Marine envenomations. Part 2: Invertebrates.

As more people travel to the oceans for sport diving and other marine-related activities, the incidence of marine envenomations has risen. This article is designed to give the emergency medicine physician an overview of varying marine envenomations, their clinical presentation, and recommended treatment. Part 1 of this article dealt with general wound management and vertebrate envenomations. Part 2 addresses invertebrate envenomations.


Variations in prion protein and glial fibrillary acidic protein mRNAs in the brain of scrapie-infected newborn mouse.

To begin to understand the molecular basis of cases of Creutzfeldt-Jakob disease recently described in young children, the expression of prion protein and glial fibrillary acidic protein (GFAP) mRNAs was investigated during the development of the brain of scrapie-infected newborn mice. Changes in the time course of expression were identified by Northern blot quantification between days 1 and 172. Although scrapie-infected and control animals showed no detectable changes in brain development (first 56 days of life), GFAP mRNAs were found to increase significantly as early as day 84. A 10-fold increase in the level of GFAP mRNA was observed in brain between day 112 and death (day 172).


Dynamic observation of dopamine autoreceptor effects in rat striatal slices.

Fast-scan cyclic voltammetry has been used to measure dopamine (DA) synaptic overflow in slices of rat caudate nucleus induced by electrical stimulation with one-, two-, and 50-pulse, 10-Hz trains. Synaptic overflow in this preparation is shown to be the result of the competing effects of release and cellular uptake. Release caused by all pulses was attenuated by the D2 agonist quinpirole (1 microM). The rapid time response of the measurements (100 ms) allows the autoinhibition induced by endogenous, released DA to be resolved in real time. The concentration of DA released during the second pulse of a train was 58% of that released by the first pulse, an effect that is partially blocked by the addition of 2 microM sulpiride, a D2 antagonist, to the perfusion buffer. DA release during the first stimulus pulse is unaffected by 2 microM sulpiride, suggesting that autoreceptors are not normally occupied in this preparation. Release caused by the third pulse was 14% of the first pulse and also could be partially enhanced by 2 microM sulpiride. The duration of the inhibition of release induced by endogenous DA was estimated by varying the interval between one-pulse stimulations until the overflow of DA induced by the second pulse was equal to that on the first; a half-time of approximately 17 s was found. The addition of picrotoxin (100 microM) and glutamate (10 microM) to the perfusion buffer did not affect stimulated release of DA, although the addition of atropine (100 microM) attenuated overflow for all the trains tested.


gamma-Glutamylaminotransferase and transglutaminase in subcellular fractions of rat cortex and in cultured astrocytes.

The subcellular distribution of gamma-glutamylamino-transferase (EC 2.3.2.2) and transglutaminase (EC 2.3.2.1) has been investigated in rat brain tissue fractionated by a centrifugation and sedimentation technique. Neither of these enzymes was enriched in the synaptosomal fraction. Comparing the in vitro grown astrocytes with synaptosomes, we find that both of these enzymes may possibly be more important in the glial element of the synaptic region. gamma-Glutamylaminotransferase is most abundant in capillaries, confirming previous reports.


Glutamate and gamma-aminobutyric acid neurotransmitter systems in the acute phase of maple syrup urine disease and citrullinemia encephalopathies in newborn calves.

Cerebral cortex tissue was obtained at autopsy from neonatal Poll Hereford calves with clinically confirmed maple syrup urine disease (MSUD), neonatal Holstein-Friesian calves with clinically confirmed citrullinemia, and matched controls. From this, synaptosomes were prepared for studies of neurotransmitter amino acid uptake and stimulus-induced release, and synaptic plasma membranes were obtained for studies of associated postsynaptic receptor binding sites. As well as having abnormal brain tissue concentrations of the pathognomic plasma amino acids (markedly increased levels of the branched-chain compounds valine, isoleucine, and leucine in MSUD; marked elevation of citrulline levels in citrullinemia), both groups of diseased animals showed reduced brain tissue concentrations of each of the transmitter amino acids glutamate, aspartate, and gamma-aminobutyric acid (GABA). Nontransmitter amino acids were generally unaffected in either disease. Citrullinemic calves showed a marked increase in brain glutamine concentration; in calves with MSUD, the glutamine concentration was raised, but to a much lesser extent. The Na(+)-dependent synaptosomal uptake of both glutamate and GABA was markedly reduced (to less than 50% of control values in both cases) in citrullinemic calves but was unaltered in calves with MSUD. Whereas synaptosomes from normal calves showed the expected stimulus-coupled release of transmitter amino acids, especially glutamate and aspartate, and no response to stimulus of nontransmitter amino acids, there was no increased release of transmitter amino acids in response to depolarization in synaptosomes from citrullinemic calves.(ABSTRACT TRUNCATED AT 250 WORDS)


Excitatory amino acid-induced phosphoinositide turnover in guinea pig cerebral cortical slices: selective enhancement by spermine of the response to DL-1-aminocyclopentane-trans-1,3-dicarboxylate.

In the presence of 1 mM spermine, accumulations of 3H labelled inositol phosphates elicited by quisqualate (100 microM) and 1-aminocyclopentane-trans-1,3-dicarboxylate (t-ACPD, 300 microM) were significantly enhanced by 21 and 26%, respectively, without a significant alteration in the accumulation elicited by L-glutamate (10 mM) or DL-alpha-amino-3-hydroxy-5-methyl-4-isoxalone propionate (10 microM). Analysis of concentration-response data indicated that the presence of spermine led to an increase in the maximal response to t-ACPD without altering the EC50 value. The stimulatory effect of spermine on the accumulation of t-ACPD-elicited 3H-inositol phosphates was not reversed by ifenprodil or diethylenetriamine (putative polyamine site antagonists), by agents that activate or inhibit protein kinase C, or by calcium channel blockade, but was abolished in the presence of elevated extracellular calcium ion concentration. We conclude that spermine enhances the phosphoinositide turnover in guinea pig cerebral cortical slices elicited by the "metabotropic" excitatory amino acid receptor. The site through which the action of spermine is mediated remains to be defined, but it is apparently distinct from that suggested to modulate N-methyl-D-aspartate receptor activity.


Abnormalities in amino acid neurotransmitters in discrete brain regions of genetically dystonic hamsters.

The concentrations of 11 amino acids, including the neurotransmitters gamma-aminobutyric acid, glutamate, aspartate, glycine, and taurine, were determined by HPLC in 12 brain regions of genetically dystonic (dtSZ) hamsters and age-matched nondystonic controls. Since dystonia in mutant dtSZ hamsters is transient and disappears after about 70 days of age, amino acids were determined at the age of maximum severity of dystonia (30-40 days) and after disappearance of the disease, to examine which neurochemical changes were related to dystonia. In dtSZ hamsters with the maximum severity of dystonia, significant changes in concentrations of the neurotransmitters gamma-aminobutyric acid, glutamate, aspartate, and taurine were found in several regions involved in motor functions, e.g., cerebellum, thalamus, and corpus striatum. Most of these changes were not permanent but disappeared in parallel with dystonia, implicating a causal relationship between altered aminoacidergic neurotransmission and dystonia in mutant dtSZ hamsters.


Potent inhibition of scrapie-associated PrP accumulation by congo red.

Transmissible spongiform encephalopathies (prion diseases), Alzheimer's disease, and other amyloidoses result in the accumulation of certain abnormally stable proteins that are thought by many to play central roles in disease pathogenesis. Using scrapie-infected neuroblastoma cells as a model system, we found that Congo red, an amyloid-binding dye, potently inhibits the accumulation of the scrapie-associated, protease-resistant isoform of protein PrP without affecting the metabolism of the normal isoform. Growth of the cells with submicromolar concentrations of Congo red for 5 days reduced the amount of protease-resistant PrP detected in the cultures by greater than 90%. This activity of Congo red suggests that it selectively disrupts the conversion of PrP to the protease-resistant isoform or destabilizes this isoform once it is made. Potential therapeutic applications of Congo red are discussed.


Characterization of descending facilitation and inhibition of spinal nociceptive transmission from the nuclei reticularis gigantocellularis and gigantocellularis pars alpha in the rat.

1. Descending influences produced by focal electrical stimulation in the nuclei reticularis gigantocellularis (NGC) and gigantocellularis pars alpha (NGC alpha) on spinal nociceptive transmission and the dorsoventral region of spinal white matter mediating stimulation-produced modulation were examined in pentobarbital sodium-anesthetized, paralyzed rats. Spinal units studied responded to mechanical stimuli and noxious heating (50 degrees C) of cutaneous receptive fields confined to the glabrous skin of the ipsilateral hind foot. Recording sites were located in laminae I-VI of the L3-L5 spinal segments. 2. Electrical stimulation in the NGC or NGC alpha produced both facilitation and inhibition of responses of spinal units to noxious heating of the skin. At 33 of 57 stimulation sites in the NGC and NGC alpha, electrical stimulation produced biphasic effects, facilitating responses at lesser intensities (5-25 microA) and inhibiting responses at greater intensities (50-100 microA). At 21 other sites in the NGC and NGC alpha, electrical stimulation (5-100 microA) only inhibited, and at 3 sites stimulation (5-100 microA) only facilitated responses of spinal units to noxious heating of the skin. 3. Electrical stimulation in the NGC or NGC alpha contralateral to the spinal recording site produced the same magnitude of facilitation/inhibition or inhibition of spinal nociceptive transmission as did stimulation in the ipsilateral NGC and NGC alpha. 4. The latencies to descending facilitation and inhibition of spinal nociceptive transmission from the NGC and NGC alpha were estimated by a cumulative sum technique to be 232 and 80 ms, respectively. 5. Responses of spinal units to graded heating (42-50 degrees C) of the skin exhibited positively accelerating stimulus-response functions (SRF) throughout the temperature range tested. Electrical stimulation at lesser, "facilitating" intensities produced a parallel, leftward shift of the SRF, whereas stimulation at greater, "inhibitory" intensities significantly decreased the slope of the SRF without affecting the threshold for response. 6. To determine whether activation of cell bodies in the NGC or NGC alpha were capable of replicating the effects of electrical stimulation, L-glutamate was microinjected into sites where electrical stimulation facilitated at lesser and inhibited at greater intensities the responses of spinal units to 50 degrees C heating of the skin. L-Glutamate (5 nmol) produced a rapid onset, short-lasting and reproducible facilitation of nociceptive transmission; glutamate microinjection into the same site at a greater dosage (50 nmol) inhibited responses of the same spinal units.(ABSTRACT TRUNCATED AT 400 WORDS)


APB suppresses synaptic input to retinal horizontal cells in fish: a direct action on horizontal cells modulated by intracellular pH.

