensate for the reduced glomerular filtration rate, suggesting that tubular (as opposed to glomerular) function was not impaired.


Non-Michaelis-Menten type hepatic uptake of liposomes in the rat. The objective of this study was to verify the methodology for measuring uptake clearance of liposomes and to characterize kinetically the saturable hepatic uptake of liposomes-through phagocytosis. The correction of vascular space was important in the evaluation of hepatic uptake. The efflux of liposomes from liver was shown to be negligible, by a repeated dose study, and thus, hepatic clearance can be obtained by the hepatic uptake divided by the area under the blood concentration-time curve (AUC). The determinant parameter which describes the saturability of uptake clearance of liposomes, independent of infusion rate, was investigated, using the data of an in-vivo constant infusion study, where infusion rate-dependent saturable hepatic clearance was observed. The mean blood concentration failed to obtain an infusion rate-independent function. On the other hand, the AUC could explain the saturability of hepatic clearance for every infusion rate by a unique relationship. The hepatic uptake amount could also explain this saturability, independent of infusion rate. These kinetic characteristics are inconsistent with Michaelis-Menten type kinetics, therefore a new model is required to describe the saturable hepatic clearance in the disposition of liposomes.


A novel prodrug of salicylic acid, salicylic acid-glycylglycine conjugate, utilizing the hydrolysis in rabbit intestinal microorganisms. The hydrolysis of salicylic acid-glycylglycine conjugate (salicyl-glycylglycine) following oral, intravenous, intracaecal and rectal administration (434, 72, 36 and 36 mumol kg-1, respectively: equivalent to salicylic acid) was examined in rabbits to develop a novel prodrug of salicylic acid. Salicylic acid was detected in the blood 2 h after oral administration of salicyl-glycylglycine and it reached a maximum level (55.6 micrograms mL-1) at 15 h, whereas a small amount of salicyl-glycylglycine was found in the blood. In contrast, unchanged salicyl-glycylglycine was found mainly in the blood following its intravenous administration, suggesting the involvement of presystemic deconjugation in the hydrolysis of salicyl-glycylglycine. Immediate and very extensive salicyclic acid formation in the caecum was observed following intracaecal administration of salicyl-glycylglycine, suggesting that the intestinal microorganisms were responsible for the biotransformation of this compound. In-vitro incubation of salicyl-glycylglycine with caecal content showed that salicyl-glycylglycine was hydrolysed efficiently in the caecum. Consequently, the blood concentration of salicylic acid was prolonged extensively following rectal administration of salicyl-glycylglycine, indicating the usefulness of salicyl-glycylglycine as a prodrug of salicylic acid.


Improvement of intestinal absorption of thyrotropin-releasing hormone by chemical modification with lauric acid. Intestinal absorption of 125I-labelled lauryl thyrotropin-releasing hormone (Lau-TRH), a novel lipophilic derivative of TRH, was examined by rat in-situ closed intestinal loops. At a dose of 1 mumol per rat into the small intestine, a significant increase in percent of dose in plasma radioactivity of Lau-TRH was observed in comparison with that of TRH. A dose-dependent decrease in percent of dose in plasma radioactivity of TRH was noted, suggesting a saturable process of TRH transport. In contrast, the percent of dose in plasma radioactivity of Lau-TRH increased with increasing dose of Lau-TRH. The stability of TRH and Lau-TRH was studied in plasma and rat small intestinal homogenates. Lau-TRH was more stable than TRH in rat plasma. These results suggest that chemical modification of TRH with lauric acid may not only increase the lipophilicity of TRH but also reduce the degradation of TRH, resulting in the increased plasma radioactivity of TRH. On the other hand, Lau-TRH was gradually converted to TRH in the intestinal mucosal homogenate. These findings indicate that chemical modification of TRH with lauric acid might be a useful approach for improving the intestinal absorption of this peptide.


The pH dependent uptake of enoxacin by rat intestinal brush-border membrane vesicles. The mechanism of the intestinal transport of enoxacin, an orally active fluoroquinolone antibiotic, has been investigated using brush-border membrane vesicles isolated from rat small intestine. The initial rate and time-course of enoxacin uptake were considerably dependent upon the medium pH (pH 5.5 greater than pH 7.5) and upon the percent ionization of the carboxyl group (pKa 6.2, anionic charge), namely, the degree of uptake of cationic form was higher than that of the zwitterionic form. There was evidence of transport into the intravesicular space as shown by the effect of extravesicular medium osmolarity on enoxacin uptake at steady state (30 min). This transport across the brush-border membrane was stimulated by the valinomycin-induced K(+)-diffusion potential (interior negative) and an outward H(+)-diffusion potential. Furthermore, changing the pH of the medium from 5.5 to 7.5 significantly decreased the effect of valinomycin-induced K(+)-diffusion potential on the enoxacin uptake. These results suggest that the uptake behaviour of the cationic form of enoxacin plays an important role in the intestinal absorption process of enoxacin.


Alpha 1-adrenoceptor subtypes in bovine prostate. The object of this study was to examine the existence and characteristics of alpha 1-adrenoceptor subtypes in the bovine prostate using the radioligand binding assay method. [3H]Prazosin was used as the radioligand and its binding sites in bovine prostate were classified into two subtypes. One subtype showed a high affinity (alpha 1High, Kd: 101.1 pM and Bmax: 11.8 fmol (mg protein)-1) and the other had a low affinity (alpha 1Low, Kd: 3371.4 pM and Bmax: 50.5 fmol (mg protein)-1). Although the same pKi values of clorethylclonidine, p-aminoclonidine, benoxathian and dibenamine to both alpha 1High and alpha 1Low binding sites in bovine prostate tissue were observed, other alpha 1 antagonists used in this study had different pKi values for the two alpha 1-adrenoceptor subtypes. The existence and binding characteristics of alpha 1-adrenoceptor subtypes in bovine prostate were clarified. It is possible that agents selective for one site may contribute to the development of better drugs for the treatment of bladder outlet obstructions of men with benign prostatic hyperplasia.


Inhibitory effects of tiamulin on contractile and electrical responses in isolated thoracic aorta and cardiac muscle of guinea-pigs. The inhibitory effect of tiamulin, an antibiotic produced by Pleurotus mutilis, on contractile and electrical responses in isolated thoracic aorta and cardiac muscle of guinea-pigs was studied. In the thoracic aorta, tiamulin with an IC50 of 9.7 x 10(-6) M inhibited sustained contractions induced by isosmotically added 60 mM KCl. The inhibitory effect of tiamulin on a Ca(2+)-induced contraction in a depolarized muscle was competitively antagonized by raising external Ca2+ concentration. Bay K 8644 (10(-7) M) antagonized tiamulin's inhibition of the Ca(2+)-induced contraction. Tiamulin (2 x 10(-5) M) decreased the elevated cytoplasmic Ca2+ level measured by the fura 2 AM method in the depolarized muscle. In high K(+)-isoprenaline-treated left atria, tiamulin (2 x 10(-5)-2 x 10(-4) M) produced negative inotropic effects. On the other hand in the membrane action potential of papillary muscles, tiamulin (2 x 10(-6)-2 x 10(-4) M) produced decreases in action potential and durations and 2 x 10(-4) M tiamulin depressed the slow response action potential in depolarized muscles. Tiamulin produced prolongations of the PR interval in ECG, negative chrono- and inotropic effects, and an increase in perfusion flow in guinea-pig isolated and perfused hearts. These effects of tiamulin on the aorta or cardiac muscle were similar to those of verapamil and nifedipine. These results suggest that both the inhibitory action of tiamulin on the high K(+)-induced contraction in the aorta and the negative inotropic effect of tiamulin on the cardiac muscle are due to an inhibition of Ca2+ entry through the voltage-dependent Ca2+ channels of cells of both these muscles.


Feverfew extracts and parthenolide irreversibly inhibit vascular responses of the rabbit aorta. Samples prepared from chloroform extracts of fresh leaves of feverfew (Tanacetum parthenium) strongly inhibited responses of rabbit aortic rings to phenylephrine, 5-hydroxytryptamine, thromboxane mimetic U46619 (9,11-dideoxy-11 alpha,9 alpha-epoxy-methano-PGF2 alpha), and angiotensin II, but the inhibition to contractions induced by potassium depolarization was much less. The inhibition was concentration- and time-dependent, non-competitive, and irreversible, and also occurred in endothelium-denuded preparations. The feverfew extracts also caused a progressive loss of tone of pre-contracted aortic rings and appeared to impair the ability of acetylcholine to induce endothelium-dependent relaxations of the tissue. These effects were mimicked by a purified preparation of an alpha-methylenebutyrolactone, parthenolide, obtained from the extract. Our results demonstrate a nonspecific and potentially toxic response to feverfew on the vasculature.


