Role of nitric oxide from the endothelium on the neurogenic contractile responses of rabbit pulmonary artery.

The effects of L-NG-nitro arginine (L-NO2Arg), a stereospecific inhibitor of nitric oxide formation, on the responsiveness of rabbit pulmonary artery to transmural electrical stimulation were studied. The contractile response evoked by electrical stimulation at 4 Hz was abolished by tetrodotoxin (10(-7) M) and depressed to approximately 10% by bunazosin (10(-6) M), an alpha 1-antagonist. Pretreatment with L-NO2Arg (10(-5) M) significantly potentiated the response to electrical stimulation without changing the resting tension. D-NO2Arg (10(-5) M) did not show such a potentiating action. In endothelium-denuded arteries, L-NO2Arg did not potentiate the response to electrical stimulation. The effect of L-NO2Arg on endogenous noradrenaline release in response to electrical stimulation was also examined by HPLC with electrochemical detection; L-NO2Arg did not affect noradrenaline release. The contractions induced by exogenous noradrenaline (10(-6)-10(-5) M) were enhanced by L-NO2Arg, but not by D-NO2Arg. These results suggest that the vasoconstriction induced by sympathetic nerve stimulation in the rabbit pulmonary artery is modulated by endogenous nitric oxide or nitric oxide-like substances released from endothelial cells.


(-)-Penbutolol as a blocker of central 5-HT1A receptor-mediated responses.

Brain 5-HT1A and 5-HT1B receptors are important targets for drug-induced modulation of 5-HT function in vivo. However, very few compounds are available that are effective antagonists at 5-HT1 receptors, thus hampering the progress of fundamental as well as clinical research in this area. The present study assessed the usefulness of the beta-adrenolytic agent (-)-penbutolol (and its (+)-counterpart) as a 5-HT1A receptor-blocking agent. The compound was found to counteract, in a stereospecific fashion, not only the behavioural and hypothermic but also the in vivo 5-HT synthesis/turnover-reducing effects of the specific 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). These findings indicate that (-)-penbutolol is an antagonist at both postsynaptic receptors and somatodendritic autoreceptors of the 5-HT1A subtype. Thus, (-)-penbutolol represents a useful addition to the array of pharmacological tools available for the study of central 5-HT1 receptor-mediated functions.


gamma-Butyrolactone-sensitive and -insensitive dopamine release, and their relationship to dopamine metabolism in three rat brain regions.

Some data suggest dopamine (DA) release from neuronal terminals is partially independent of impulse flow. We examined the changes in tissue DA and its major metabolite levels 30 and 90 min after treatment with y-butyrolactone (750 mg/kg). Accumulation of 3-methoxytyramine within 10 min of pargyline injection (75 mg/kg) was used as an index of DA release. Thirty minutes after gamma-butyrolactone injection, DA content was increased maximally in the frontal cortex, nucleus accumbens and striatum (by 91%, 80% and 73%, respectively). 3-Methoxytyramine rates of accumulation were reduced by 77%, 77%, and 92%, respectively. Ninety minutes after the treatment, DA levels remained high in all three areas, while DA release was persistently low in the striatum and nucleus accumbens, but had returned to baseline in the frontal cortex. Changes in 3,4-dihydroxyphenyl-acetic acid and homovanillic acid levels were not synchronized with changes in DA release in the striatum and nucleus accumbens, and were absent in the frontal cortex. The data suggest that an impulse flow-independent mechanism contributes to approximately one tenth and one fourth of the basal DA release in the terminals of DA neurons originating in the substantia nigra and ventral tegmental area, respectively. The acidic DA metabolite levels become at best poorly associated with DA release during blockade of the DA neuronal firing, probably because of the increased in situ metabolism of newly synthesized DA.


Platelet activating factor interaction with tumor necrosis factor and myocardial depressant factor in splanchnic artery occlusion shock.