1. Membrane potentials and cone-driven light responses were recorded from the H1-type horizontal cells in isolated retinas. Membrane potentials and intracellular pH were recorded also in enzymatically dissociated solitary horizontal cells. 2. In isolated retinas the glutamate analogue 2-amino-4-phosphonobutyrate (APB) hyperpolarized horizontal cells and reduced their light responses in a dose-dependent manner (5-200 microM). 3. The action of APB depended on the formulation of the saline; APB was effective in L-15 saline buffered with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) but not in a commonly used, nominally CO2-free bicarbonate/Tris-buffered saline. 4. The major factor controlling the potency of APB was intracellular pH. APB was ineffective during retinal perfusion with NH4Cl-containing or CO2-free bicarbonate saline, both of which are known to alkalinize cells. In contrast, APB was effective in salines formulated to acidify the retinal neurons. These included both HEPES and Tris-buffered salines containing a weak acid and bicarbonate/Tris-buffered saline gassed with CO2. 5. APB reduced the size of glutamate-evoked depolarizations in solitary horizontal cells but had no independent action in the absence of glutamate. This reduction of glutamate-induced depolarization was observed in salines formulated to block voltage-dependent calcium and potassium currents. 6. The magnitude of APB's antagonistic action on solitary horizontal cells increased in a dose-dependent manner from 10 to 200 microM. The antagonism was increased by intracellular acidification and was reduced or eliminated by alkalinization. 7. We conclude that APB can reduce glutamate-evoked and, by inference, the photoreceptor neurotransmitter-evoked depolarization of horizontal cells by acting directly on the horizontal cells. This effect of APB is modulated by intracellular pH.


Large aspiny cells in the matrix of the rat neostriatum in vitro: physiological identification, relation to the compartments and excitatory postsynaptic currents.

1. Large aspiny neurons (20-60 microns diam) in the neostriatum were studied in an in vitro rat slice preparation by whole-cell recording to reveal physiological identification from medium-sized spiny projection cells (10-20 microns diam), relation to the patch and matrix compartments, and excitatory synaptic inputs. Recorded cells were identified by intracellular biocytin staining. Compartmental identification was made by calbindinD28K immunohistochemistry in fixed slices. 2. Large stained neurons were morphologically heterogeneous and had aspiny or sparsely spiny dendrites and dense local axonal branches. They were located in the matrix or on the patch-matrix border. Axonal branches of the large aspiny cells were preferentially distributed in the matrix and gave off terminal boutons there. Some of the secondary dendrites arising from stem dendrites running along the border, however, crossed compartment boundaries and made fine branches in a patch. 3. Large aspiny cells had less negative resting membrane potentials and lower thresholds for spike generation than medium spiny cells. They showed longer-duration and larger-amplitude afterhyperpolarizations (AHPs) than medium spiny cells. During hyperpolarizing current pulses, apparent resistance slowly reduced, and a prominent sag was observed in the voltage record, which was absent in medium spiny cells. The large aspiny cells showed no spontaneous firing but had a tendency to fire repetitive spikes in response to depolarizing current pulses, although spike interval tended to increase in later spikes. Spike frequency of large aspiny cells increased less with current intensity than that of medium spiny cells. 4. Most large aspiny cells were considered to belong to a single physiological class, although one large aspiny cells showed shorter-duration AHPs than both most other large aspiny cells and medium spiny cells, and little spike-frequency adaptation. 5. Excitatory postsynaptic currents (EPSCs) of large aspiny cells induced by intrastriatal stimulation had two components. An early, linear component was blocked by 10 microM 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX), a selective antagonist of non-N-methyl-D-aspartate (NMDA) receptors. A later component with a nonlinear current-voltage (I-V) relationship was blocked by 50 microM DL-2-amino-5-phosphonovaleric acid (DL-APV), a selective antagonist of NMDA receptors. 6. From these results, four conclusions can be drawn. 1) Most large aspiny neostriatal cells in the matrix, although they take heterogeneous shapes, belong to one physiological class with long-duration AHPs and a strong time-dependent component of anomalous rectification.(ABSTRACT TRUNCATED AT 400 WORDS)


Effects of opioid and alpha 2-adrenoceptor agonists on the isolated ileum of morphine-dependent guinea-pigs during withdrawal and after clonidine treatment.

The present study was undertaken to investigate the effect of clonidine administration to opiate-dependent guinea-pigs after morphine withdrawal on subsequent twitch responses of the longitudinal muscle-myenteric plexus preparations to electrical field stimulation. The results indicate that clonidine, administered immediately after morphine removal, causes tolerance to the inhibition exerted by opioid and alpha 2-adrenoceptor agonists on the electrically-evoked twitches. Such a finding suggests that the mechanism of action of clonidine involves not only its well-known effects on locus coeruleus neurons but also that it has specific actions on the myenteric plexus. This work shows the existence of interactions between opioid and alpha 2-adrenoceptor on the cholinergic neurons present in the isolated ileum.


Contractile response of human omental arteries to endothelin.

The effects of endothelin have been studied in isolated arterial segments (0.8-1 mm in external diam.) of human omental arteries obtained during the course of abdominal operations (15 patients, 7 men and 8 women). Paired segments, one normal and the other de-endothelized, were mounted for isometric recording of tension in organ baths. Endothelin produced concentration-dependent contractions with an EC50 value of 5.4 x 10(-9) M. Removal of endothelium did not affect significantly endothelin-induced contractions (EC50, 6.7 x 10(-9) M). Removal of extracellular calcium or addition of the calcium channel blocker nicardipine (10(-6) M) diminished but did not abolish responses to endothelin. These results indicate that endothelin exerts powerful contractile effects on human isolated omental arteries which are independent of the presence of an intact endothelial cell layer; this contraction cannot be explained solely by voltage-dependent calcium channels.


Further characterization of the protective effect of 8-cyclopentyl-1,3-dipropylxanthine on glycerol-induced acute renal failure in the rat.

In the rat, treatment with the alkylxanthine 8-cyclopentyl-1,3-dipropylxanthine (CPX) at a dose of 0.1 mg kg-1 antagonizes adenosine-induced falls in renal blood flow and reduces the severity of glycerol-induced acute renal failure. Treatment of glycerol-injected rats with 0.03 mg kg-1 of CPX resulted in no significant improvements in a range of indices of renal function. However, treatment with 0.1 or 0.3 mg kg-1 doses of CPX did significantly ameliorate acute renal failure although there were no significant differences in the degree of protection of renal function afforded by these two doses. In glycerol-injected rats, 0.1 or 0.3 mg kg-1 CPX administered either as a single dose or repeated doses every 12 h for two days did not inhibit renal phosphodiesterase. Thus the beneficial effects of CPX can be produced by doses that have no effect on renal phosphodiesterase activity whereas 0.1 mg kg-1 of CPX has been shown previously to antagonize the actions of adenosine. The findings provide further evidence that the beneficial effect of CPX in glycerol-induced acute renal failure is a consequence of adenosine antagonism and not phosphodiesterase inhibition.


Inhibition of dapsone-induced methaemoglobinaemia by cimetidine in the presence of trimethoprim in the rat.

Administration of dapsone in combination with trimethoprim and cimetidine to male rats resulted in a marked decrease (P less than 0.05) in measured methaemoglobin levels (46.2 +/- 24% Met Hb h) compared with administration of dapsone alone (124.5 +/- 24.4% Met Hb h). The elimination half-life of dapsone (814 +/- 351 min) was more than doubled in the presence of trimethoprim and cimetidine compared with control (355 +/- 160 min, P less than 0.05). However, there were no significant differences in AUC and clearance when dapsone was administered in combination with trimethoprim and cimetidine compared with dapsone alone. Co-administration of trimethoprim with dapsone in the absence of cimetidine did not affect either methaemoglobin formation, AUCs, half-lives, or clearance values of dapsone compared with control. There was a threefold increase in the AUC of trimethoprim (6296 +/- 2249 micrograms min mL-1) in the presence of dapsone compared with trimethoprim alone (2122 +/- 552 micrograms min mL-1). There was also a corresponding decrease in the clearance of trimethoprim in the presence of dapsone compared with control (19.1 +/- 6.9 vs 60.8 +/- 21.0 mL min-1). However, there was no change in the elimination half-life of trimethoprim between the two experimental groups (273 +/- 120 vs 292 +/- 54 min). The AUC of trimethoprim increased more than threefold in the presence of cimetidine (7100 +/- 1501 micrograms min mL-1) compared with trimethoprim alone (2122 +/- 552 micrograms min mL-1).(ABSTRACT TRUNCATED AT 250 WORDS)


Improved transdermal delivery of prostaglandin E1 through hairless mouse skin: combined use of carboxymethyl-ethyl-beta-cyclodextrin and penetration enhancers.

The optimal prescription of transdermal preparations of prostaglandin E1 (PGE1) for treatment of peripheral vascular diseases has been investigated. The chemical stability of PGE1 in fatty alcohol/propylene glycol (FAPG) ointment was markedly improved by carboxymethyl-ethyl-beta-cyclodextrin (CME-beta-CyD). Application of a PGE1 ointment containing the penetration enhancer, 1-dodecylazacycloheptane-2-one (Azone) or 1-[2-(decylthio)ethyl]azacyclopentane-2-one (HPE-101), onto the skin of hairless mice showed the increase of blood flow in the skin due to the vasodilating action of PGE1. In particular, the ointment containing a PGE1-CME-beta-CyD complex supplemented with HPE-101 showed the most prominent increase of the blood flow. Compared with other ointments, this ointment was found to show significantly greater transfer of HPE-101 into in-vitro preparations of the skin of hairless mice. Transfer of PGE1 into the skin was thought to be facilitated by this increased transfer of HPE-101. These results suggest that a combination of CME-beta-CyD and HPE-101 is useful for designing PGE1 ointments for topical application with good chemical stability and percutaneous permeability.


Absorption kinetics of cyclosporin in the rat.

Cyclosporin was administered (6 mg kg-1, i.v.) over 15 min, or (10 mg kg-1) by gavage, to two groups of 5 rats. Following i.v. infusion, cyclosporin exhibited triphasic behaviour with mean +/- s.e.m. disposition half-lives of 9.0 +/- 1.3 min, 4.0 +/- 0.5 h and 16.0 +/- 1.7 h. Following oral administration, peak blood concentration (Cmax) of 1290 +/- 93 ng mL-1 was reached after 5 h, when cyclosporin absorption essentially ceased. The absolute bioavailability (F) of cyclosporin was 24.0%. Standard laboratory rat chow consisting of 2% corn oil did not appear to alter cyclosporin absorption kinetics.


Toloxatone pharmacokinetics in the plasma and cerebrospinal fluid of the rabbit.

The pharmacokinetics of toloxatone (5 and 10 mg kg-1, i.v.) was studied in anaesthetized rabbits. There was a biexponential decline in plasma concentration with time. No differences were observed in the pharmacokinetic parameters with the increase of the dose. The terminal half-life was short (47.4 +/- 2.8 and 41.5 +/- 4.2 min for 5 and 10 mg kg-1, respectively). The total clearance was 79 +/- 18 mL min-1 after a dose of 5 mg kg-1 and 106 +/- 40 mL min-1 after a dose of 10 mg kg-1. The volume of distribution was 5.8 +/- 2.8 (5 mg kg-1) and 5.4 +/- 1.8 L (10 mg kg-1). The average percentage of toloxatone bound to plasma protein was 30% and was not affected by concentrations within the investigated range. In cerebrospinal fluid (CSF), the highest concentrations of toloxatone were obtained within 15 min after the end of the injection. The CSF level of toloxatone was equal to that of plasma unbound toloxatone and declined at a rate similar to toloxatone in plasma. These results suggest that the toloxatone passage through the blood-brain barrier may be completed by passive diffusion. In addition, the unbound plasma concentration would accurately reflect the toloxatone concentration in CSF and could be a useful tool for drug monitoring.