Relationship between paracetamol plasma levels and its analgesic effect in the rat. The relationship between plasma levels of paracetamol and its analgesic effect was studied in the rat using a model of pain-induced functional impairment (PIFI). Female Wistar rats received an intraarticular injection of 30% uric acid in the knee of the right hind limb, inducing its dysfunction. Animals then received oral paracetamol at doses of 178, 316 or 562 mg kg-1 and the recovery of functionality over time was considered as an expression of analgesia. Paracetamol plasma levels were determined by HPLC. Results showed that there is a direct relationship between paracetamol plasma levels and its analgesic effect that follows a sigmoidal model according to the Hill equation. The PIFI model appears to be a useful tool to establish pharmacokinetic/pharmacodynamic relationships for non-narcotic analgesics.


Nicotinate esters: their binding to and hydrolysis by human serum albumin. Nicotinate esters were studied for their binding to, and hydrolysis by, human serum albumin. Some esters (ethyl, isopropyl, t-butyl, cyclohexyl, benzyl) were bound but not hydrolysed, while others (2-chloroethyl, 2-butoxyethyl) displayed the opposite behaviour; 1-carbamoylethyl ester was neither bound nor readily hydrolysed. Only p-methoxyphenyl nicotinate was both a ligand and a substrate, and its rate constants of binding and hydrolysis were calculated in a stepwise procedure using a kinetic model.


Single- and multiple-dose pharmacokinetics of nefiracetam, a new nootropic agent, in healthy volunteers. The pharmacokinetic profile of nefiracetam (N-(2,6-dimethylphenyl)-2-(2-oxo-1-pyrrolidinyl)acetamide), a new nootropic agent, was studied in healthy Japanese male volunteers. Nefiracetam was administered orally at doses of 10-200 mg in the single-dose studies, and at doses of 200 mg three times a day for seven days in the multiple-dose study. An HPLC method was used to determine the concentrations of nefiracetam in serum, urine and faecal samples. Linear kinetic behaviour was obtained after single oral administration. Serum concentrations of nefiracetam reached maximum values (Cmax) within 2 h for all dosage groups, and declined monophasically after Cmax with half-lives of 3-5 h. The area under the concentration-time curve (AUC infinity) and Cmax were linearly related to the dose. The apparent clearance (CL) values were 94.4-140.3 mL min-1. Urinary excretion of nefiracetam was independent of the administered dose, and less than 10% of the dose was recovered in urine as the unchanged form within 24 h after administration. Renal clearance (CLR) did not change significantly as dose increased from 10 to 1200 mg. Faecal excretion of nefiracetam was less than 0.1% of the dose up to 24 h after a 300 mg oral dose. Food intake delayed the absorption of nefiracetam but did not significantly modify its pharmacokinetics. No clinically significant accumulation of nefiracetam in the body was observed during and after multiple doses.


Phenobarbitone population pharmacokinetics from routine clinical data: role of patient characteristics for estimating dosing regimens. Routine clinical pharmacokinetic data collected from patients receiving phenobarbitone have been analysed to evaluate the role of patient characteristics for estimating dosing regimens. The data were analysed using NONMEM, a computer program designed for population pharmacokinetic analysis that allows pooling of data. The pharmacokinetic model of phenobarbitone was described using a one-compartment steady-state model. The effect of a variety of developmental and demographic factors on clearance was investigated. NONMEM estimates indicated a nonlinear function of total body weight as the optimum adjustment of phenobarbitone clearance. Concomitant administration of phenobarbitone and other antiepileptic drugs showed a decrease of phenobarbitone clearance in young children. The dosing method based on clearance values obtained by NONMEM analysis allowed the prediction of the steady-state concentration as a function of maintenance dose with acceptable error for therapeutic drug monitoring.


Digoxin population pharmacokinetics from routine clinical data: role of patient characteristics for estimating dosing regimens. Routine clinical pharmacokinetic data collected from patients receiving digoxin have been analysed to evaluate the role of patient characteristics for estimating dosing regimens. The data were analysed using NONMEM, a computer program designed for population pharmacokinetic analysis that allows pooling of data. The pharmacokinetic model of digoxin was described using a one-compartment steady-state model. The effect of a variety of developmental and demographic factors on clearance was investigated. NONMEM estimates indicate that digoxin clearance was influenced by the demographic variables of age, total body weight, serum creatinine and sex. The interindividual variability in digoxin clearance was modelled with additive error with an estimated standard deviation of 46.15 L day-1 and the intraindividual variability, or residual error was 0.209 ng mL-1. The dosing method based on clearance values obtained by NONMEM analysis allowed the prediction of the steady-state concentration as a function of maintenance dose with acceptable error for therapeutic drug monitoring.


Distribution of antipyrine in the rat liver. The rate and extent of hepatic distribution of antipyrine was examined in the rat isolated perfused liver. Tritiated water and [14C]antipyrine were injected simultaneously into the portal vein as a bolus using either Krebs-Ringer bicarbonate or rat plasma as the perfusate. The effluent profiles of each compound using the two perfusates were superimposable, a finding expected for water and consistent for antipyrine, which was negligibly bound in rat plasma. Although full recovery (97%) of administered material was achieved with both compounds, the fractional output profile for antipyrine peaked at a lower value (0.10 mL-1) and at a later time (24 s) than water (0.14 mL-1, 17.5 s), due to antipyrine having a larger volume of distribution (water 0.61 mL (g liver) -1); antipyrine 0.81 mL (g liver)-1). This observation is explained by antipyrine binding to, or partitioning into cellular components. Nonetheless, like water, distribution of antipyrine into hepatic cells is perfusion rate limited as evidenced by the superimposition of the dimensionless plots of fractional output vs time normalized to mean residence time.


Dissolution and absorption of caffeine from guarana. The rate of release of caffeine from capsules of guarana was compared with that from capsules containing an equivalent amount of caffeine using the British Pharmacopoeia dissolution test apparatus. Determinations were carried out in media of pH 2 and 6.8 and caffeine concentrations in the dissolution fluid were determined by HPLC. No significant differences in release rates were found between the two preparations at either pH. The rate of absorption of caffeine across rat intestine using the everted gut was also compared for a guarana suspension and a solution containing an equivalent amount of caffeine. Experiments were carried out using fluids of pH 4.0 and 7.4. No significant differences in absorption between the two preparations were observed. These results show that the release and uptake of caffeine from guarana is the same as for preparations containing free caffeine.


PAF formation by human gastrointestinal mucosa/submucosa in-vitro: release by ricinoleic acid, and inhibition by 5-aminosalicylic acid. Human isolated gastrointestinal mucosa/submucosa incubated with ricinoleic acid (12.5-100 micrograms mL-1) or the calcium ionophore A23187 (10 micrograms mL-1) released platelet-activating factor (PAF) as determined by a scintillation proximity assay after extraction and purification. 5-Aminosalicylic acid (25-100 micrograms mL-1) inhibited PAF release by ricinoleic acid in a concentration-dependent manner, and 50 micrograms mL-1 reduced the effect of A23187. We suggest that PAF may play a role in the laxation and mucosal damage by ricinoleic acid released from castor oil.


Nifedipine-morphine interaction: a further investigation on nociception and locomotor activity in mice. Nociception and locomotor activity were tested in mice (C57BL/6 and DBA/2 strains), receiving the dihydropyridine calcium-channel blocker nifedipine, alone or combined with morphine. The calcium antagonist did not change the reaction time to thermal stimulation (tail-flick test), when administered alone, but combinations of nifedipine and morphine prolonged tail-flick latencies less than did the opiate alone. Nifedipine decreased locomotion in both strains, reduced the hypermotility induced by morphine in C57 mice, and enhanced the locomotor depression induced by the opiate in DBA mice. A comparison of the effects of nifedipine with those of the non-calcium antagonist vasodilator, hydralazine, suggests that the interactions with morphine were not exclusively related to neuronal changes produced by calcium channel blockade, but also to haemodynamic factors. In fact, except for the lack of interference with morphine-induced hypermotility in C57 mice, hydralazine, given alone or in combination with morphine, produced effects similar to those of nifedipine.