Anaesthetized rats, subjected to total occlusion of the superior mesenteric artery and the celiac trunk for 45 min, developed a severe shock state (splanchnic artery occlusion shock) resulting in a fatal outcome within 75-90 min after release of the occlusion. Shocked rats, treated with an intravenous bolus of L-659,989, a specific platelet activating factor (PAF) receptor antagonist (12.5, 25 or 50 nmol/kg, 4 min after reperfusion followed, 8 min thereafter, by a continuous infusion of 125, 250 or 500 nmol/kg for 30 min), maintained post-release mean arterial blood pressure at significantly higher values than did rats receiving the vehicle. Treatment with L-659,989 significantly increased survival rate, blunted the rise in plasma myocardial depressant factor activity and lowered serum and macrophage levels of tumor necrosis factor (TNF-alpha). In addition, the drug completely restored macrophage phagocytosis, improved macrophage killing and significantly inhibited leukopenia. To investigate the interaction between PAF, TNF-alpha and myocardial depressant factor, the blood levels of these three mediators were evaluated: shocked rats exhibited increased PAF levels with a peak at 30 min. The plasma levels of PAF peaked earlier than did either serum TNF-alpha or plasma myocardial depressant factor. Both peaks occurred 75 min after the release of occlusion. The results of this study therefore suggest that PAF is a key mediator of splanchnic artery occlusion shock and plays a permissive role in inducing the release of other factors (i.e. TNF-alpha and myocardial depressant factor) that are relevant to shock.


The distribution of 5-HT1D and 5-HT1E binding sites in human brain.

Total 5-HT1, 5-HT1D and 5-HT1E binding sites were measured in homogenates of human frontal cortex, hippocampus, amygdala, globus pallidus, caudate and putamen. Combined 5-HT1D/1E sites were the predominant 5-HT1 subtype (66-95% of total 5-HT1 sites in all regions except hippocampus (38% of total 5-HT1 sites). Globus pallidus contained the highest density and the highest proportion of 5HT1D sites (74% of total 5-HT1 sites). 5HT1D sites in the other brain areas accounted for 19-27% of the total 5-HT1 sites. The highest densities and the highest proportions of 5-HT1E sites were in caudate (72%) and putamen (64%) and the lowest density and lowest proportion in hippocampus (16%).


Effect of Rb+ on cromakalim-induced relaxation and ion fluxes in guinea pig trachea.

The effects of cromakalim, verapamil and salbutamol have been examined in guinea pig trachealis smooth muscle in both Krebs physiological salt solution and Krebs solution where K+ has been replaced by Rb+. Cromakalim-induced relaxation in the presence of Rb+ was reduced in extent and became transient, whilst the relaxation response to verapamil was enhanced and that to salbutamol unaffected. The transient relaxation occurring in Rb+ was blocked by quinidine and glibenclamide. The presence of extracellular Rb+ also prevented cromakalim-stimulated efflux of both 86Rb+ and 42/43K+. There was, however, no effect on cromakalim-stimulated 86Rb+ uptake. It is proposed that cromakalim is opening two populations of potassium channel in guinea pig tracheal smooth muscle, one of which is susceptible to blockade by Rb+ and one of which is not. The latter channel appears to play the dominant role in cromakalim-stimulated uptake, and is responsible for the transient relaxation response in the presence of rubidium, whilst the former is responsible for the maintained relaxation.


Tianeptine attenuates stress-induced morphological changes in the hippocampus.

Repeated 6-h daily restraint stress over 21 days reduces length and number of branch points of hippocampal CA3c pyramidal dendrites in the hippocampal formation of adult male rats. This effect is mimicked by daily injections of 40 mg/kg corticosterone. Daily treatment with tianeptine (15 mg/kg) prior to stress sessions or the corticosterone treatment prevented these effects of stress or corticosterone, respectively. Tianeptine treatment did not prevent the effects of stress to increase adrenal/body weight ratio, nor did it prevent the effects of stress to decrease body weight gain, indicating that its actions are not mediated solely by effects on stress-induced secretion of corticosterone. Because tianeptine is known to enhance neural uptake of serotonin, these results suggest that the serotonergic system may be involved in modulating stress and corticosterone effects on dendritic morphology.


Effects of velnacrine, tacrine and physostigmine on tetanic twitch responses at the rat neuromuscular junction.

The effects of velnacrine (1-hydroxytacrine), tacrine and physostigmine on indirectly elicited twitch at low and high stimulation frequencies were analyzed in the rat phrenic hemidiaphragm preparation. At 0.2 Hz, velnacrine and physostigmine behaved in a similar manner, the latter showing a higher potentiating effect. This potentiation was observed at 3-100 microM velnacrine, whereas a slight depression appeared at higher concentrations. When tetanic responses were studied, the drug concentrations needed to depress tetanic tension and tetanic fade were quite different in the case of velnacrine (depression of tetanic tension from 1 microM and tetanic fade from 170 microM), whereas physostigmine and tacrine were able to affect these parameters at very similar concentrations. The results suggest that some effects of velnacrine could differ from those of tacrine in spite of the chemical similarity.