Stomach outline visualization in gastrointestinal transit studies using scintigraphy.

A major disadvantage of gamma scintigraphy in gastrointestinal transit studies is the inability to provide adequate delineation of anatomical details. As an aid to the important requirement of outlining the stomach to ensure accurate quantification of the time at which material empties from this organ, a new technique is described, using short half-life 81mKr gas to provide clearer identification of the stomach outline and position.


Contractions induced by phenylephrine and noradrenaline are differently affected by endothelium-dependent relaxation in rat aorta.

In rings of rat aorta precontracted with phenylephrine (10 microM) or noradrenaline (10 microM), addition of carbachol (10 microM) produced an endothelium-dependent relaxation. However, regardless of the concentration of agonist tested, both the intensity and duration of the relaxation were significantly less when noradrenaline, rather than phenylephrine, was used as the precontracting agent. The different responses observed do not appear to be related to destruction of endothelium-derived relaxing factor by autoxidation of noradrenaline since neither EDTA (30 microM) nor superoxide dismutase (30 units mL-1) improved the relaxation to carbachol. In addition, in endothelium-free rings, the noradrenaline (1 microM)-induced contraction was less sensitive than the phenylephrine (1 microM)-induced contraction to sodium nitroprusside (0.1 microM) or to 8-Br-cGMP (300 microM). With phenylephrine-, but not noradrenaline-, induced contraction, the relaxation triggered by carbachol was significantly reduced by pretreatment of the aortic rings with chloroethylclonidine (50 microM), which inactivates a subpopulation of alpha 1-adrenoceptors. Thus, the results confirm that both alkylation sensitive and resistant alpha 1-adrenoceptors exist in rat aorta and indicate that EDRF may discriminate between these two alpha 1-adrenoceptor subtypes which are differently affected by phenylephrine and noradrenaline.


Analysis of the effects of adenosine in skinned bovine coronary artery.

We have investigated the mechanism of adenosine-induced relaxation in relation to its effects on intracellular organelles in Triton X-100- and saponin-skinned bovine coronary arteries. In intact coronary arteries, high K+ and prostaglandin F2 alpha caused sustained contractions, whereas caffeine produced transient contractions. Triton X-100 treatment abolished these contractions. However, Triton X-100-skinned coronary arteries were responsive to added free calcium. There was no significant difference between calcium concentration-response curves obtained in the absence and presence of adenosine (50 microM). Unlike Triton X-100, in saponin-skinned arteries, caffeine produced transient contractions but high K+ and prostaglandin F2 alpha did not. Adenosine had no effect on caffeine-induced contractions in saponin-skinned coronary arteries. These data suggest that adenosine had no direct inhibitory effect on either the contractile apparatus or calcium release from sarcoplasmic reticulum in coronary arteries.


An in-vitro study of chloroquine transport in the rat submaxillary gland.

The uptake and efflux of chloroquine by the rat submaxillary gland in-vitro were studied under various incubation conditions. Variations in the extracellular pH significantly affected both the uptake and efflux of the drug. Increasing chloroquine concentration significantly decreased the uptake. Uptake was also decreased significantly (P less than 0.05) when compared with control conditions (pH 7.40, 37.5 degrees C, O2 aeration, 6 x 10(-6) M chloroquine) by the following experimental variations: aeration of the incubation medium with N2 instead of O2; decrease of bath temperature from 37.5 to 4 degrees C; addition of metabolic inhibitors, cyanide (10(-3) M), iodoacetic acid (10(-3) M) and o-nitrophenol (10(-3) M). Cimetidine (10(-3) M), a known organic cationic inhibitor, had no significant effect on chloroquine uptake when compared with control values. These results show that the uptake of chloroquine by the rat submaxillary gland in-vitro is concentration-dependent and this is indicative of possible saturable binding sites for the drug in the gland. These results suggest that the transfer of chloroquine across the submaxillary gland may be mediated by an active transport process. On the other hand, it is possible that the apparent active transport process implicated in this study could be a consequence of chloroquine ion trapping in the acidic interior of lysosomes.


Influence of inhibition of extraneuronal uptake and of O-methylation on the hyperglycaemia caused by sympathomimetic amines in depancreatized rats.

This study aimed at testing whether the O-methylating system (extraneuronal uptake + O-methylation) modulates, in-vivo, beta-adrenoceptor-mediated responses. The influences of U-0521 (3,4-dihydroxymethylpropiophenone, an inhibitor of catechol-O-methyl transferase (COMT)) and hydrocortisone (an inhibitor of extraneuronal uptake) on the hyperglycaemia evoked by isoprenaline and adrenaline were compared. Both inhibitors enhanced the increase of the plasma glucose level induced by either isoprenaline (0.36 nmol kg-1 min-1) or adrenaline (0.55 nmol kg-1 min-1). The enhancement caused by U-0521 developed faster than that caused by hydrocortisone, but was of the same magnitude. This is the first report of supersensitivity to sympathomimetic amines caused by inhibition of either COMT or extraneuronal uptake in-vivo and for a response not involving smooth muscle cells.


Supersensitivity of isolated atria from diabetic rats to adenosine and methacholine: modulation by pertussis toxin.

The chronotropic response of isolated right atria obtained from rats made diabetic 14-15 weeks previously by streptozotocin, was compared with age-matched controls. Diabetic rat atria are significantly more sensitive to the negative chronotropic actions of adenosine and of methacholine. Pretreating both control and diabetic rats with 2.5 mg kg-1 pertussis toxin attenuated the negative chronotropic effects of methacholine and adenosine on isolated atria, although diabetic atria still displayed a significantly greater sensitivity to these agonists (P less than 0.05-0.001). The negative chronotropic effects of methacholine and adenosine on both control and diabetic atria were abolished following pretreatment with higher doses of pertussis toxin (10 mg kg-1). These results suggest that pertussis toxin-sensitive G proteins may be involved in the supersensitivity of diabetic hearts to methacholine and adenosine.


Inhibitory effect of 2-hydroxypropyl-beta-cyclodextrin on crystal-growth of nifedipine during storage: superior dissolution and oral bioavailability compared with polyvinylpyrrolidone K-30.

To prevent the crystal-growth of nifedipine during storage, 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CyD) was employed as a hydrophilic drug carrier and compared with polyvinylpyrrolidone K-30 (PVP). Amorphous nifedipine powders were prepared by spray-drying with HP-beta-CyD or PVP, and their crystal-growing behaviour at accelerated storage conditions were examined by X-ray diffraction analysis and microscopy. Although PVP initially retarded the crystallization of nifedipine, it failed to control the increase of crystal size after prolonged storage at 60 degrees C, 75% r.h., resulting in a remarkable decrease in dissolution rate in water. In sharp contrast, a relatively fine and uniform size of nifedipine crystals was maintained in the HP-beta-CyD system even after accelerated storage conditions. The enhanced dissolution observed for all the HP-beta-CyD systems in a dissolution medium containing 0.1% non-ionic surfactant HCO-60 were clearly reflected in the in-vivo absorption of nifedipine following oral administration to dogs. These results suggest that HP-beta-CyD is particularly useful in solving problems encountered on storage of amorphous nifedipine in solid dosage forms.


Effect of physical and chemical properties on drug release from selected thermosoftening vehicles.

The release profile of several drugs, (chlorpheniramine maleate, salicylic acid, hydrochlorothiazide, p-hydroxy benzoic acid, sulphafurazole, anhydrous theophylline) and the marker (D&C yellow No. 10) was detailed to determine the effect of physical and chemical properties on release from selected thermosoftening matrices (Gelucire 50/02 and 50/13). At a concentration of drug or marker of 2.5% w/w, hydrochlorothiazide showed the slowest release from G50/02, due to its low aqueous solubility, while theophylline showed the highest release owing to its low mol. wt and moderate aqueous solubility. Release reflected two of the selection criteria, aqueous solubility and mol. wt, set forth for the drug/markers used in the study. The hydrophobic matrix, G50/02, offered no enhancement in drug release and functioned in a manner commensurate with other hydrophobic matrices. No hydrogen bonding was noted between any of the drugs or markers and the matrix. As drug or marker concentration increased from 2.5 to 15% w/w, potential hydrogen bonding was noted between p-hydroxy benzoic acid and the matrix. Theophylline no longer had the highest release being replaced by chlorpheniramine maleate and D&C yellow No. 10. With Gelucire excipient G50/13, chlorpheniramine maleate showed the highest release; it dissolved within the matrix at experimental temperature and lowered the matrix melting point. The matrix swelled upon exposure to the dissolution medium and it was from this swollen layer that release occurred. Sulphafurazole, hydrochlorothiazide, salicylic acid and p-hydroxy benzoic acid exerted a similar effect to chlorpheniramine maleate on the matrix. No hydrogen bonding was observed between the drugs and matrix.(ABSTRACT TRUNCATED AT 250 WORDS)


Effect of chronic nicotine treatment and withdrawal on rat striatal D1 and D2 dopamine receptors.

The effects on rat striatal dopamine receptors after chronic nicotine administration (3 and 12 mg kg-1 day-1), and after withdrawal from chronic nicotine (12 mg kg-1 day-1), were studied. After 21 days of continuous minipump infusion, the control (saline) and nicotine-treated rats were killed. The nicotine-withdrawal rats were killed on day 28, 7 days after pump removal. Radioligand studies were performed to determine D1 ([3H]SCH23390) and D2 ([3H]spiperone) striatal dopamine receptor affinity (Kd) and maximum binding (Bmax). Dopamine inhibition of antagonist binding at 3 concentrations and the effect of 0.3 mM GTP on binding affinity were examined. No statistically significant differences between control and nicotine treatment or withdrawal groups were noted in either D1 or D2 receptor Kd or Bmax. Although nicotine has been shown to affect nigrostriatal dopamine release, chronic treatment does not appear to alter overall striatal dopaminergic receptor binding parameters.


Calcium-calmodulin-dependent activation of adenylate cyclase in prostaglandin-induced electrically-monitored intestinal secretion in the rat.

The calcium-calmodulin antagonist 5-iodo-C8-W7 inhibited the PGE2-induced stimulation of cAMP production by isolated enterocytes from rat small intestine. It also reduced the secretory response of intestinal sheets to PGE2, measured as a rise in short-circuit current. It did not however, inhibit the electrical responses to forskolin and dibutyryl cAMP, nor to acetylcholine, a secretagogue whose effect is not mediated by cAMP. It is concluded that the receptor-mediated activation of adenylate cyclase and the subsequent secretory response are dependent upon calcium-calmodulin.