Stimulation of postsynaptic 5-HT1A receptors is responsible for the anticonflict effect of ipsapirone in rats. Ipsapirone (1.25-10 mg kg-1), a non-benzodiazepine anxiolytic drug with high affinity for 5-hydroxytryptamine1A (5-HT1A) receptors, increased dose-dependently the number of punished licks in the drinking conflict test (Vogel test) in rats. The anticonflict effect of the drug administered at a dose of 5 mg kg-1 was not modified in animals with lesions of 5-HT neurones, produced by p-chloroamphetamine (PCA, 2 x 10 mg kg-1). The anticonflict effect of ipsapirone in PCA-pretreated rats was antagonized by the 5-HT1A receptor and alpha 1-adrenoceptor antagonist NAN-190 (1-(2-methoxyphenyl)-4-[4-(2-phthalimmido)]butylpiperazine hydrobromide; 0.5-1 mg kg-1), but not by the selective alpha 1-adrenoceptor blocker prazosin (0.5 mg kg-1). Neither NAN-190 nor prazosin affected the punished response in PCA-pretreated rats. The present results indicate that the anticonflict effect of ipsapirone depends on stimulation of postsynaptic 5-HT1A receptors.


BQ-153, a novel endothelin (ET)A antagonist, attenuates the renal vascular effects of endothelin-1. Endothelin (ET)-1, leukotriene D4 and the thromboxane analogue, U-44069, were all shown to produce dose-dependent reductions in renal blood flow after direct injection into the renal artery of anaesthetized pigs. The effects of ET-1 differed from the other two mediators in that ET-1 caused a transient vasodilator followed by a prolonged vasoconstrictor response. The pressor response was not mediated by the secondary release of either leukotriene D4 or thromboxane A2 as evidenced by the lack of effect of appropriate receptor antagonist MK571 (3-[-2(7-chloro-2 quinolinyl) ethenyl]phenyl[3-(dimethylamino-3-oxopropyl)thio]methyl thio propionic acid) and L-670,596 respectively. This response, however, could be inhibited in a dose-dependent fashion by the selective ETA antagonist, BQ-153 (cyclo-D-sulphalanine-L-Pro-D-Val-L-Leu-D-Trp-). Following blockade by BQ-153 the vasodilator response was unaffected and a residual pressor response remained, suggesting that either or both of these effects were mediated either through an ETB or a novel, as yet undefined, endothelin receptor.


The P-glycoprotein gene family of Caenorhabditis elegans. Cloning and characterization of genomic and complementary DNA sequences. P-glycoproteins, encoded by families of evolutionarily conserved genes, can confer a multidrug-resistant phenotype to mammalian tumor cells. To obtain more information on their functions in normal cells we have cloned genomic and complementary DNA sequences of four P-glycoprotein gene homologs of the genetically well-characterized nematode Caenorhabditis elegans, termed pgp-1, pgp-2, pgp-3 and pgp-4, respectively. The genes were physically mapped on chromosome IV (pgp-1), I (pgp-2) and X (pgp-3 and pgp-4). Phenotypic mutants corresponding to these loci have not yet been described. Two of the genes, pgp-1 and pgp-3, were analyzed in detail. They are predicted to encode ATP-binding membrane-spanning proteins of 1321 and 1254 amino acid residues, respectively, with the characteristic features shared by most P-glycoproteins described thus far. Intra-species divergence of P-glycoprotein genes is more pronounced in C. elegans than in mammals. Only 40% of the amino acids of pgp-1 and pgp-3 are identical, in contrast to 77% identity between human MDR1 and MDR3. pgp-1 consists of 14 exons, pgp-3 of 13. The two genes share only one intron position, whereas they share four (pgp-1) and five (pgp-3) intron positions with mammalian P-glycoprotein genes. pgp-1, pgp-2, and pgp-3 are transcribed into low abundance mRNAs in wild-type nematodes. pgp-1 and pgp-3 mRNAs have the trans-spliced leader SL1 at their 5' ends. Arsenite, emetine and actinomycin D drugs did not increase the steady state levels of pgp mRNA, unlike in some mammalian cell types. Heat shock disturbed trans as well as cis-splicing of pgp-1 and led to the accumulation of partially processed pgp-1 RNA. Thus, in C. elegans these genes are not induced in the context of a general stress response, as has been proposed for mammalian P-glycoprotein genes in certain tissues.


Cold-induced alterations in the binding of adrenomedullary nuclear proteins to the promoter region of the tyrosine hydroxylase gene. It is well documented that cold stress induces a rapid trans-synaptically mediated increase in the relative abundance of rat adrenomedullary tyrosine hydroxylase (TH) mRNA. To investigate the transcriptional mechanisms regulating the cold stress response, we have employed a gel mobility shift assay, using DNA fragments prepared from the proximal 5' flanking region of the bovine TH gene as a heterologous molecular probe. In pilot studies, this region of the bovine TH promoter (nucleotides -246 to +21) was fused to the bacterial reporter gene, chloramphenicol acetyltransferase, and the chimeric construct transfected into human neuroblastoma SK-N-BE(2)-C, hepatoma HepG2, and rat pheochromocytoma PC-12 cells. Results of this analysis indicate that the proximal 5' flanking region of the bovine TH gene contains sufficient information to drive transient reporter gene expression in both human and rat catecholaminergic clonal cell lines. The findings derived from the gel mobility shift studies demonstrate that cold exposure causes rapid and selective alterations in the binding of adrenomedullary nuclear proteins to the proximal 5' flanking region of the TH gene. The most striking cold stress-induced alteration in DNA/nucleoprotein binding occurs in a region of the TH promoter (nucleotides -246 to -189) which contains an element bearing marked sequence similarity to an AP1 binding site and is highly conserved among animal species. This alteration occurs within 1 hr of cold exposure and persists for up to 48 hr after the onset of stress. The results of adrenal denervation experiments indicate that the cold-induced change in DNA/nucleoprotein binding is neurally mediated, requiring intact sympathetic innervation of the gland.(ABSTRACT TRUNCATED AT 250 WORDS)


Tau protein induces bundling of microtubules in vitro: comparison of different tau isoforms and a tau protein fragment. Expression of tau protein in non-neuronal cells can result in a redistribution of the microtubule cytoskeleton into thick bundles of tau-containing microtubules (Lewis et al.: Nature 342:498-505, 1989; Kanai et al.: J Cell Biol 109:1173-1184, 1989). We reconstituted microtubule bundles using purified tubulin and tau in order to study the assembly of these structures. Taxol-stabilized tubulin polymers were incubated with various concentrations of recombinant human tau and examined by electron microscopy. With increasing concentrations of tau 3 (tau isoform containing three microtubule binding domains) or tau 4 (isoform containing four microtubule binding domains) the microtubules changed orientation from a random distribution to loosely and tightly packed parallel arrays and then to thick cables. In contrast, tau 4L, the tau isoform containing four microtubule binding domains plus a 58-amino acid insert near the N-terminus, showed minimal bundling activity. tau 4-induced bundling could be inhibited by the addition of 0.5 M NaCl or 0.4 mM estramustine phosphate, conditions which are known to inhibit tau binding to microtubules. A tau construct that contained only the microtubule binding domains plus 19 amino acids to the C-terminus was fully capable of bundling microtubules. Phosphorylation of tau 3 with cAMP-dependent protein kinase had no effect on its ability to induce microtubule bundling. These results indicate that tau protein is directly capable of bundling microtubules in vitro, and suggests that different tau isoforms differ in their ability to bundle microtubule filaments.


Identification of mouse type-2-like astrocytes: demonstration of glutamate and GABA transmitter activated responses. We have identified mouse type-2-like astrocytes and examined some of their electrophysiological properties. Cultures were prepared from P4 mouse neopallia. We demonstrate that mouse type-2-like astrocytes can be identified using the following criteria: presence of glial fibrillary acidic protein (GFAP), presence of chondroitin sulfate polysaccharide, and presence of gamma-aminobutyric acid (GABA). A2B5-binding is not a sufficient criterion to identify O2A lineage cells in mouse neopallial glial cultures since the monoclonal antibody A2B5 binds not only to O2A lineage cells but also to a subpopulation of large, flat type-1-like astrocytes. Mouse type-2-like astrocytes have resting membrane potentials of -76.2 +/- 2.1 mV-i.e., similar to that of mouse type-1-like astrocytes. The input resistance of 44.2 +/- 0.5 M omega is an order of magnitude greater than that of type-1-like astrocytes suggesting the type-2-like astrocytes are not extensively electrically coupled either to each other or to type-1-like astrocytes. Glutamate application caused an 8.8 +/- 1.7 mV depolarization of type-2-like astrocytes. Application of glutamate to barium treated astrocytes caused a fast depolarization with a peak amplitude of 21.4 +/- 1.8 mV; the cells repolarized from this peak by about 10 mV and upon removal of glutamate returned to its pre-glutamate value. Application of GABA caused a transient depolarization of 14.0 +/- 1.7 mV. The presence of barium resulted in a steady-state GABA-induced depolarization of 10.3 +/- 2.0 mV. Neither SITS nor beta-alanine interfered with the amplitude of the glutamate and GABA responses.