No effect of D-myo-inositol-1,2,6-triphosphate on vasoconstriction evoked by neuropeptide Y and non-adrenergic sympathetic nerve stimulation.

The effects of the proposed neuropeptide Y (NPY) antagonist, D-myo-inositol-1,2,6-triphosphate (PP56), on vasoconstrictor responses evoked by NPY and non-adrenergic sympathetic nerve stimulation were investigated in the pig in vivo. Under control conditions, exogenous NPY evoked a dose-dependent increase in arterial blood pressure and vasoconstriction in spleen, kidney and skeletal muscle. After administration of PP56 (50 mg/kg), which transiently reduced systemic blood pressure by 18 +/- 5 mm Hg, the vascular responses evoked by NPY did not differ from those observed under control conditions. Stimulation of the splenic nerve and the lumbar sympathetic chain with 20-Hz burst activity in reserpine-pretreated pigs, which are devoid of their noradrenaline content, decreased splenic and hindlimb vascular conductance by 67 +/- 7 and 57 +/- 7%, respectively, under control conditions. In the presence of PP56 the nerve stimulation-evoked reductions in splenic and hindlimb vascular conductance were slightly but not significantly reduced to 59 +/- 9 and 48 +/- 7%, respectively. It is concluded that PP56 in the presently used high dose, which causes non-selective inhibition of vasoconstriction in the rat, cannot be used as an antagonist of vasoconstrictor responses evoked by NPY or non-adrenergic sympathetic nerve stimulation in the pig.


Modulation of vascular reactivity by vasoactive peptides in aortic rings from hypercholesterolemic rabbits.

The effect of moderate elevation in serum cholesterol on vascular reactivity to epidermal growth factor (EGF), endothelin (ET-1) and thrombin, vasoactive peptides present at sites of vascular injury, was examined in isolated aortic rings from rabbits fed either a casein-rich or a control diet for 10-12 weeks. In rings from hypercholesterolemic rabbits, development of maximal isometric tension to all peptide agonists was increased 22 +/- 0.6% while the EC50 for contraction was decreased. Vasorelaxant responses to nitroprusside, an endothelium-independent dilator, were largely intact, while those to A231897, an endothelium-dependent agent, were attenuated. These data suggest that elevation in serum cholesterol in the absence of atherosclerotic lesions is sufficient to increase vascular reactivity to peptide vasoactive mediators, an effect which may predispose arteries to vasospasm.


SDZ ENS 163, a selective muscarinic M1 receptor agonist, facilitates the induction of long-term potentiation in rat hippocampal slices.

The effect of SDZ ENS 163; (+)-(3S,cis)-3-ethyldihydro-4-[(1-methyl-1H-imidazol-5-yl)methyl-2 (3H)- thiphenonedihydrogenphosphate], a selective muscarinic M1 agonist, on long-term potentiation (LTP) was studied in a rat hippocampal slice preparation. LTP was induced by theta-burst stimulation (TBS) delivered to the Schaffer/commissural fibers. In untreated slices delivery of 8 or 10 trains at 100 Hz induced a 27 +/- 8.3 and 54 +/- 7.2% potentiation of the amplitude of the excitatory postsynaptic potential (epsp), respectively (calculated as percentage of the pre-LTP amplitude). In slices pretreated with SDZ ENS 163 (2 x 10(-6) M, -30 min) delivery of 8 or 10 trains at 100 Hz induced a 62 +/- 8.4 and 54 +/- 7.1% potentiation of the epsp amplitude, respectively. In addition, treatment with SDZ ENS 163 (2 x 10(-6) M) increased the N-methyl-D-aspartate receptor-induced component of the epsp response to TBS from 21 +/- 3 (control) to 33 +/- 2%. Pretreatment with the muscarinic antagonist, scopolamine (6 x 10(-8) M), or with the M1 selective muscarinic receptor antagonist, pirenzepine (6 x 10(-8) M), did not affect LTP in untreated slices but inhibited the enhancement of LTP by SDZ ENS 163 (2 x 10(-6) M) completely. AF-DX 116 (10(-6) M, -60 min), a selective muscarinic M2 receptor antagonist did not affect LTP in control slices nor in slices treated with SDZ ENS 163 (2 x 10(-6) M). These data suggest that activation of muscarinic M1 receptors by SDZ ENS 163 facilitates the induction of LTP.