Alpha 1-adrenoceptor subtypes in the rat ventricular muscle.

Scatchard analyses of [3H]prazosin binding in rat ventricular muscle membranes showed biphasic curves, which identified alpha 1High- and alpha 1Low-affinity sites. The alpha 1High-affinity site was completely inhibited by 1 microM phenoxybenzamine. The displacement potencies of alpha 1-adrenergic antagonists were characterized by [3H]prazosin binding to alpha 1High- and alpha 1Low-affinity sites in the absence and presence of 1 microM phenoxybenzamine. The affinities of most chemicals for alpha 1Low-affinity sites were significantly lower than those for alpha 1High-affinity sites, but WB-4101 (2-(2,6-dimethoxy-phenoxyethyl)aminomethyl-1,4-benzodioxane), arotinolol, cinanserin, nifedipine, and p-aminoclonidine had the same affinities for both alpha 1Low- and alpha 1High-affinity sites. These results show that two alpha 1-adrenoceptor subtypes, alpha 1High- and alpha 1Low-affinity, are present in the rat heart, and that there are physical variations in alpha 1-adrenoceptor binding sites, based on their selectivity to antagonists.


Polyarteritis nodosa confined to calf muscles.

Two patients who developed pain and tenderness in their calf muscles, without other organ system involvement, are described. A gastrocnemius biopsy disclosed necrotizing muscular arteritis. Five other reported cases are reviewed and we conclude that there is a limited form of polyarteritis nodosa localized to calf muscles, with a differentiated and more benign clinical course, a differential diagnosis restricted to local pathological processes and prompt response to treatment with corticosteroids alone.


Combined effect of doxazosin and pindolol on blood pressure control and lipid concentrations in patients with essential hypertension selected from general practice. Hunter Hypertension Research Group.

Doxazosin, an alpha-adrenergic antagonist with potentially favourable effects on lipid status was evaluated in 25 otherwise healthy general practice patients with mild to moderate hypertension. A mean dose of 5.7 mg produced a significant fall in lying and standing systolic and diastolic blood pressure over an 11 week period as well as an 8% increase in HDL cholesterol and a 7.5% decrease in the total cholesterol/HDL cholesterol ratio. Following this, the doxazosin dose was halved and pindolol, a beta-blocker with intrinsic sympathomimetic activity, was added (mean dose 7.8 mg) to maintain blood pressure control. At the completion of 11 weeks of combined alpha- and beta-blockade, HDL cholesterol and triglyceride levels were found to be unaltered when compared to pretreatment; however there was a small but significant fall in total cholesterol. This study demonstrates that this combination of alpha- and beta-blocking antihypertensive therapy can produce potentially favourable blood lipid changes.


Comparison of antihypertensive and lipid actions of terazosin and atenolol in essential hypertension.

Terazosin is a selective alpha 1-adrenoceptor antagonist; its actions on the serum lipoprotein profile were compared with those of the cardioselective beta-adrenoceptor antagonist atenolol in 40 patients with mild to moderate hypertension. Atenolol and terazosin were titrated over six weeks until blood pressure control (diastolic blood pressure less than 90 mmHg or greater than 10 mmHg fall in blood pressure) or a maximum dose of atenolol 100 mg or terazosin 10 mg had been achieved. Patients not controlled were then prescribed additional diuretic therapy (cyclopenthiazide 0.5 mg and potassium 1200 mg). At each visit blood pressure and adverse events were recorded; plasma lipids were measured at baseline, six and 12 weeks. During titration there was a linear decrease in systolic (-29 mmHg on atenolol and -24 mmHg on terazosin) and diastolic blood pressure (-17 and -12 mmHg) in both groups without subsequent change over the next six weeks; atenolol reduced heart rate (-11 bpm) without change on terazosin. During the initial six weeks the total cholesterol fell in both groups; however, there were significant between-treatment differences in triglyceride responses with a fall on terazosin and a rise on atenolol. Comparing atenolol and terazosin over the total 12 week study (irrespective of thiazide treatment) increased triglyceride levels and reduced cholesterol ratios and HDL were demonstrated on atenolol, contrasting with reduced triglyceride levels and elevated HDL and cholesterol ratios of terazosin.(ABSTRACT TRUNCATED AT 250 WORDS)


Synaptosomal plasma membrane transport of excitatory sulphur amino acid transmitter candidates: kinetic characterisation and analysis of carrier specificity.

The transport kinetics of the excitatory sulphur-containing amino acid (SAA) transmitter candidates, L-cysteine sulphinate (L-CSA), L-cysteate (L-CA), L-homocysteine sulphinate (L-HCSA), and L-homocysteate (L-HCA), together with their plasma membrane carrier specificity, was studied in cerebrocortical synaptosome fractions by a sensitive high performance liquid chromatographic assay. A high affinity uptake system could be demonstrated for L-CSA (Km = 57 +/- 6 microM; Vmax = 1.2 +/- 0.1 nmol/min/mg protein) and L-CA (Km = 23 +/- 3 microM; Vmax = 3.6 +/- 0.1 nmol/min/mg protein), whereas L-HCSA (Km = 502 +/- 152 microM; Vmax = 6.1 +/- 1.3 nmol/min/mg protein) and L-HCA (Km = 1550 +/- 169 microM; Vmax = 10.3 +/- 1.1 nmol/min/mg protein) exhibited much lower affinity as transport substrates. In all cases, only a single, saturable Na(+)-dependent component of uptake could be identified, co-existing with a non-saturable, Na(+)-independent influx component. Plasma membrane carrier specificity of the SAAs was established following comparison with other high-affinity neurotransmitter systems. High-affinity L-CSA and L-CA transport and low-affinity L-HCSA and L-HCA transport demonstrate strong positive correlations in inhibition profiles when compared against each other or individually against the high-affinity transport of L-[3H]glutamate, L-[3H]aspartate, or D-[3H]aspartate. Moreover, the transport systems for the excitatory SAAs exhibited a negative correlation when compared in inhibition profiles with the high affinity transport of both [3H] gamma-aminobutyric acid (GABA) and [3H]taurine.(ABSTRACT TRUNCATED AT 250 WORDS)


[Recovery of hematopoiesis after high-dose chemotherapy and peripheral blood stem cell autografts in children].

Hematopoietic recovery kinetics were evaluated in 34 children with therapy-refractory malignant tumors who underwent a total of 35 peripheral blood stem cell autografts (PBSCT) after marrow-ablative chemotherapy without total body irradiation. A negative correlation was found between the numbers of colony-forming units of granulocyte-macrophage (CFU-GM) infused per kilogram of the patients' body weight and the time of achieving an absolute granulocyte count (AGC) of 0.5 x 10(9)/l or a platelet count of 50 x 10(9)/l (granulocyte: r = -0.631, p less than 0.001, platelet: r = -0.590, p less than 0.001). The patients were classified into three groups; 14 patients who received less than 1 x 10(5) CFU-GM/kg (group A), 7 patients who received 1-3 x 10(5) CFU-GM/kg (group B), and 14 patients who received greater than or equal to 3 x 10(5) CFU-GM/kg (group C). The AGC recovered to 0.5 x 10(9)/l by 21 day in group A, 14 day in group B, and 10 day in group C. The platelet count recovered to 50 x 10(9)/l 102 in group A, 23 in group B, and 16 day in group C patients. The final platelet infusion was on day 60 in group A, day 12 in group B, and day 12 in group C. Transient decrease in the blood cell count developed in all patients 3 to 7 weeks after transplantation, and two cases developed reversible ITP. In the remaining patients, the recovered hematopoietic function was sustained for 1-48 months after transplant action.(ABSTRACT TRUNCATED AT 250 WORDS)


Inhibition of viral and cellular promoters by human wild-type p53.

Mutation of the p53 tumor suppressor gene is a recurring event in a variety of human cancers. Wild-type p53 may regulate cell proliferation and has recently been shown to repress transcription from several cellular promoters. We studied the effects of wild-type and mutant human p53 on the human proliferating-cell nuclear antigen promoter and on several viral promoters including the simian virus 40 early promoter-enhancer, the herpes simplex virus type 1 thymidine kinase and UL9 promoters, the human cytomegalovirus major immediate-early promoter-enhancer, and the long terminal repeat promoters of Rous sarcoma virus, human immunodeficiency virus type 1, and human T-cell lymphotropic virus type I. HeLa cells were cotransfected with a wild-type or mutant p53 expression vector and plasmids containing a chloramphenicol acetyltransferase reporter gene under viral (or cellular) promoter control. Expression of wild-type p53 correlated with a consistent and significant (6- to 76-fold) reduction of reporter enzyme activity. A mutation at amino acid 143 of p53 releases this inhibition significantly with all the promoters studied. Expression of a p53 mutated at any one of the five amino acid positions 143, 175, 248, 273, and 281 also correlated with a much smaller (one- to sixfold) reduction of reporter enzyme activity from the herpes simplex virus type 1 thymidine kinase promoter. These mutant forms of p53 are found in various cancer cells. Thus, failure of tumor suppression correlates with loss of the promoter inhibitory effect of p53.


[Microalbumin excretion in the group of patients with asymptomatic hematuria].

Urine albumin was determined in patients with chronic glomerular injuries with normal renal function, who had shown positive test of microhematuria but negative test of proteinuria at any time of our renal clinic. The subjects were divided into 4 groups: (1) IgA nephropathy (IgAN); 13, (2) asymptomatic hematuria (AS); 18, (3) nephrotic syndrome in complete remission (CR); 21 and (4) age matched normal subjects; 44. Urine albumin concentration was measured with radioimmunoassay in the ambulatory urine, and, in some cases, in the urine obtained after supine position for 30 minutes to demonstrate the effect of ambulatory physical movement on albumin excretion. Also urine alanine aminopeptidase (AAP) and N-acetyl-beta-D-glucosaminidase (NAG) were estimated by monitoring the absorbance of products released by the enzyme, as the indices of tubular function. The results indicated that urine albumin were 8.4 +/- 7.3 mg/g Cr (Mean +/- SD) in normal subjects, and 8.8 +/- 8.9 mg/g Cr in CR (vs. controls: N.S.), 18.9 +/- 14.5 mg/g Cr in AS (P = 0.0071), and 22.2 +/- 14.9 mg/g Cr in IgAN (P = 0.0063). The albumin excretion had no relation with the grade of microhematuria and also with the ambulatory physical movement. Moreover, AAP and NAG excretion in each group had shown no significant alterations. These results indicate that urine albumin increases in IgAN and AS with normal renal function and with microhematuria alone, but not in CR. Urine albumin is probably glomerular origin, since no abnormality is found in the tubular functions.


[A clinical study of the long-term therapeutic effects of low-dose erythromycin in diffuse panbronchiolitis--with special reference to changes in tumor-associated carbohydrate antigens in serum].