S-100 beta and insulin-like growth factor-II differentially regulate growth of developing serotonin and dopamine neurons in vitro. To study the phenotypic specificity of S-100 beta and insulin-like growth factor II (IGF-II) for developing monoamine neurons, serotonin (5-HT) neurons from the embryonic day 14 (E14) rostral raphe or dopamine (TH) neurons from the substantia nigra/ventral tegmental area were cultured for 3 days in vitro (3 DIV) in the presence of these factors. Neuronotrophic effects were analyzed by computer-assisted morphometry of 5-HT and TH-immunoreactive neurons. S-100 beta and IGF-II differentially regulated the growth of 5-HT and TH neurons but did not affect their survival. S-100 beta significantly increased several parameters of neurite outgrowth by 5-HT neurons but inhibited the spatial extent (field area) of TH neurites. IGF-II promoted growth of cell bodies of both phenotype, but only stimulated neurite outgrowth by TH neurons. S-100 beta and IGF-II differentially affected the number of GFAP immunoreactive cells from raphe and substantia nigra, but these effects did not correlate with the specificity of neuronotrophic effects. S-100 beta and IGF-II immunoreactivities were expressed in glial cultures derived from the same brain regions, raising the possibility that these factors have autocrine effects on glia as well as paracrine actions on neurons. The results of this study suggest that specificity of neurotrophic factors for particular embryonic neurons may be correlated with their neurotransmitter phenotype.


Recombinant human ciliary neurotrophic factor alters the threshold of hippocampal pyramidal neuron sensitivity to excitotoxin damage: synergistic effects of monosialogangliosides. Ciliary neurotrophic factor (CNTF) is a multifunctional protein which not only promotes neuronal survival in vitro and in vivo but also controls cell division of neuronal precursors, transmitter differentiation, and glial cell differentiation. Recent studies have indicated that neurotrophic factors can alter hippocampal neuronal threshold to excitotoxin sensitivity. To examine such a role for CNTF, cultures of rat embryonic hippocampal neurons were maintained with recombinant human CNTF for different times, prior to exposure to a toxic dose of glutamate at 5 days in vitro for a further 24 hr. The cytotoxic action of 200 microM glutamate (approximately 40% of pyramidal neurons remaining after 24 hr) was reduced in a concentration-dependent manner in cultures receiving a prior exposure to CNTF within the first 3 days of cell plating: 30 ng/ml CNTF permitted about 75% of the initial number of pyramidal neurons to survive. Presentation of CNTF less than 48 hr before glutamate challenge was ineffective at up to 100 ng/ml. When pyramidal neurons were cultured with a subthreshold concentration (2 ng/ml) of CNTF together with 10 microM of the monosialoganglioside GM1 (or its inner ester form) in the same paradigm, the resulting neuronal survival was similar to that seen with 30 ng/ml CNTF in the face of a glutamate challenge. Such low doses of either CNTF or ganglioside alone were ineffective. The ability of trophic factors to influence the threshold of neuronal sensitivity to excitatory amino acid injury suggests that these proteins could play an important role in the reparative capacity of acutely traumatized central neurons and in neurodegenerative diseases linked to an excitotoxic mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)


Effect of acute trauma on the levels of intracellular cAMP, cGMP and DNA: studies on endocrinology and metabolism. In this experiment, we used morgrel dogs to study the effect of trauma on the neuro-endocrine and metabolic organs and the effects of hormones on metabolism by determining the levels of intracellular cAMP, cGMP and DNA. From the changes in intracellular nucleotides, we learned that many neuro-endocrine organs and the main metabolic organs are characteristic of predominance in alpha-adrenoreceptor after trauma, thereby leading to high blood sugar.


The effect of alpha 1-blocker, bunazosin on a murine model of congestive heart failure induced by viral myocarditis. The purpose of this study was to investigate the therapeutic effect of an alpha 1-blocker, bunazosin, using an experimental murine model of congestive heart failure induced by viral myocarditis. This model is characterized by a high incidence of severe myocarditis and subsequent congestive heart failure, and is suitable for the evaluation of the effect of drugs. To estimate myocardial damage objectively and quantitatively, we used antimyosin monoclonal antibody in addition to histopathological grading. Four-week-old BALB/c mice were inoculated with encephalomyocarditis virus. The mice were injected daily with bunazosin or saline as a placebo from the day of viral inoculation until day 7 (protocol-I) or day 14 (protocol-II), or from day 4 to day 14 (protocol-III). They were then injected with 1.5 microCi of indium-111 labeled antimyosin antibody and were killed 24 h later. The antimyosin cardiac uptake was counted and histopathological grading was performed. The heart-weight to body-weight ratio, left ventricular dimension, histopathological grades and antimyosin cardiac uptake were significantly lower in the bunazosin group than in the placebo group in protocol-II, but not in protocol-I or protocol-III. Bunazosin showed a protective effect against viral myocarditis only when it was started early after infection and continued until the stage of congestive heart failure.


[The assessment of clinical usefulness of 131I-MIBG scintigraphy for localization of tumors of sympathetic and adrenomedullary origin--a report of multicenter phase III clinical trials]. A phase III clinical study of 131I-metaiodobenzylguanidine (131I-MIBG) was performed in 66 patients with tumors of sympathetic and adrenomedullary origin, including 32 patients with suspected pheochromocytoma, 25 with suspected neuroblastoma, 7 pre- or postoperative medullary carcinoma of the thyroid and each with carcinoid and suspected Sipple's syndrome. A total of 150 sites which were confirmed for presence (72 sites) or absence (78 sites) of tumors were examined on 131I-MIBG scintigrams. True positive ratio of the scintigraphy was 84.7% (61/72) and true negative ratio was 94.9% (74/78). Positive scintigraphy was obtained in 86.5% (32/37) of pheochromocytoma, 78.6% (22/28) of neuroblastoma and 100% (6/6) of medullary carcinoma of the thyroid. Accumulation of 131I-MIBG was seen in 16.8% of normal adrenal glands. Neither adverse reactions nor abnormal laboratory findings were noted in relation to 131I-MIBG injections. Our study indicates that 131I-MIBG is a safe and clinically useful radiotracers for the visualization and localization of tumors of sympathetic and adrenomedullary origin.


[Radionuclide therapy of Sipple syndrome using iodine-131 metaiodobenzylguanidine]. A 40-year-old female who had lung and liver metastases from malignant pheochromocytoma was treated with 3.7 GBq 131I-MIBG (metaiodobenzylguanidine). After the treatment, 131I-MIBG showed increased uptake in the metastatic lesions of the lung and liver. The size of tumor was no significant change on CT and MRI, but the intensity of liver metastases decreased gradually on MRI. Prior to the treatment, the levels of adrenaline and noradrenaline were high. One to three days after treatment, the level of these laboratory data further increased, but they gradually decreased in 1 to 3 months. These changes may be due to necrosis of tumor tissue.


Autoreactive kidney-infiltrating T-cell clones in murine lupus nephritis. T-cells have been implicated in autoimmune renal injury. To examine the role of T-cells in lupus nephritis we propagated T-cell clones from the cortical interstitium of MRL/lpr mice. All isolated kidney-infiltrating (KI) T-cell clones [6] express surface markers identical to the T-cells regulated by the lpr gene (Thy 1.2+, TCR alpha/beta +, Lyt-2-, L3T4-, B220+). Although KI T-cell clones have the same surface markers as lymph node-infiltrating (LNI) T-cells, they differ functionally. KI T-cells, but not LNI T-cells, are autoreactive and kidney-specific, exclusively proliferating to renal tubular epithelial (TEC) and mesangial cells. In addition, unlike LNI T-cell supernatants (SN), KI T-cell clones SN induce class II and ICAM-1 on cultured TEC. When KI T-cell clones are injected under the renal capsule, class II is increased on TEC. All clones transcribe mRNA for cytokines capable of inducing class II and ICAM-1 (IL-4, TNF-alpha, IFN-gamma). Anti-IFN-gamma mAb prevents the induction of class II and ICAM-1 on cultured TEC. Since class II and ICAM-1 expression on TEC precedes renal injury, the ability to propagate autoreactive, kidney-specific T-cell clones that induce these molecules provides evidence for their role in initiating renal injury in MRL/lpr mice.