The antihypertensive effect of acute intracerebroventricular administration of captopril in Dahl salt-sensitive rats.

The ability of centrally administered angiotensin converting enzyme (ACE) inhibitors to lower mean arterial pressure (MAP) has been demonstrated in numerous animal models of hypertension. In the present study, we assessed the effect of intracerebroventricular (i.c.v.) injection of the ACE inhibitor captopril (10 micrograms) on MAP in conscious, freely moving hypertensive inbred Dahl salt-sensitive (DS/JR) rats and their normotensive control inbred Dahl salt-resistant (DP/JR) rats. Both DS/JR and DR/JR rats were maintained on an 8% salt diet from 4 weeks of age until experimentation at 7-8 weeks of age, at which time DS/JR pressures were significantly elevated as compared to DR/JR rats (185 +/- 6 vs. 99 +/- 2 mm Hg, respectively). Following i.c.v. administration of captopril, a significant depressor response lasting for several hours was observed in DS/JR rats, with a maximum reduction of 17.6 +/- 4.1 mm Hg. The same treatment had no effect on the MAP of DR/JR rats. Mean arterial pressures in both groups were not significantly affected by i.c.v. administration of vehicle alone or by intravenous (i.v.) administration of 100 micrograms of captopril. These findings indicate that i.c.v. captopril lowers MAP in hypertensive DS/JR rats. Further studies will be necessary to elucidate the mechanism of this antihypertensive effect.


Drug discrimination analysis of ethanol as an N-methyl-D-aspartate receptor antagonist.

Ethanol has been shown to antagonize N-methyl-D-aspartate (NMDA) receptor-mediated neurotransmission in a number of in vitro systems. Drug discrimination procedures in rats were used to evaluate ethanol as an antagonist of NMDA discrimination and for its ability to produce discriminative stimulus effects similar to those of competitive and noncompetitive NMDA antagonists. Ethanol (300-1500 mg/kg i.p.) failed to antagonize the stimulus effects of 30 mg/kg NMDA, nor did it substitute fully for either the competitive antagonist NPC 12626 nor the noncompetitive antagonist phencyclidine (PCP). A maximum average of 55.4% PCP-lever responding provided evidence for partial substitution in this model. The effects of ethanol on NMDA discrimination are distinct from those previously reported for competitive NMDA antagonists but similar to those of noncompetitive antagonists. On the other hand, ethanol can be distinguished from both competitive and PCP-like noncompetitive NMDA antagonists using drug discrimination procedures.


Adenine nucleotide analogues, including gamma-phosphate-substituted analogues, are metabolised extracellularly in innervated frog sartorius muscle.

The metabolism of adenine nucleotides and of their analogues by ecto-enzymes in the innervated frog sartorius muscle was investigated with HPLC. The breakdown of beta, gamma-methylene-ATP was also evaluated by studying the ability of the adenosine uptake inhibitor, dipyridamole, and of the adenosine receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), to modify the effect of beta, gamma-methylene-ATP on nerve-evoked twitches. ATP-gamma-S at low (10 microM) but not at high (> or = 100 microM) concentrations was quickly metabolised into a compound with a higher negative charge. L-ATP, homo-ATP and 2-methylthio-ATP were metabolised into compounds with a lower negative charge. Beta-gamma-Imido-ATP and gamma-anilino-ATP were only metabolised slightly. As determined by HPLC, beta, gamma-methylene-ATP was not metabolised. In contrast, this ATP analogue inhibited nerve-evoked twitch responses, an effect which was potentiated by dipyridamole and antagonised by DPCPX. Alpha, beta-Methylene-ATP was dephosphorylated into alpha, beta-methylene-ADP, which was virtually resistant to metabolism in the absence of ATP. In the presence of ATP, alpha, beta-methylene-ADP was transiently phosphorylated into alpha, beta-methylene-ATP. Formation of ATP from ADP was observed even in the absence of an exogenous phosphate donor, and was prevented by the adenylate kinase inhibitor, P1P5-di-(adenosine-5')pentaphosphate (AP5A). AP5A caused only partial inhibition of AMP formation from ADP. The results suggest that some ATP analogues with substitutions in the gamma-phosphate, such as ATP-gamma-S and beta, gamma-methylene-ATP, are metabolised in the innervated frog sartorius muscle. The ADP analogue, alpha, beta-methylene-ADP, might be a substrate for an ecto-nucleoside diphosphate kinase. ADP, besides being dephosphorylated, is also a substrate for an ecto-adenylate kinase in innervated frog sartorius muscle.