We investigated the long-term (3-30 months) therapeutic effects of low-dose (300-600 mg/day) erythromycin in 26 patients with diffuse panbronchiolitis (DPB). Significant improvements of pulmonary functions especially in %VC and PaO2 as well as respiratory symptoms were shown. However, erythromycin treatment was not associated with a significant change in surface phenotypes on peripheral blood lymphocytes (CD4, CD8, CD4/CD8). It is well known that serum levels of tumor-associated carbohydrate antigens such as SLX (sialylated Lewis X-i) and CA19-9 (sialylated Lewis(a)) are significantly elevated in patients with DPB. In the present study, 68.4% (13/19) of DPB patients showed marked elevation of SLX and 52.9% (9/17) showed marked elevation of CA19-9 levels in serum. These positive ratios were significantly decreased by erythromycin treatment to 31.6% (6/19) in SLX and 23.4% (4/17) in CA19-9. The mean values of each marker were also significantly decreased after erythromycin administration from 54.9 +/- 26.9 U/ml to 39.5 +/- 22.1 U/ml for SLX and from 70.5 +/- 77.4 U/ml to 28.8 +/- 37.4 U/ml for CA19-9.


[Sialyl Lewis X-i (SLX) in the bronchoalveolar lavage fluid from patients with lung cancer].

The measurement of serum SLX is thought to be a useful aid in the diagnosis of malignant diseases, particularly adenocarcinoma of the lung. In the present investigation, we measured and compared SLX values in BALF from affected and normal bronchi, obtained from 83 patients. They consisted of 64 males and 19 females, with mean age of 60 years, consisting of 8 normal controls, 19 cases of benign lung disease, and 56 cases of primary lung cancer. SLX value in BALF from normal bronchi was significantly higher in patients with lung cancer than in normal controls, but there was no significant difference in SLX value between lung cancer and benign lung disease. On the other hand, SLX value from affected bronchi was significantly higher in patients with lung cancer than in normal controls and patients with benign lung disease. The rate of elevated SLX in BALF from affected bronchi was significantly higher in patients with lung cancer than in those with benign lung disease. These results suggest that measurement of SLX levels in BALF from affected bronchi may be a useful method for differential diagnosis of primary lung cancer.


Amino acid neurotransmitters in hypertension.

There is compelling evidence for the participation of excitatory and inhibitory amino acids in the neural regulation of blood pressure in the normotensive rat. This is most clearly evident in the neural pathways which form the baroreceptor reflex arc. Excitatory amino acids are contained in baroreceptor afferents, neurons in the nucleus tractus solitarius (NTS) and neurons in the rostral ventrolateral medulla (RVLM). Inhibitory neurons in the caudal ventrolateral medulla (CVLM) contain gamma-aminobutyric acid. Electrophysiological and pharmacological evidence indicates that amino acid neurotransmitters are critically important to the normal function of these integrative sites in the baroreceptor reflex. Spontaneously hypertensive rats (SHR) differ from Wistar Kyoto (WKY) controls in their responses to stimulation, inhibition or lesions of neurons in the baroreceptor arc. One week after baroreceptor denervation, blood pressure is elevated in WKY but not in SHR. Stimulation of the CVLM results in a greater fall in pressure in SHR than WKY, whereas injection of tetrodotoxin into the CVLM results in a smaller increase in pressure in SHR. Blockade of glutamate receptors in the spinal cord attenuates the response to stimulation of the RVLM in both SHR and WKY, but reduces resting blood pressure in SHR only. These experiments suggest that altered activity in amino acid pathways contributes to the pathogenesis of hypertension in SHR.


Clonal analysis of Escherichia coli serotype O6 strains from urinary tract infections.

A total of 36 Escherichia coli urinary tract isolates (UTI) of serotype O6, with different combinations of capsule (K) and flagellin (H) antigens, were analysed according to the outer membrane pattern (OMP), serum resistance properties, mannose-resistant hemagglutination using various types of erythrocytes, and also for the genetic presence and the expression of P-fimbriae, S fimbriae/F1C fimbriae, Type 1 fimbriae, aerobactin and hemolysin. Twenty selected strains were further analysed by pulsed field gel electrophoresis (PFGE), elaborating genomic profiles by XbaI cleavage and subsequent Southern hybridization to virulence-associated DNA probes. It could be shown that O6 UTI isolates represent a highly heterogeneous group of strains according to the occurrence and combination of these traits. Relatedness on the genetic and the phenotypic level was found for some of the strains exhibiting the same O:K:H:F serotype. DNA long-range mapping further indicated some interesting features, according to the copy number and the genomic linkage of virulence genes.


DNA sequence polymorphism within hominoid species exceeds the number of phylogenetically informative characters for a HOX2 locus.

Within- and between-species variability was examined in a noncoding 238-bp segment of the HOX2 cluster. DNA of 4-26 individuals of four species (Pongo pygmaeus, Pan troglodytes, Gorilla gorilla, and Homo sapiens) was PCR amplified and electrophoresed in a denaturing gradient gel to screen for variability. Coupled amplification and sequencing was used to determine the complete sequence for each of the different alleles identified, one each in humans and orangutans, two in chimpanzees, and four in gorillas. Maximum-parsimony methods were used to construct a gene tree for these sequences. Alleles in all four species cluster into groups consisting of only one species (i.e., alleles within a species are monophyletic). The number of base-pair differences observed among alleles within P. troglodytes and within G. gorilla is larger than the number of base-pair substitutions that phylogenetically link Pan with Homo. Given these and other published data, it is premature to accept any particular phylogenetic tree that relates these three genera through two separate speciation events.


Mitochondrial DNA reveals formation of nonhybrid frogs by natural matings between hemiclonal hybrids.

The European water frog Rana esculenta (RL), a natural hybrid between R. ridibunda (RR) and R. lessonae (LL), reproduces by hybridogenesis: haploid gametes usually contain an intact chromosome set of R. ridibunda (R); the lessonae nuclear genome (L) is lost from the germ line. Hybridity is restored in the next generation, via fertilization by syntopic R. lessonae. Matings between two hybrids (RL x RL) usually give inviable R. ridibunda (RR) progeny. The adult R. ridibunda subpopulation of Trubeschloo, a gravel pit in northern Switzerland, consists only of females. Fragment patterns for mitochondrial DNA (mtDNA) of these R. ridibunda were identical with those of syntopic R. esculenta and of local populations of R. lessonae; they differed from the patterns in eastern European populations of R. lessonae and of R. ridibunda mtDNAs (3.7% and 9.3% estimated sequence divergence, respectively). In contrast, mtDNAs of two R. ridibunda from an introduced Swiss population with both sexes, although different (2.7% divergence) from each other, were typical R. ridibunda rather than R. lessonae mtDNAs. These data, together with unisexuality, demonstrate conclusively that the all-female R. ridibunda population at Trubeschloo originated from matings between two R. esculenta. The formation of independently reproducing R. ridibunda populations via such hybrid x hybrid matings is precluded because progeny of these matings are unisexual. Recombination in the regenerated fertile R. ridibunda females, followed by matings with R. lessonae, nevertheless provides a mechanism for meiotic reshuffling of genetic material in ridibunda haplotypes that is not typically available in hemiclonal lineages.


Patterns of alcohol consumption in the Kimberley aboriginal population.

To estimate patterns of alcohol consumption and alcohol-related problems among adult Aborigines in the Kimberley region of Western Australia. A community survey of adult Aborigines. A stratified random sample of 516 Aboriginal men and women over the age of 15 years in the Kimberley. Participants' reports of their frequency and quantity of alcohol consumption, and their lifetime experience of alcohol-related problems; and the laboratory measure gamma-glutamyltranspeptidase. Aborigines in the Kimberley were more likely to be non-drinkers than non-Aborigines in the Australian population, but the majority of drinkers consumed hazardous amounts of alcohol: 85% (95% Cl, 82% to 88%) of drinkers in the population were estimated to be drinking above the level defined by the National Health and Medical Research Council (NHMRC) as harmful. Alcohol abuse among Aborigines in the Kimberley is a major public health problem which requires urgent action.


The pharmacological relief of pain--contemporary issues.

To provide a general description of the pharmacological principles of pain management particularly the management of severe pain with opioid analgetic agents. Literature updating previous reviews by the authors in this Journal and the Proceedings of the VIth World Congress on Pain, Adelaide, 1-6 April 1990. Studies were selected under the subheadings: opioid receptors; endogenous opioids; development of new agents; recognition of the need to improve analgesia; pharmacokinetics and pharmacodynamics of opioids; and novel drug delivery methods. The extracted information was more descriptive than quantitative. The pharmacological relief of pain involves treatment of patients with drugs characterised by extremely large interpatient variability in pharmacokinetics and pharmacodynamics by way of an extremely complex interaction with endogenous control mechanisms. It is not surprising that management of pain in many patients is less than adequate. Although much vigorous interdisciplinary research is being undertaken to develop a scientific basis for understanding pain and analgesia, improvements in the clinical management of pain can only occur if practitioners recognise the need for individualised methods of pain relief and treat their patients accordingly.


Drug-induced pulmonary disease.

To review some of the types of drug-induced pulmonary disease reported in Australia. Reports of pulmonary side effects made to the Australian Adverse Drug Reaction Advisory Committee between December 1972 and March 1991. Reports of drug-induced pulmonary disease from other countries are also reviewed. Only reports involving large patient numbers are included in the review to limit the bias likely from limited patient numbers. The incidence of adverse responses to a range of commonly used drugs from other countries is compared with reports to the Australian Adverse Drug Reaction Advisory Committee in the last 19 years. Of adverse drug reactions reported in Australia in the last 19 years 7.7% were of a pulmonary side effect and 55% of these involved a syndrome of airway dysfunction. Bronchospasm and cough were the two most common symptoms of airway dysfunction and beta-blocking drugs and radio contrast media were the drug groups most commonly reported to cause them. These figures are similar to data reported from other countries. Some of the more commonly occurring types of drug-induced disease affecting either the airways or the lung interstices are reviewed. Medical practitioners should maintain a high index of clinical suspicion that any unexplained pulmonary disease could be caused by a drug. Most of these reactions are reversible if the drug is withdrawn in time and if additional appropriate measures are taken as required.


Changing patterns of medical treatment in acute myocardial infarction. Observations from the Perth MONICA Project 1984-1990.

Descriptive study of trends in the drug therapy for acute myocardial infarction. Population-based register of acute coronary events compiled for the years 1984 to 1990 in the course of the Perth MONICA project. 5294 cases meeting clinical criteria for acute myocardial infarction. Striking changes were seen in the use of aspirin before admission to hospital (from 4% to 18%). During the stay in hospital the use of beta-blockers increased steadily from 52% to 76%, while the use of aspirin increased 3.5-fold from 25% to 88% and the use of streptokinase increased 13.5-fold from 2.4% to 32.4%. The proportion of patients prescribed beta-blockers on discharge from hospital increased from 46% to 65% and that for aspirin rose from 16% to 83%. There were also major relative increases in the use of lipid-lowering agents and declines in the use of antiarrhythmic drugs. These trends in the pharmacological management of myocardial infarction mirror the emerging evidence from clinical trials, although the increases in the use of certain types of drugs antedated publication of the results of major randomised studies. The changes in therapy would partly explain observed improvements in case fatality and may have contributed to the decline in coronary mortality observed in the Perth community.