Analysis of complement C4 loci in Caucasoids and Japanese with idiopathic membranous nephropathy. Deletion of the HLA class III complement gene, C4A, has been linked with susceptibility to a number of autoimmune diseases. In this study, we show a strong positive association between C4A gene deletion and development of idiopathic membranous nephropathy (IMN) in European Caucasoids [patients, 17/27 (63%); healthy controls, 13/65 (20%); RR 6.8; P = 0.003]. To clarify whether C4A deletion is an independent risk factor for IMN or is increased secondarily to the Caucasoid HLA A1, B8, DR3 extended haplotype, we examined the frequency of C4A deletion in Japanese patients, in whom the disease is associated with another HLA haplotype (DR2-DQw1). Analysis of 31 Japanese patients and 46 healthy controls showed that C4A deletion was present in only one patient (3%) and one control (2%). In addition, examination of the C4B locus in Japanese patients showed that there was no significant increase in the estimated frequency of C4B deletion in patients against controls (31 vs. 27%) and no difference in the frequency of the C4B long gene (73 vs. 87%) or C4B short gene (77 vs. 78%). We conclude that although C4A deletion confers significant risk of IMN in Caucasoids, there is no significant association between C4 polymorphism, as detected here, and risk of IMN in Japanese. This suggests that either C4A deletion is irrelevant to the pathogenesis of IMN or that more than one genetic mechanism is involved.


Analysis and isolation of renal tubular cells by flow cytometry. The cells of the renal cortex have rich heterogeneity of structure and function. Flow cytometry, the technique of rapid laser-based single cell analysis, can give information about cellular mixtures not obtainable by any other means. We examined a variety of fluorescent markers to identify populations of renal cells by flow cytometry. Cellular digests of rat cortex were fluorescently stained with either enzymatic activity probes, or polyclonal antibodies. Fluorescent staining for the proximal marker gamma-glutamyl-transpeptidase (tau-GT) was an order of magnitude brighter than autofluorescence, and stained 71 +/- 11% of the cells. Second, we colocalized enzymatic and antibody markers. There was tight colocalization of tau-GT enzyme activity, detected with fluorogenic substrates, with specific surface binding of tau-GT antibodies. Third, populations of fluorescently labelled cells can be rapidly isolated by flow cytometry sorting. Flow cytometry sorting isolated 10(7) cells positive for the proximal tubular marker tau-GT in a little under one hour. The sorted cells were viable with 99 +/- 2% trypan blue exclusion (N = 8). Sodium-dependent phloridzin-inhibitable glucose uptake was present in sorted cells, with greater uptake/mg protein than in unsorted controls. The sorted cells grew in culture as a monolayer of tightly adherent cuboidal cells. Hence, flow cytometry allows us to quantitate the heterogeneity in mixed renal cellular digests. Flow cytometry allows us to rapidly isolate millions of cells according to fluorescently tagged markers. The isolated cells are viable, retain sodium-dependent transport properties, and grow in culture.


Mesangial cell killing by leukocytes: role of leukocyte oxidants and proteolytic enzymes. Mesangial cells from human and rat kidney were examined for sensitivity to killing by neutrophils. Cells from both species were sensitive to killing by phorbol myristate acetate-stimulated neutrophils. Catalase was highly protective while superoxide dismutase was less protective and a number of protease inhibitors were not protective. Strong protection was also observed with the iron chelators, deferoxamine and phenanthroline, and with the hydroxyl radical scavengers, dimethylthiourea and 5,5-dimethyl-1-pyrroline N-oxide. Pretreatment of the mesangial cells with deferoxamine followed by washing also provided protection. Mesangial cells were also killed by reagent hydrogen peroxide (H2O2) but were much less sensitive to injury by direct application of proteolytic enzymes. The ability of H2O2 to injure mesangial cells was prevented by pre-incubation of the H2O2 with human leukocyte myeloperoxidase. These data suggest that killing is due primarily to the generation of H2O2 by the stimulated neutrophils and its further reduction in an iron-catalyzed reaction. The hydroxyl radical may be the reduction product that actually mediates lethal injury but lack of scavenger specificity prevents definitively concluding this. Mesangial cell killing by activated neutrophils could be significantly inhibited by monoclonal antibodies to CD11/CD18 molecules, suggesting that close contact between the target and effector cells is required for cytotoxicity. Although qualitatively similar to endothelial cells, the mesangial cells appeared to be quantitatively more oxidant sensitive than previously examined human and rat endothelial cells. Taken together, these data show that mesangial cells from rat and human are sensitive to leukocyte-induced injury and that injury results via an oxidant pathway.(ABSTRACT TRUNCATED AT 250 WORDS)


Complete prevention of myocardial stunning, contracture, low-reflow, and edema after heart transplantation by blocking neutrophil adhesion molecules during reperfusion. This study tested the hypothesis that preventing neutrophil adhesion during reperfusion, by blocking either the neutrophil membrane CD18 integrin complex or its endothelial and myocyte ligand, intercellular adhesion molecule-1 (ICAM-1), would reduce myocardial inflammation and edema and improve reflow and ventricular function after heart preservation and transplantation. After cardioplegia and insertion of a left ventricular balloon, rabbit hearts were heterotopically transplanted into recipient rabbits either immediately (immediate, n = 12) or after preservation in 4 degrees C saline (3 hours of ischemia, n = 33). Forty-five minutes before reperfusion, recipients of preserved hearts received intravenous infusions of either saline (vehicle, n = 13), anti-CD18 monoclonal antibody (Mab) R15.7 (2 mg/kg) (anti-CD18, n = 10), or anti-ICAM-1 Mab R1.1 (2 mg/kg) (anti-ICAM, n = 10). During 3 hours of reperfusion the slope of the peak-systolic pressure-volume relation and its volume-axis intercept, the exponential elastic coefficient of the end-diastolic pressure-volume relation, the unstressed ventricular volume, and the time constant of the exponential left ventricular pressure decay after dP/dtmin were serially measured. Myocardial blood flow was measured with microspheres from which coronary vascular resistance was calculated. After explanation, the degree of myocardial inflammation, estimated by tissue neutrophil sequestration (myeloperoxidase assay) and myocardial water content were determined. Within each group no significant differences in measurements made at 1, 2, and 3 hours of reperfusion were noted. Compared with the immediate transplantation group, the vehicle group demonstrated a significant increase in myeloperoxidase activity (3380 +/- 456 versus 1712 +/- 552 microU/gm, p < 0.05), coronary vascular resistance (115.5 +/- 13.4 versus 70.5 +/- 10.6 U/gm, p < 0.05), and myocardial water content (79.8% +/- 0.4% versus 75.6% +/- 1.3%, p < 0.05), a significant decrease in unstressed ventricular volume (a leftward shift in the end-diastolic pressure-volume relation) (-0.49 +/- 0.24 versus 0.28 +/- 0.21 ml, p < 0.05), and a marked prolongation in exponential left ventricular pressure delay after dP/dtmin (156.64 +/- 3.81 versus 37.25 +/- 3.34 msec, p < 0.01).(ABSTRACT TRUNCATED AT 400 WORDS)


Double-blind trial of aspirin in primary prevention of myocardial infarction in patients with stable chronic angina pectoris. The Swedish Angina Pectoris Aspirin Trial (SAPAT) Group. Clinical trials have demonstrated a prophylactic role for aspirin in myocardial infarction and in unstable angina pectoris. The Swedish Angina Pectoris Aspirin Trial (SAPAT) is the first prospective study of aspirin in stable angina. 2035 patients were randomised double-blind to treatment with aspirin 75 mg daily or placebo. All patients were treated with sotalol for control of symptoms. The median duration of follow-up was 50 months. Compared with the placebo+sotalol group, the aspirin+sotalol group had a 34% (81 vs 124 patients) reduction in primary outcome events (myocardial infarction and sudden death; 95% confidence interval 24-49%; p = 0.003) and the reduction observed in secondary outcome events (vascular events, vascular death, all cause mortality, stroke) ranged from 22% to 32%. Treatment withdrawal caused by adverse events occurred in 109 patients in the aspirin+sotalol group and 100 in patients in the placebo+sotalol group; major bleeds, including haemorrhagic stroke, occurred in 20 and 13 patients, respectively (not significant). The addition of a low dose of aspirin to sotalol treatment showed significant benefit in terms of cardiovascular events, including a significant reduction in the incidence of first myocardial infarction in patients with symptoms of stable angina pectoris.


Effect of negative pressure ventilation in severe chronic obstructive pulmonary disease. The hypothesis that patients with chronic obstructive pulmonary disease (COPD) have chronic inspiratory muscle fatigue was tested in an effectiveness trial in which negative pressure ventilation (NPV) was used to produce inspiratory muscle rest. In a double-blind study 184 patients with severe COPD were randomly allocated active or sham NPV treatment for a 12-week period of home use. The distance walked in a 6 min walk test was the primary outcome variable. Secondary outcome measures were cycle exercise endurance time, severity of dyspnoea, quality of life, arterial blood gas tensions, and respiratory muscle strength. The percentage reduction in amplitude of the diaphragmatic electromyographic signal multiplied by hours of NPV was used to reflect the dose of NPV so we could examine dose-response relations. Analysis was based on intention to treat. We found no evidence of a clinically or statistically significant difference in any outcome measure between active and sham groups. No dose-response relation was observed. Moreover, the intervention was poorly accepted despite substantial clinical support. We conclude that NPV as used in this study is difficult to apply and ineffective when used with the aim of resting the respiratory muscles in patients with stable COPD.


Impaired endothelium-dependent vasodilation of forearm resistance vessels in hypercholesterolaemia. Endothelium-dependent vasodilation in response to acetylcholine is impaired in the coronary microvasculature of hypercholesterolaemic subjects. Outside the coronary circulation, however, it has been suggested that hypercholesterolaemia results in a functional abnormality of vascular smooth muscle rather than in endothelial dysfunction. We examined vasodilator responses to acetylcholine, methacholine, and the endothelium-independent vasodilator sodium nitroprusside in the forearm resistance vessels of 12 men with primary hypercholesterolaemia and 12 normocholesterolaemic male controls. Endothelium-dependent vasodilation in response to acetylcholine was impaired in hypercholesterolaemic patients compared with controls: at the highest dose of drug (15 micrograms per min) mean blood flow in the forearms of the hypercholesterolaemic group was only 52% (95% Cl 31-88%) of that in the control group. Responses to sodium nitroprusside and to methacholine in the two study groups were not significantly different. These results indicate that endothelial dysfunction in hypercholesterolaemic subjects is generalised and extends to vascular beds outside the coronary circulation. Selective impairment to acetylcholine suggests that, at a molecular level, the defect is limited to a specific pathway.


Polymerase chain reaction for diagnosis of meningococcal meningitis. Meningococcal disease is normally suspected on clinical grounds but confirmed by isolation of Neisseria meningitidis from blood or cerebrospinal fluid (CSF), or by detection of gram-negative diplococci in CSF. After parenteral antibiotics are started the isolation rate of meningococci from blood cultures drops from 50% to less than 5% and the chances of CSF being positive by culture or microscopy are also reduced. We used the polymerase chain reaction (PCR) in a blinded study to detect meningococcal DNA in 54 CSF samples from patients with meningococcal disease or from controls. The PCR primers were specific for the meningococcal insertion sequence IS1106. The sensitivity and specificity of this PCR for diagnosis of meningococcal meningitis were both 91%. Sensitivity was not affected by prior antibiotic treatment. The IS1106 PCR is a rapid and sensitive test for confirmation of the diagnosis of meningococcal meningitis.


Renal biopsy findings in hypertensive patients with proteinuria. 27 patients with hypertension and persistent proteinuria were investigated by renal biopsy. The 13 patients without structural glomerular abnormalities were younger and had less proteinuria than the other 14, but otherwise the two groups had similar clinical features. 6 of the 14 had diffuse glomerular abnormalities; the other 8 had segmental sclerosing lesions, which were mainly in the hilum of the glomeruli, as seen in states of glomerular overload. Glomeruli in all groups were larger than those in normotensive people. It is possible that hypertension causes glomerular enlargement, proteinuria, and segmental glomerular lesions because of loss of functioning glomeruli due to ischaemia.


Molecular evaluation of residual disease as a predictor of relapse in acute promyelocytic leukaemia. Acute promyelocytic leukaemia (APL) is characterised by a unique fusion transcript, PML/RAR alpha. We tested for this transcript in 35 APL patients who were in apparent remission after various treatments. 11 of 13 patients who tested positive 4 months after achieving remission were in relapse 1-4 months later. All 22 patients who tested negative at 4 months were disease-free after a further 3 months to five years. The test may therefore prove useful in determining the need for additional treatment during clinical remission.


Acute nicotine administration increases somatostatin content and binding in the rat hypothalamus. Within 4 minutes a single, intravenous injection of nicotine (0.3 mg/Kg) induced increases in somatostatin-like immunoreactivity concentrations in the rat hypothalamus but not in the striatum. These changes were associated with a significant increase in the specific binding of somatostatin to putative receptor sites in hypothalamic membranes, while no significant changes were found in striatum. The enhancement of somatostatin binding resulted from a rapid increase in the number of available receptors rather than a change in receptor affinity. This effect appears to be mediated by nicotinic cholinergic receptors, because pretreatment with a centrally active nicotinic receptor antagonist, mecamylamine (5.0 mg/Kg i.v.), prevented the nicotine-induced changes in somatostatin content and binding in the hypothalamus. Mecamylamine alone had no observable effect on the hypothalamic somatostatinergic system. These results suggest that the rat hypothalamic somatostatinergic system can be regulated by nicotine-like acetylcholine receptors.


Chronic clonidine treatment and its termination: effects on penile erection and ejaculation in the dog. The effects of chronic administration (4 weeks) of the alpha-2 adrenoceptor agonist clonidine (CL) and its termination on penile erection and ejaculation were investigated in male dogs. Penile erection and ejaculation were elicited by manual penile stimulation (for 5 min). CL (10 micrograms/kg/hr, s.c.) was delivered via osmotic minipump (Alza, 2ML-4). 3 or 7 days after the minipump implantation, CL caused a significant decrease in the amount of ejaculate produced by the genital stimulation without affecting the erectile potency. Ejaculatory ability returned to pretreatment levels despite continued CL administration, becoming evident in tests 14 days after initiation of treatment. Further, chronic CL (23 days) antagonized the inhibitory effects of acute administration of CL (0.05 mg/kg, i.p.). These data indicate tolerance to continued delivery of low doses as well as to acute administration of a higher dose. In the acute drug experiments, the ejaculatory inhibition elicited by CL (0.05 mg/kg, i.p.) was completely antagonized by pretreatment with yohimbine (0.05 and 0.10 mg/kg, i.p.), an alpha-2 adrenoceptor antagonist, but not with naloxone (1.0 mg/kg, i.p.), an opioid receptor antagonist. Furthermore, DG-5128 (1.0 and 2.0 mg/kg, i.p.), a selective alpha-2 adrenoceptor antagonist that poorly penetrates the blood-brain barrier, failed to antagonize the CL-induced ejaculatory inhibition. This study suggests that functional alterations in the central alpha-2 adrenoceptor mechanism may be related to the changes in the ejaculatory capacity during chronic treatment with CL.


The effect of beta blocking drugs on lipid peroxidation in rat heart in vitro. Beta-adrenergic receptor blocking drugs include a structurally related class of drugs that are employed clinically to treat a variety of cardiovascular disorders. Since these drugs exert additional nonspecific effects including membrane stabilization, representative samples including atenolol, dilevolol, labetalol, metoprolol and propranolol were studied to determine their influence on lipid peroxidation. Homogenates or liposomes of adult rat hearts were incubated in the presence of various concentrations of propranolol or equivalent concentrations of dilevolol, labetalol, metoprolol or atenolol. Lipid peroxidation was stimulated with 50 microM FeSO4, 5 microM t-butyl hydroperoxide (homogenates) or 0.2 mM citrate FeSO4 (liposomes) plus O2. Lipid peroxidation, as assessed by both the thiobarbituric acid reaction and chemiluminescence, was reduced in a dose-dependent manner as the propranolol concentration was increased from 1 to 10 mM. The five beta-adrenergic receptor blocking drugs reduced lipid peroxidation both in crude homogenates and in liposomes; their effectiveness was related to their lipophilicity. Dilevolol, propranolol, labetalol and metoprolol at a concentration of 20 mM reduced lipid peroxidation by 45%, 37%, 35% and 28%, respectively. The hydrophilic blocker atenolol was ineffective in reducing lipid peroxidation even at elevated concentrations. Lipophilic beta-blocking drugs apparently are capable of exerting an antioxidant effect in protecting membrane lipids against peroxidation.