[A case with Graves' disease whose TBII index was decreased after heart surgery].

This case report describes a patient with Graves' disease who underwent heart surgery and we studied changes in thyroid hormone and antithyroid autoantibodies. TBII index of this case, which was high value before surgery, was decreased after surgery for two years, and the patient did not need any antithyroid drug. It is suggested that these changes may be due to cardio-pulmonary bypass and administration of steroids during heart surgery. This case indicates that heart surgery may influence the clinical course of Graves' disease.


Separation of factors required for cleavage and polyadenylation of yeast pre-mRNA.

Cleavage and polyadenylation of yeast precursor RNA require at least four functionally distinct factors (cleavage factor I [CF I], CF II, polyadenylation factor I [PF I], and poly(A) polymerase [PAP]) obtained from yeast whole cell extract. Cleavage of precursor occurs upon combination of the CF I and CF II fractions. The cleavage reaction proceeds in the absence of PAP or PF I. The cleavage factors exhibit low but detectable activity without exogenous ATP but are stimulated when this cofactor is included in the reaction. Cleavage by CF I and CF II is dependent on the presence of a (UA)6 sequence upstream of the GAL7 poly(A) site. The factors will also efficiently cleave precursor with the CYC1 poly(A) site. This RNA does not contain a UA repeat, and processing at this site is thought to be directed by a UAG...UAUGUA-type motif. Specific polyadenylation of a precleaved GAL7 RNA requires CF I, PF I, and a crude fraction containing PAP activity. The PAP fraction can be replaced by recombinant PAP, indicating that this enzyme is the only factor in this fraction needed for the reconstituted reaction. The poly(A) addition step is also dependent on the UA repeat. Since CF I is the only factor necessary for both cleavage and poly(A) addition, it is likely that this fraction contains a component which recognizes processing signals located upstream of the poly(A) site. The initial separation of processing factors in yeast cells suggests both interesting differences from and similarities to the mammalian system.


Comparative analysis of the intracellular localization of c-Myc, c-Fos, and replicative proteins during cell cycle progression.

In eukaryotic cells, nucleus-cytoplasm exchanges play an important role in genomic regulation. We have analyzed the localization of four nuclear antigens in different growth conditions: two replicative proteins, DNA polymerase alpha and proliferating cell nuclear antigen (PCNA), and two oncogenic regulatory proteins, c-Myc and c-Fos. A kinetic study of subcellular localization of these proteins has been done. In cultures in which cells were sparse, these proteins were detected in the nucleus. When proliferation was stopped by the high density of culture cells or by serum starvation, these proteins left the nucleus for the cytoplasm with different kinetics. DNA polymerase alpha is the first protein to leave the nucleus, with the PCNA protein, c-Fos, and c-Myc leaving the nucleus later. In contrast, during serum stimulation c-Fos and c-Myc relocalize into the nucleus before the replicative proteins. We also noticed that in sparse cell cultures, 10% of the cells exhibit a perinuclear staining for the DNA polymerase alpha, PCNA, and c-Myc proteins but not for c-Fos. This peculiar staining was also observed as an initial step to nuclear localization after serum stimulation and in vivo in Xenopus embryos when the G1 phase is reintroduced in the embryonic cell cycle at the mid-blastula stage. We suggest that such staining could reflect specific structures involved in the initiation of the S phase.


Targeted misexpression of Hox-4.6 in the avian limb bud causes apparent homeotic transformations.

In the limb bud the 5' members of the Hox-4 gene cluster are expressed in a nested set of overlapping domains which are progressively restricted in the posterior and distal directions. These domains arise early in limb bud development and come to approximate the primordia of the major structural elements of the limb along the anterior/posterior axis (Fig. 1). This pattern, and the fact that surgical manipulations which lead to mirror image duplications along the anterior/posterior axis give rise to mirror image duplications of the domains of expression of these genes, have led to the proposal that these transcription factors specify positional identity along the anterior/posterior axis. Here we test this hypothesis directly using replication-competent retroviral vectors to expand the domain of expression of the Hox-4.6 gene anteriorly during limb development in vivo. We report that alteration of the domain of expression of the Hox-4.6 gene in the developing limb leads to reproducible pattern alterations consistent with a posterior homeotic transformation.


Repetitive injections of L-glutamic acid, in contrast to those of N-methyl-D,L-aspartic acid, fail to elicit sustained hypothalamic GnRH release in the prepubertal male rhesus monkey (Macaca mulatta).

The purpose of the present study was to examine whether repetitive intravenous injections of L-glutamic acid (Glu), like those of N-methyl-D,L-aspartic acid (NMA), are able to elicit a sustained train of gonadotropin releasing hormone (GnRH) discharges from the hypothalamus of the prepubertal male monkey. In order to utilize pituitary luteinizing hormone (LH) secretion as a bioassay of hypothalamic GnRH release, the responsiveness of the gonadotroph of the prepubertal animals was enhanced prior to the study with a chronic intermittent intravenous infusion of the synthetic decapeptide (0.1 microgram/min for 3 min every h). Sequential intravenous injections of Glu (150 mg/kg BW) were administered at 3-hour intervals for 6 or 24 h. Although the first injection of this acidic amino acid elicited a robust discharge of GnRH, subsequent stimulation with Glu resulted in GnRH discharges with progressively decreasing magnitudes, and by the 9th injection Glu-induced GnRH release was abolished. Peak concentrations of circulating Glu following the 1st and 4th Glu injection were indistinguishable (3,959 +/- 437 vs. 4,139 +/- 72 nmol/ml, respectively). Interestingly, the failure of repetitive intravenous injections of Glu to sustain pulsatile GnRH release was not associated with a loss of responsiveness to NMA administration, nor was it accompanied by a corresponding decrement in Glu induced growth hormone (GH) discharges. As previously demonstrated, repetitive intravenous administration of NMA (2-5 mg/kg BW) every 3 h for 9 h sustained pulsatile GnRH secretion without decrement. A similar intermittent infusion of kainic acid (KA; 1 mg/kg BW every 3 h for 6 h), however, elicited a GnRH response that mimicked that observed in response to intermittent Glu treatment.(ABSTRACT TRUNCATED AT 250 WORDS)


Transmitter content and afferent connections of estrogen-sensitive progestin receptor-containing neurons in the primate hypothalamus.

Progestin receptor-containing cells in the hypothalamus of the adult female green monkey (Cercopithecus aethiops) were examined by double-label immunocytochemical methods to determine their anatomical location, neurotransmitter content and afferent connections. Animals were ovariectomized and administered either estradiol valerate or the oil injection vehicle, and were sacrificed after 10 days of treatment. Using a monoclonal antibody raised against rabbit uterine progestin receptor (PR), the distribution of PR-immunoreactive cells in the mediobasal hypothalamus and the effect of estrogen treatment on this distribution was determined. PR-immunoreactive cells were found throughout the ventromedial nucleus (VMN), in the area between the VMN and fornix, and in the medial portion of the infundibular nucleus. Estrogen treatment dramatically increased both the number of labeled cells and the intensity of immunoreaction product in these regions. In double-immunostained sections, boutons immunoreactive for antigens indicative of serotonin, pro-opiomelanocortin derived peptides, GABA, catecholamine, neuropeptide Y, substance P, cholecystokinin, and somatostatin were demonstrated to establish synaptic contact with the soma of PR-immunoreactive hypothalamic neurons. In colchicine-pretreated animals, all PR-containing neurons in the mediobasal hypothalamus were found to contain immunoreactivity for glutamic acid decarboxylase, the enzyme required for synthesis of GABA. No evidence of colocalization with other antigens, including LHRH, was observed. Because LHRH neurons are known to receive a rich GABAergic innervation PR-containing GABAergic cells may represent steroid-sensitive sites of integration for inputs from other neural systems involved in the control of gonadotropin secretion.


Autoradiographic localization of receptors for glucagon-like peptide-1 (7-36) amide in rat brain.

Glucagon-like peptide-1 (GLP-1) has a sparse but well defined distribution in the rat brain where it is co-localized with glucagon-like immunoreactivity due to other fragments of the glucagon precursor. We have investigated the localization of GLP-1 receptors in rat brain using mono-125I-iodinated GLP-1(7-36) amide, the biologically active form of the peptide that occurs in brain, as the tracer for binding and autoradiographic studies of tissue sections. Displaceable binding of the label was sharply localized to discrete areas, being high in mamillary nuclei, the arcuate nucleus, nucleus of the solitary tract and the pretectal area, intermediate in the lateral septal nuclei, olfactory bulb, dorsal tegmental nuclei and the interpenduncular nucleus, and low in other regions. These results indicate areas where GLP-1(7-36) amide may have a role as a neurotransmitter or neuromodulator.


Membrane currents induced by L-homocysteic acid in mouse cultured hippocampal neurons.

The concentration-response relationship of membrane currents induced by L-homocysteic acid was studied on mouse embryonic hippocampal neurons in culture (n = 56). In the majority of neurons two phases in the dose-response relationship could be distinguished. The first was characterized by responses to 3-100 microM L-homocysteic acid which desensitized with a time-constant greater than 1 s in a concentration-dependent manner and were antagonized by 30 microM D-L-2-amino-5-phosphonovaleric acid indicating activation of the N-methyl-D-aspartate receptors. At higher concentrations of L-homocysteic acid this component was strongly depressed. The second phase was characterized by sustained responses that were concentration-dependent (1 mM L-homocysteic acid maximum concentration tested) and were not blocked by D-L-2-amino-5-phosphonovaleric acid indicating activation of non-N-methyl-D-aspartate receptors. Eight neurons did not exhibit these two-phase characteristics in the concentration-response relationship at the beginning of the recording. The magnitude of responses to L-homocysteic acid was positively related to concentration and the responses were partially blocked by D-L-2-amino-5-phosphonovaleric acid. In these neurons, however, repeated applications of L-homocysteic acid at concentrations 30 microM up to 300 microM resulted in a long-lasting, three- to four-fold increase of the membrane current. This increase was completely blocked by D-L-2-amino-5-phosphonovaleric acid (50-100 microM) suggesting that it was produced by activation of receptors.(ABSTRACT TRUNCATED AT 250 WORDS)


Effects of 6-hydroxydopamine lesions of the prefrontal cortex on tyrosine hydroxylase activity in mesolimbic and nigrostriatal dopamine systems.

The effects of prefrontal cortical dopamine depletion on subcortical dopamine function in the rat were examined. 6-Hydroxydopamine lesions of the dopaminergic innervation of the prefrontal cortex did not alter concentrations of dopamine or its metabolite 3,4-dihydroxyphenylacetic acid in either the striatum or nucleus accumbens. Similarly, the activity of the catecholamine biosynthetic enzyme tyrosine hydroxylase in the striatal complex was not changed in animals with prefrontal cortical lesions. Animals sustaining neurotoxic lesions of the prefrontal cortex were challenged with haloperidol in order to activate submaximally tyrosine hydroxylase activity. The magnitude of the haloperidol-induced increase in enzyme activity in the nucleus accumbens was significantly greater in lesioned subjects than in control animals. These data suggest that lesions of the prefrontal cortical dopamine innervation do not result in significant alterations in basal dopaminergic function in the striatal complex. However, lesions of the dopaminergic innervation of the prefrontal cortex significantly increase the responsiveness of mesolimbic dopamine afferents to pharmacological challenge.


Host dopaminergic afferents affect the development of DARPP-32 immunoreactivity in transplanted embryonic striatal neurons.

Homotopic transplantation provides an interesting way to observe the relationships between developing cells and ingrowing host afferents. We have performed a complete and selective elimination of the mesostriatal dopaminergic system in adult rats to observe the influence of its absence on the development and chemical differentiation of embryonic striatal cells. Cell suspensions from striatal primordia of 14-15-day-old embryos were transplanted into host striata that were (i) neuron-depleted by kainic acid (control group) or (ii) deprived of dopamine by 6-hydroxydopamine prior to the neuronal depletion by kainic acid (experimental group). The expression of dopamine- and adenosine 3',5'-monophosphate-regulated phosphoprotein (DARPP-32) by transplanted cells was observed in correlation with their innervation by host dopaminergic afferents which in turn were identified by tyrosine hydroxylase immunohistochemistry. Observations were made between four days and three months after transplantation. Four days after transplantation, no immunoreactivity for DARPP-32 was observed in transplants of control animals despite the presence of tyrosine hydroxylase-immunopositive fibers growing from the host to discrete cell clusters in the transplant. DARPP-32-labeled cells appeared soon afterwards. Six days after transplantation they displayed varying intensities of immunoreaction, ranging from just detectable to normal levels and were specifically targeted by developing tyrosine hydroxylase-immunopositive fibers. The number of DARPP-32-labeled cells increased rapidly and they formed increasingly compact clusters. Fourteen days after transplantation and afterwards, all the DARPP-32-labeled cells displayed an intensity of immunoreaction and a distribution comparable to that observed in long-term transplants. Transplants in the experimental hosts displayed the same organization and developmental features as the control transplants with the exception of DARPP-32 labeling which was not detected before eight days after transplantation. Ten days after transplantation, the distribution and intensity of DARPP-32 labeling was similar to that observed at six days in the control group. The evolution of DARPP-32 labeling after 10 days in the experimental group paralleled that observed six days post-transplantation and beyond in the control group. Dopaminergic mesostriatal host afferents are able to provide developing cells in grafted striatal tissues with normal innervation very rapidly. Despite this rapidity, the innervation does not seem to have any trophic influence on the general development of the transplant but does affect the onset time of the expression of neurochemical markers that are directly related to its synaptic function.(ABSTRACT TRUNCATED AT 400 WORDS)


A mechanism for glutamate toxicity in the C6 glioma cells involving inhibition of cystine uptake leading to glutathione depletion.

We have demonstrated that addition of L-glutamate in millimolar amounts to a culture of C6 glioma cells induced cell death within 24 h. The glutamate-induced toxicity in the C6 glioma cells was completely suppressed by adding L-cystine (0.4-1.0 mM), while the C6 cells degenerated in L-cystine-deprived culture medium. Kinetic studies of [35S]cystine and [3H]glutamate uptake showed that cystine competitively inhibited glutamate uptake, and conversely glutamate inhibited cystine uptake competitively, suggesting that C6 cells have a cystine/glutamate antiporter (system CG or Xc) similar to that already described in the periphery. Exogenous cystine (1 mM) stimulated a release of endogenous glutamate from C6 cells in a Na(+)-independent Cl(-)-dependent fashion. Thus, the antiporter normally transports glutamate out of and cystine into the cells. With the glutamate analogues tested, there was a good correlation between cytotoxicity and inhibition of cystine uptake. The de novo synthesis of glutathione was largely dependent upon the uptake of extracellular cystine. Intracellular levels of glutathione were dramatically decreased within 8-10 h by culture in glutamate-added or cystine-free medium. Vitamin E (100 microM), an antioxidant, rescued the death of C6 cells induced by glutamate exposure or by culture in cystine-deprived medium, but did not restore the apparent decrease of intracellular glutathione. Taken together, the present data strongly indicate that glutamate-induced cell death is initially due to inhibition of cystine uptake through the antiporter Xc system; such inhibition leads to glutathione depletion exposing the cells to oxidative stress. Excess of extracellular glutamate introduced from endogenous or exogenous roots might disorder this mechanism, resulting in cell death.


FAMC Intensive Stuttering Treatment Program: ten years of implementation.

An intensive stuttering treatment program for military service members is described. Over a 10-year period, 117 stutters have been treated in a program in which graded airflow, tension/relaxation, electromyographic biofeedback, and a modified hierarchical desensitization procedure have been used to obtain and maintain normally fluent speech. All patients treated have met the criterion of less than 1% stuttered words. Of 57 patients followed for from 3-36 months after treatment, 42 (74%) have maintained normally fluent speech.


[Effects of therapy with bis-hemisuccinate of ursodeoxycholic acid bisodium salt in patients with chronic hepatitis].

It has recently been shown that ursodeoxycholic acid administration improves liver function tests in patients with chronic liver diseases. Aim of the present study was to evaluate an ursodeoxycholic acid derivative (bis-hemisuccinate bisodic salt Ursodamor, Farmaceutici Damor, Napoli) in patients with chronic hepatitis. Forty patients (15 M, 25 F) with biopsy proven chronic liver disease participated to the study. Patients were randomly allocated to two treatment groups. Twenty patients (4 PBC, 11 CAH/CPH, 5 cirrhosis) received the ursodeoxycholic acid derivate at the dose of 600 mg/day, while 20 patients (1 PBC, 11 CAH/CPH, 8 cirrhosis) received a placebo. For both groups the treatment period was six months. ALT serum levels were significantly reduced in the treated group (from 84 +/- 14 to 62 +/- 14 p less than 0.0005) while no significant change was observed in the placebo group. In the treated group but not in the placebo group alkaline phosphatases and gamma-GT were also significantly reduced (from 268 +/- 56 to 160 +/- 23 p less than 0.0005 and from 79 +/- 21 to 45 +/- 10 p less than 0.0005). In conclusion, our results suggest that the administration of the ursodeoxycholic acid derivate, bis-hemisuccinate, bisodic salt, improves liver function tests in patients with chronic liver hepatitis. Similarly to ursodeoxycholic acid this new derivate probably interferes with bile acid pool composition by replacing the more detergent and probably more toxic endogenous bile acid.


Adenosine inhibits the synaptic potentials in rat septal nucleus neurons mediated through pre- and postsynaptic A1-adenosine receptors.

Intracellular and voltage-clamp recordings were made from neurons in rat brain slices containing dorsolateral septal nucleus (DLSN), in vitro. Bath application of adenosine (100 microM) produced a hyperpolarization (2-15 mV) in 46% of DLSN neurons (AH-neurons); in the remaining 54% neurons (non-AH-neurons), no hyperpolarization to adenosine was observed. Adenosine (1-300 microM) depressed not only the excitatory postsynaptic potential (EPSP) but also the inhibitory postsynaptic potential (IPSP) and the late hyperpolarizing potential (LHP) evoked by stimulation of the hippocampal CA3 area or the fimbria/fornix pathway in both AH- and non-AH-neurons. In non-AH-neurons, adenosine did not block current responses resulting from glutamate, muscimol or baclofen applied directly to DLSN neurons. In AH-neurons, adenosine partially depressed the baclofen-induced outward current. Adenosine did not block the directly-evoked IPSP (monosynaptic IPSP) as well as the glutamate-induced (hyperpolarizing) postsynaptic potential (PSP) that is mediated by GABA released from interneurons. These results suggest that adenosine does not directly inhibit the release of GABA. The effects of adenosine was mimicked by selective A1-receptor agonists and was blocked by selective A1-receptor antagonists. Pertussis toxin (PTX) blocked the hyperpolarization induced by adenosine or baclofen applied exogenously. Adenosine consistently produced presynaptic inhibition of the EPSP even in DLSN neurons treated with PTX. We conclude that adenosine inhibits neurotransmission between the hippocampus and septum through activation of pre- and postsynaptic A1-receptors which couple with G-proteins of different PTX-sensitivity or with distinct transduction processes at pre- vs. postsynaptic sites.


Escherichia coli virulence factors and 99mTc-dimercaptosuccinic acid renal scan in children with febrile urinary tract infection.

Correlation of virulence factors of Escherichia coli with renal inflammation documented by 99mTc-dimercaptosuccinic acid renal scan was undertaken in 59 children with febrile urinary tract infections to identify more accurately the role of bacterial virulence factors in the development of pyelonephritis. P fimbriae were present in 63% of isolates from the positive scan group and 83% of those from the negative scan group (P = 0.126). Multivariate regression analysis showed no significant role for established E. coli virulence factors in the development of pyelonephritis. The pap genome was independently associated with negative scan (P less than 0.007) and with the absence of reflux (P = 0.031). E. coli pyelonephritogenic clone O16:K1:H6 was isolated from negative scan patients and did not produce hemolysin. We conclude that P fimbriae are important in the development of febrile urinary tract infection regardless of the level of infection. Virulent E. coli clones described in prior Scandinavian urinary tract infection studies were not common causes of pyelonephritis in our patient population.


Otitis media in children born to human immunodeficiency virus-infected mothers.

Acute otitis media (AOM) is thought to occur frequently in children infected with human immunodeficiency virus (HIV). We compared experience with AOM of 28 HIV-infected children with that of 33 children who seroreverted to HIV antibody negative status by age 18 months. The mean number of episodes/year of AOM for children who seroreverted decreased from 1.33 in the first year of life to 0.13 in the third year, whereas the mean number of episodes/year in HIV-infected children increased from 1.89 to 2.40. By age 3 years, all HIV-infected children had experienced 1 or more episodes of AOM, and 80% had experienced 6 or more, whereas 75% of children who seroreverted had experienced 1 or more episodes, and none had had 6 or more. HIV-infected children with normal T4 lymphocyte counts had a mean of 1.18 episodes of AOM in the first year of life compared with 2.35 episodes in HIV-infected children with decreased counts (P = 0.023). HIV-infected children with low counts had a nearly 3-fold increased risk of recurrent AOM (47% vs. 18%).


Effect of a beta-adrenergic agonist on glucose transport and insulin-responsive glucose transporters (GLUT4) in brown adipose tissue of control and obese fa/fa rats.

A beta-adrenergic agonist specific for brown adipose tissue, Ro 16-8714, was administered to control and obese insulin-resistant fa/fa rats and glucose utilisation measured in brown adipose tissue using the euglycaemic hyperinsulinaemic clamp combined with the injection of 2-deoxyglucose. Treatment with the beta-agonist increased basal and insulin-stimulated glucose utilization in both groups, resulting in an increased effect of the hormone in treated animals. This effect is specific for brown adipose tissue and is not found in other insulin-sensitive tissue. The total number of insulin-responsive glucose transporters (GLUT4) measured in crude membrane preparations was similar in the two groups when expressed per total tissue. They were, however, decreased in the fa/fa group when expressed per milligram of tissue. Acute treatment with the beta-adrenergic agonist increased the total number of GLUT4 in both groups. The agonist also increased the amount of mRNA coding for GLUT4 suggesting an effect on the transcription and/or on the stability of GLUT4 mRNA.