Nucleotide sequences of the major subunits of F9 and F12 fimbriae of uropathogenic Escherichia coli. An Eco RV-Cla I fragment containing the gene encoding the F9 fimbrial subunit of the human uropathogenic Escherichia coli strain C1018 and a PstI-PstI fragment containing the F12 fimbrial subunit gene of the dog uropathogenic strain 1442 have been cloned and the nucleotide sequence of the fragments determined. The structural gene of the F9 fimbriae (FniA) codes for a protein of 165 amino acid residues with a signal peptide of 25 amino acids. The F12 fimbrial gene (FtwA) codes for a protein of 155 amino acids which is preceded by a single peptide of 21 amino acids. The amino acid sequences of the FniA and FtwA proteins deduced from the nucleotide sequence were compared with sequences of other known P-fimbrial subunit proteins. As expected, most differences between the various proteins were found in the hypervariable regions defined by van Die et al. (1987). The N-terminal sequence of the FtwA protein differs from the one published by Klemm et al. (1983). FtwA contains two deletions found in comparison to the other fimbrial subunits.


Effect of attachment factors (pili plus Opa) on Neisseria gonorrhoeae invasion of human fallopian tube tissue in vitro: quantitation by computerized image analysis. Pili (P) and opacity-associated proteins (Opa) facilitate Neisseria gonorrhoeae attachment to human fallopian tube epithelium. Subsequent effects on invasion are unproven. Computerized image analysis was used to study the effects of attachment factors on invasion by comparing a P+Opa+ variant to a P-Opa- variant of strain R10 in the fallopian tube organ culture model. Gonococci in sections of infected fallopian tube tissue were identified with FITC-labelled monoclonal anti-gonococcal antibodies. Nomarski DIC microscopy was used to establish anatomic boundaries that excluded extracellular gonococci from invasion measurements. The area of intracellular fluorescence served as an index of gonococcal invasion. With conservative criteria to exclude extracellular gonococci, the per cent of the intracellular area occupied by fluorescent P+Opa+ gonococci was 18% compared to 4.7% for the P-Opa- variant (P < 0.001). Data suggest that P+Opa+ organisms invaded deeper than P-Opa- microbes over the same time period (P = 0.029). Intra-observer variation in invasion measurements was not significant (P > or = 0.85), and inter-observer correlation was high (correlation coefficient = 0.96). Computerized image analysis is a rapid, reliable means of quantifying gonococcal invasion of fallopian tube epithelium. We conclude that gonococcal attachment factors can facilitate events which enhance gonococcal invasion of fallopian tube epithelium.


Morphological and cytoskeletal changes in epithelial cells occur immediately upon interaction with Salmonella typhimurium grown under low-oxygen conditions. Salmonella typhimurium grown under oxygen-limiting conditions were found to enter into, elicit actin filament rearrangement in, and effect morphological changes upon HEp-2 cells within 15 min after infection. Video microscopy revealed that host cell morphological changes associated with entry began within 1 min of productive adherence. Polarized Caco-2 cell morphology was affected 40 s after infection with low-oxygen-grown S. typhimurium. Stationary-phase S. typhimurium did not elicit these phenomena within this time-period even when adherence was enhanced with the afimbial adhesin, AFA-I. Thus, environmental cues regulate S. typhimurium invasion factors, allowing for immediate entry into host cells. Additionally, actin filament rearrangement and morphological changes in the eukaryotic host cell are essential for entry and occur within minutes of infection.


The subcellular distribution of nickel in Ni-sensitive and Ni-resistant strains of Saccharomyces cerevisiae. Examination of the subcellular distribution of nickel in a Ni-resistant strain N08 of Saccharomyces cerevisiae showed that 70% of the nickel is distributed in the vascular fraction, which contains large amounts of histidine. The nickel taken up by cells grown in medium containing a high concentration of histidine was preferentially distributed to the vacuole. Arginine and lysine did not affect the intracellular distribution of Ni. In a Ni-sensitive strain 0605-S6, the distribution of nickel into the vacuole was lower than that observed in strain N08. Strain 0605-S6 exhibited no increase in the histidine content of the vacuolar fraction when grown in a Ni-supplemented medium. The Ni-resistant mechanism appears to involve the sequestration of nickel to the vacuole, and histidine could play an important role in the reduction of free nickel in the vacuole by the formation of histidine-nickel complexes.


Histochemical evaluation of energy metabolism in rat glioma. The key enzymes of oxidative phosphorylation and glycolysis were evaluated histochemically in rat-implanted C6 gliomas using spot densitometry. Hexokinase, the initial enzyme for the glycolysis pathway, was 40% higher within tumour than the contralateral cerebral cortex. A similar increase within tumours for 2-deoxyglucose was observed by autoradiography. Glucose-6-phosphate dehydrogenase (G6PDH), which is the first enzyme in the pentose phosphate pathway, shunting glucose towards nucleic acid synthesis, was more than 300% higher in gliomas compared with the normal cortex. In contrast, enzymes in the energy producing tricarboxylic acid cycle (succinate-, isocitrate-, and malate-dehydrogenase) and in the electron-transport system (cytochrome c oxidase) were significantly reduced in tumour (58% less than the contralateral cortex). Lactate dehydrogenase activity, which converts pyruvate to lactate, was 50% higher within tumour. Significant reductions of enzymatic activities also occurred in non-neoplastic tissue in ipsilateral hemisphere, with larger tumours. Some enzymes showed heterogeneous activity within tumours, especially G6PDH. These results suggest that: (1) energy production is more dependent on lactate production than on oxidative phosphorylation in C6 glioma, and (2) a significant part of the increased glucose utilization in glioma cells is due to increased activity of the pentose phosphate shunt for increased DNA synthesis, and not energy production.


Antivenom use in Australia. Premedication, adverse reactions and the use of venom detection kits. To analyse reports of antivenom use and sequelae in Australia from July 1 1989 to June 30 1990. The value of snake venom detection kits (VDKs) was also analysed. Information was obtained from antivenom usage reports returned to the Commonwealth Serum Laboratories and from personal letters sent to those reporting doctors. Information on VDKs was obtained from antivenom usage reports or from questionnaires packaged with VDKs. Reported antivenoms used were: red-back spider, 258 cases; funnel-web spider, 3 cases; stonefish, 26 cases; box jellyfish, 6 cases; snake, 91 cases. Immediate reactions followed administration of red-back spider antivenom in only two patients and snake antivenoms in four patients. Delayed reactions (serum sickness) followed use of red-back spider antivenom in three patients, stonefish antivenom in two, and snake antivenoms in three. No reaction was life-threatening. Premedication was used in the majority of red-back spider bites and in 66 of 86 snake bites. Oral corticosteroids were given prophylactically after some uses of snake antivenom to prevent serum sickness. VDKs were used in 181 cases of snake bite and were reported as being useful in selecting appropriate snake antivenom in 31% of cases of antivenom use. (Only 10 of these 181 were also reported on the antivenom usage report.) Appropriate first aid was given in 61% of cases. There were 50% fewer snake bites reported than 10 years ago. Antivenoms in Australia are well tolerated with few immediate or delayed reactions. The use of premedication and prophylactic oral corticosteroids for four to five days after antivenom administration may be responsible for this low reaction rate. VDK results help select the appropriate antivenom; however, in some cases positive results were obtained from urine samples from patients with no symptoms of envenomation.


Cryosurgery re-visited for the removal and destruction of brain, spinal and orbital tumours. Advances in neuroimaging and cryosurgical techniques have prompted us to re-evaluate the potential of cryosurgical techniques for the removal and the destruction of various neoplasms. We have used cryosurgical instrumentation to remove tumours in the brain, spine and orbit in 71 patients without complications. Cryosurgery was used to facilitate removal and extraction in 64 and to destroy residual neoplasms when removal was incomplete in 7. Intraoperative real time ultrasonic imaging permitted precise delimitation of tumours from surrounding tissues and allowed monitoring during the production of cryosurgical lesions thus permitting heretofore unavailable visualization of the production of cryogenic lesions in the central nervous system. New cryosurgical instrumentation was used to produce lesions up to three times larger than similar sized probes previously available. Our results reconfirm that cryosurgery facilitates the removal of tumours in the brain, spinal cord and orbit, reduces blood loss in vascular tumours, and is effective in ablating residual neoplasms involving the superior sagittal sinus, torcula and parasagittal areas. A Doppler flowmeter proved useful for monitoring sagittal sinus blood flow during the production of cryosurgical ablation of residual tumour attached to the walls of the sagittal sinus. Recent advances in ultrasonic and neuroimaging coupled with stereotactic techniques and improvements in cryosurgical instrumentation may prove useful in the future percutaneous destruction of selective intracranial neoplasms.