Purification of PCNA as a nucleotide excision repair protein.

Human cell free extracts carry out nucleotide excision repair in vitro. The extract is readily separated into two fractions by chromatography on a DEAE column. Neither the low salt (0.1 M KCl) nor the high salt (0.8 M KCl) fractions are capable of repair synthesis but the combination of the two restore the repair synthesis activity. Using the repair synthesis assay we purified a protein of 37 kDa from the high salt fraction which upon addition to the low salt fraction restores repair synthesis activity. Amino acid sequence analysis, amino acid composition and immunoblotting with PCNA antibodies revealed that the 37 kDa protein is the proliferating cell nuclear antigen (PCNA) known to stimulate DNA Polymerases delta and epsilon. By using an assay which specifically measures the excision of thymine dimers we found that PCNA is not required for the actual excision reaction per se but increases the extent of excision by enabling the excision repair enzyme to turn over catalytically.


Predicting antisense oligonucleotide inhibitory efficacy: a computational approach using histograms and thermodynamic indices.

Antisense oligonucleotides (ASOs) are designed to bind to a specific mRNA and selectively suppress its translation. To facilitate selection of optimal ASO targets, we have developed three thermodynamic indices to evaluate putative structural complexes important in ASO action. These indices are: a secondary structure score (Sscore), which estimates the strength of local mRNA secondary structures at the ASO target site; a duplex score (Dscore), which estimates the delta Gformation for the ASO:mRNA target sequence duplex; and a competition score (Cscore), which is the difference between the Dscore and the Sscore. We also present two histograms to graphically display these indices from different regions of the mRNA. The indices are compared to the inhibition reported in five studies of ASO-mediated suppression of gene expression. The Dscore is the most consistent predictor of ASO efficacy in four of the five studies (r2 from 0.44 to 0.99), while the results of the fifth study could not be predicted by any thermodynamic or physical index. Thus the Dscores and their histogram may prove useful in selection of ASO targets.


Parental bias of Ki-ras oncogenes detected in lung tumors from mouse hybrids.

A mouse strain with low lung tumor susceptibility (C3H) and a strain with high lung tumor susceptibility (A/J) were reciprocally crossed to produce C3A and AC3 F1 hybrid mice. Ki-ras oncogenes were detected in spontaneous and chemically induced lung tumors obtained from the C3A and AC3 mice. To further explore the genetics of the Ki-ras gene in mouse lung tumor susceptibility, the parental origin of Ki-ras oncogenes detected in lung tumors from the F1 hybrids was determined by a strategy based on a 37-base-pair deletion in the second intron of the A/J Ki-ras allele. Ki-ras oncogenes were derived from the A/J parent in 38 of 40 tumors obtained from C3A mice and 30 of 30 tumors from AC3 mice. The observation that the activated oncogene in hybrids originates from the susceptible parent suggests that the Ki-ras gene is directly linked to mouse lung tumor susceptibility. This finding may have implications for pulmonary adenocarcinoma development in humans, since Ki-ras oncogenes are detected in 35% of this human tumor type.


Efficient inhibition of P-glycoprotein-mediated multidrug resistance with a monoclonal antibody.

P-glycoprotein (Pgp), encoded by the MDR1 gene, is an active efflux pump for many structurally diverse lipophilic compounds. Cellular expression of Pgp results in multidrug resistance (MDR) in vitro and is believed to be a clinically relevant mechanism for tumor resistance to chemotherapy. We have developed a mouse monoclonal antibody, UIC2, that recognizes an extracellular epitope of human Pgp. UIC2 inhibited the efflux of Pgp substrates from MDR cells and significantly increased the cytotoxicity of Pgp-transported drugs, under the conditions where no effect was detectable with other anti-Pgp antibodies. Potentiation of cytotoxicity by UIC2 was observed with all the tested drugs associated with MDR (vinblastine, vincristine, colchicine, taxol, doxorubicin, etoposide, actinomycin D, puromycin, and gramicidin D) but not with any of the drugs to which MDR cells are not cross-resistant (methotrexate, 5-fluorouracil, cis-platinum, G418, and gentamicin). The inhibitory effect of UIC2 in vitro was as strong as that of verapamil (a widely used Pgp inhibitor) at its highest clinically achievable concentrations. Our results suggest that UIC2 or its derivatives provide an alternative or supplement to chemical agents for the reversal of MDR in clinical cancer.


Dual regulation of human syncytial adenylyl cyclase.

The dual (stimulatory and inhibitory) regulation of adenylyl cyclase was studied in syncytiotrophoblast basal membranes prepared from term human placenta. Stimulation of adenylyl cyclase activity with GTP, non-hydrolyzable GTP analogs, isoproterenol and PGE1 was observed, confirming the presence of an intact stimulatory pathway in these membranes. Investigations of the inhibitory pathway revealed tight coupling of the G-protein, Gi alpha, to catalytic adenylyl cyclase, with high doses of GTP producing 80 per cent inhibition of GTP/forskolin-stimulated activity. Confirming Gi alpha involvement, pertussis toxin (PTX) treatment of basal membranes augmented the responses of adenylyl cyclase to both GTP and forskolin. In addition, immunoblotting of basal membrane proteins revealed the presence of the G-protein subunits, Gs alpha, Gi alpha, and G beta/gamma. The response of adenylyl cyclase was measured to a series of agonists known to inhibit adenylyl cyclase in other tissues, however a reproducible inhibitory effect was produced only by somatostatin (approximately 80 per cent). Treatment of basal membranes with PTX caused a degree of reversal of the somatostatin-mediated adenylyl cyclase inhibition. However, the intoxication was insufficient to restore GTP/forskolin-stimulated activity.


Recombinant anti-erbB2 immunotoxins containing Pseudomonas exotoxin.

Immunotoxins were made using five different murine monoclonal antibodies to the human erbB2 gene product and LysPE40, a 40-kDa recombinant form of Pseudomonas exotoxin (PE) lacking its cell-binding domain. All five conjugates were specifically cytotoxic to cancer cell lines overexpressing erbB2 protein. The most active conjugate was e23-LysPE40, generated by chemical crosslinking of anti-erbB2 monoclonal antibody e23 to LysPE40. In addition, a recombinant immunotoxin, e23(Fv)PE40, was constructed that consists of the light-chain variable domain of e23 connected through a peptide linker to its heavy-chain variable domain, which in turn is fused to PE40. The recombinant protein was made in Escherichia coli, purified to near homogeneity, and shown to selectively kill cells expressing the erbB2 protooncogene. To improve the cytotoxic activity of e23(Fv)PE40, PE40 was replaced with a variant, PE38KDEL, in which the carboxyl end of PE is changed from Arg-Glu-Asp-Leu-Lys to Lys-Asp-Glu-Leu and amino acids 365-380 of PE are deleted. The e23(Fv)PE38KDEL protein inhibits the growth of tumors formed by the human gastric cancer cell line N87 in immunodeficient mice.


Design and synthesis of a CD4 beta-turn mimetic that inhibits human immunodeficiency virus envelope glycoprotein gp120 binding and infection of human lymphocytes.

Poor bioavailability, rapid degradation, antigenicity, and high cost often limit the use of proteinaceous pharmaceuticals. One goal of structural biochemistry is the reduction of complex molecules to small functional units that are amenable to high-resolution structural analysis and rapid modification. The dissection of complex proteins into small synthetic conformationally restricted components is an important step in the design of low molecular weight nonpeptides that mimic the activity of the native protein. We have developed a reverse-turn mimetic system to explore peptide and protein structure-function relationships. We now report the design and synthesis of a small molecule (M(r) 810, as its trifluoroacetate salt), water soluble, proteolytically stable mimetic of residues Gln40-Thr45 of the complementarity-determining 2-like region of CD4. This mimetic has a low micromolar Kd for human T-lymphotropic virus type IIIB gp120 and reduces syncytium formation.


Nitric oxide stimulates the ADP-ribosylation of a 41-kDa cytosolic protein in Dictyostelium discoideum.

Nitric oxide-releasing compounds were shown to activate an ADP-ribosyltransferase activity in the cytosol of Dictyostelium discoideum. The enzyme ADP-ribosylated a cytosolic protein of approximately 41 kDa, p41. Neither cGMP nor GTP and its analogues affected this ADP-ribosylation. p41 differs from other substrates ADP-ribosylated by cholera, pertussis, or diphtheria toxins. Treatment of ADP-ribosylated p41 with snake venom phosphodiesterase released adenosine 5'-monophosphate, indicating a mono-ADP-ribose-protein linkage. This linkage was stable to neutral hydroxylamine but was sensitive to mercury ions and iodomethane, suggesting an attachment to a cysteine residue. Treatment of intact cells with nitric oxide-releasing compounds appeared to stimulate the ADP-ribosylation of p41 and this modification was reversible.


Isolation of a gene encoding a chaperonin-like protein by complementation of yeast amino acid transport mutants with human cDNA.

A human cDNA library in lambda-yes plasmid was used to transform a strain of Saccharomyces cerevisiae with defects in histidine biosynthesis (his4-401) and histidine permease (hip1-614) and with the general amino acid permease (GAP) repressed by excess ammonium. We investigated three plasmids complementing the transport defect on a medium with a low concentration of histidine. Inserts in these plasmids hybridized with human genomic but not yeast genomic DNA, indicating their human origin. mRNA corresponding to the human DNA insert was produced by each yeast transformant. Complementation of the histidine transport defect was confirmed by direct measurement of histidine uptake, which was increased 15- to 65-fold in the transformants as compared with the parental strain. Competitive inhibition studies, measurement of citrulline uptake, and lack of complementation in gap1- strains indicated that the human cDNA genes code for proteins that prevent GAP repression by ammonium. The amino acid sequence encoded by one of the cDNA clones is related to T-complex proteins, which suggests a "chaperonin"-like function. We suggest that the human chaperonin-like protein stabilizes the NPR1 gene product and prevents inactivation of GAP.


High-efficiency receptor-mediated delivery of small and large (48 kilobase gene constructs using the endosome-disruption activity of defective or chemically inactivated adenovirus particles.

One limit to successful receptor-mediated gene delivery is the exit of the endocytosed material from the endosome. We demonstrate here the delivery of marker genes to tissue culture cells using a modification of the receptor-mediated gene delivery technique that exploits the endosomolytic activity of defective adenovirus particles. In particular, greater than 90% of the transfected-cell population is found to express a beta-galactosidase gene, and, most importantly, this high level of expression can be obtained with psoralen-inactivated virus particles. Furthermore, because the delivered gene is not carried within the genome of the adenovirus particle, the size constraints are relieved, and we can, therefore, show the delivery of a 48-kilobase cosmid DNA molecule.