Mondor's disease of the breast resulting from jellyfish sting. To present two cases of Mondor's disease of the breast resulting from jellyfish stings in Western Australia. A 30-year-old Caucasian woman presented with a palpable thickened cord in her right breast. The straightness of the cord suggested a thrombosed lymphatic. A 50-year-old Caucasian woman presented with an obvious palpable cord extending most of the length of her left breast. Mammography demonstrated no abnormality. Both women reported having been stung by jellyfish a month earlier. As Mondor's disease is a benign, self-limiting disease, the patients were reassured and reviewed routinely. In each case, the condition settled spontaneously over a period of several weeks. Jellyfish stings should be recognised as an unusual variant of the numerous causes which have been described for Mondor's disease.


Determination of the rate of cerebral oxygen consumption and regional cerebral blood flow by non-invasive 17O in vivo NMR spectroscopy and magnetic resonance imaging: Part 1. Theory and data analysis methods. Theory and novel data analysis methods of 17O inhalation measurements are presented for the calculation of CMRO2, regional cerebral blood flow (rCBF), the reflow (R), the arterial venous difference (AVD) and the partition coefficient (lambda). Several of the methods proposed for the determination of CMRO2 do not require measurements of regional cerebral blood flow and H2(17)O arterial concentration. All methods of analysis are based on the Kety-Schmidt approach.


Pathology of spinal cord lesions caused by ossification of the posterior longitudinal ligament, with special reference to reversibility of the spinal cord lesion. This report describes pathological findings of the spinal cord damage, with ossification of the posterior longitudinal ligament (OPLL), with special reference to reversibility of such lesions. Twenty-five autopsy cases associated with OPLL were examined, and the spinal cord damage was pathologically classified into four categories based on degree of destruction (stage 0-3). In stage 0 and stage 1, major pathological changes in the gray matter and the degree of compression on the spinal cord were well correlated to deformity of the anterior horn. In stage 2 and stage 3, neurons were almost completely obliterated and necrosis with cavitation were frequently observed. Destruction of the spinal cord in stage 2 and stage 3 is considered to be irreversible; therefore, surgical treatment is recommended at stage 0 or stage 1.


Plasma lipoproteins in cortical versus lacunar infarction with or without cardiac arrhythmia, and in transient ischaemic attacks: a case control study. We investigated the relation of plasma lipids to the risk for cortical infarction with (22 cases) or without (38 cases) cardiac arrhythmias, for lacunar infarction (28 cases) and transient ischaemic attacks (TAI) (15 cases). In the group of cortical infarction with or without cardiac arrhythmias, we observed a maximum increase of total cholesterol, of very low density lipoprotein (VLDL) and low density lipoprotein (LDL), triglycerides, total Apolipoprotein (Apo) B, LDL-Apo B and Apo-A1. On the contrary, we observed a decrease of total ApoE, HDL-ApoE, a distribution of LDL in a single layer and the presence of LDL of small weight. TAI is different from the former group by a low level of HDL and the lack of abnormalities of Apo-A1, and on the distribution and the weight of LDL. Finally, lacunar infarction presents a normal plasma lipoprotein profile. These data suggest that previously demonstrated differences in LDL-cholesterol levels between patients with ischaemic stroke and control subjects may apply to patients with cortical but not lacunar infarction. The presence or not of a cardiac arrhythmia doesn't give a special lipoprotein profile, and TAI has no changes on the distribution and the weight of LDL. Therefore, separation of ischaemic strokes into types based on mechanism as large vessel atherosclerosis versus small vessel atherosclerosis may help clarify lipid-related risk factors in cerebrovascular disease.


Change of xanthine dehydrogenase and xanthine oxidase activities in rat brain following complete ischaemia. We studied the activities of xanthine dehydrogenase and xanthine oxidase in rat forebrain after complete ischaemia. Complete ischaemia was induced by decapitation after transcardiac infusion with saline. The activities of xanthine dehydrogenase and xanthine oxidase immediately after ischaemia were 93.3 +/- 38.7 and 18.8 +/- 7.7 microU/mg protein, respectively, and at 24 h after ischaemia were 183.5 +/- 75.1 and 60.8 +/- 15.2 microU/mg protein, respectively. The ratios of xanthine dehydrogenase/xanthine oxidase immediately and 24 h after ischaemia were 5.04 +/- 1.03 and 3.04 +/- 0.99, respectively. These data indicate that xanthine dehydrogenase and xanthine oxidase activities were maintained even 24 h after complete ischaemia. Conversion of xanthine dehydrogenase to xanthine oxidase proceeds slowly during complete ischaemia.


Can somatosensory evoked potential monitoring predict energy dynamics during controlled hypotension? Somatosensory evoked potentials and redox (reduction/oxidation ratio) of cytochrome a,a3 were studied simultaneously before, during, and after controlled hypotension used for arteriovenous malformation resection. These studies were also conducted before and after ED-IC (external-internal carotid) bypass procedures for treatment of patients with transient ischaemic attacks. The former served as an acute model and the latter as a chronic model of low blood flow (ischaemia). The use of non-invasive reflection spectrophotometry (for redox studies) in conjunction with somatosensory evoked potentials (SSEP) has demonstrated that metabolic and electrophysiological changes parallel each other during "controlled" hypotension. The authors conclude that an analysis of SSEP changes are valuable in the study of metabolic and functional states of the brain during controlled hypotension.


DNA synthesis and intracellular calcium elevation in porcine cerebral arterial smooth muscle cells by cerebrospinal fluid from patients with subarachnoid haemorrhage. To understand the molecular mechanism of the pathogenesis of cerebral vasospasm following subarachnoid haemorrhage, we analysed the effect of cerebrospinal fluid from patients with subarachnoid haemorrhage on DNA synthesis and cytosolic-free calcium elevation in cultured porcine cerebral smooth muscle cells. Cerebrospinal fluid from patients on day 2 after subarachnoid haemorrhage induced transient elevation in cytosolic-free calcium levels. In contrast, the maximal elevation of cytosolic-free calcium levels induced by cerebrospinal fluid from control patients (without subarachnoid haemorrhage) was significantly lower than that induced by cerebrospinal fluid from patients with subarachnoid haemorrhage. In cultured porcine cerebral arterial smooth muscle cells, cerebrospinal fluid from patients with subarachnoid haemorrhage promoted levels of [3H]-thymidine incorporation (DNA synthesis) more than 2.5-fold higher than that promoted by cerebrospinal fluid from control patients without subarachnoid haemorrhage. However, in cultured aortic smooth muscle cells, there was no significant difference in [3H]-thymidine incorporation between cerebrospinal fluid from patients with subarachnoid haemorrhage and that by control cerebrospinal fluid. From these results in cerebral arterial smooth muscle cells, cerebrospinal fluid from patients following subarachnoid haemorrhage may play not only constrictive functions, evidenced by cytosolic-free calcium elevations, but also proliferative functions, demonstrated by promotion of [3H]-thymidine incorporation. The relevance of these factors to vasospasm will be discussed.


Correction of ischaemic brain acidosis with SQ29,548/1-benzylimidazole. Thromboxane A2 (TXA2) is a proaggregatory vasoconstrictor that is synthesized and released during reperfusion of ischaemic brain. We administered a TXA2 receptor antagonist, SQ29,548, and a thromboxane A synthase inhibitor, 1-benzylimidazole (1-BI), to rats subjected to 30 min of reversible forebrain ischaemia. Cerebral thromboxane B2 (TXB2), the stable metabolite of TXA2, measured after 60 min of reperfusion was 0.37 +/- 0.08 ng/mg brain protein in animals treated with SQ29,548/1-BI compared with 1.20 +/- 0.16 in ischaemic controls (p < 0.05). Cerebral pH determined by 31P magnetic resonance spectroscopy was higher in treated animals, 7.06 +/- 0.04, than in ischaemic controls, 6.5 +/- 0.01, after 20 min of reperfusion (p < or = 0.01). The significant elevation of cerebral pH in treated animals persisted at 30 (7.17 +/- 0.05 vs. 6.5 +/- 0.01; p < or = 0.01), 35 (7.17 +/- 0.05 vs. 6.44 +/- 0.04; p < or = 0.01), and 40 min of reperfusion (7.06 +/- 0.06 vs. 6.37 +/- 0.01; p < or = 0.05). We conclude that SQ29,548/1-BI reduces thromboxane levels and promotes resolution of tissue acidosis in ischaemic brain. The combination of a TXA2 receptor antagonist with a thromboxane A synthase inhibitor deserves further study as a potential treatment for acute cerebral infarction.