Analogues of F8Famide resistant to degradation, with high affinity and in vivo effects.

Four analogues of Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2, a mammalian FMRFamide-like peptide with antiopiate properties, were synthesized with N-terminus modifications and were shown to have high affinity for F8Famide binding sites. The degradation rate of these analogues in mouse brain slices was 3 times lower than that of the natural peptide. One analogue, (2DME)Y8Fa (D.Tyr-D.Leu-[N-Me]Phe-Gln-Pro-Gln-Arg-Phe-NH2), produced a clear hyperalgic effect and inhibited morphine analgesia in the mouse tail-flick test at lower doses than did the parent compound. (3D)Y8Fa (D.Tyr-D.Leu-D.Phe-Gln-Pro-Gln-Arg-Phe-NH2) and (2D)Y8Fa (D.Tyr-D.Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2) in contrast did not decrease morphine analgesia but were analgesic alone. The analgesic effects of 22 nmol (2D)Y8Fa and (3D)Y8Fa were decreased by (1DME)Y8Fa (D.Tyr-Leu-[N-Me]Phe-Gln-Pro-Gln-Arg-Phe-NH2) or (2DME)Y8Fa and were reversed by naloxone. These results indicate opioid modulating properties of F8Famide. These analogues may prove to be useful tools for studying the modulation of pain by F8Famide.


Vasopressin receptors in the area postrema differentially modulate baroreceptor responses in rats.

This study examined the effects of microinjection of [Arg8]vasopressin (AVP) into the area postrema (AP) on baroreceptor reflex control of heart rate (HR) and sympathetic efferent discharge (SED) in anesthetized rats. Comparable increments in blood pressure evoked by systemic AVP, as opposed to phenylephrine, were associated with significantly greater reflex bradycardia. Similarly, AVP augmented the baroreflex-mediated sympathoinhibition; however, this effect was evident only with the lower increments in arterial pressure (< 45 mm Hg) i.e. following systemic administration of small doses of AVP. Beyond 45 mm Hg there was no further augmentation of baroreflex-mediated sympathoinhibition showing the non-linearity of the response compared to phenylephrine which was linear over a wide range of induced pressure increases. Microinjection of AVP into the AP produced a differential effect on HR and SED responses to baroreceptor activation by systemically administered phenylephrine, the baroreflex slope of HR response was attenuated whereas that of SED was enhanced. Microinjection of the V1 antagonist AVPX (d(CH2)5Tyr(Me)-AVP) into the AP abolished the inhibitory effect of AVP on the baroreceptor-HR response suggesting that V1 receptors are involved in this response. Further, AVPX inhibited the baroreceptor-SED response suggesting that V1 receptors in the AP are tonically involved in modulating the baroreceptor reflex control of SED. Qualitatively similar but smaller responses were obtained following microinjection of AVP into the nucleus tractus solitarii (NTS) suggesting involvement of neural input from the AP to the NTS in AVP-evoked responses in the AP. It is concluded that the AVP receptors in the AP differentially modulates the baroreceptor reflex control of HR and SED.


Relationship between tissue content of TC-81 and relaxation of rat aorta.

This study dealt with the relationship between the relaxant action of TC-81, a new Ca2+ antagonist, and its distribution in rat aorta depolarized by high K+ (65.4 mM). The inhibitory effect by TC-81 on K(+)-induced contraction and 45Ca2+ uptake was strongly time-dependent. TC-81 and nicardipine, each 10(-9) M, produced gradual relaxation of high-K(+)-induced contraction. The tissue contents of TC-81 and nicardipine increased with time courses that reflected decreasing tension. Both the maximum tissue content of TC-81 and the maximum relaxation were significantly greater than those for nicardipine. Also, the relaxations produced by TC-81 and nicardipine were correlated with the logarithm of the content of each drug in muscle tissues. However, the dissociation of TC-81 from the tissues was slower than that of nicardipine. The data suggest that the action of TC-81 is closely related to a gradual distribution of drug into muscle tissue, resulting in a slow onset of action. Also, the saturation time of TC-81 was longer than that of nicardipine, thereby accounting for the greater relaxation with TC-81.


Attenuation of ischemic and postischemic damage to brain metabolism and circulation by a novel Ca2+ channel antagonist, NC-1100, in spontaneously hypertensive rats.

We investigated the effect of a newly synthesized Ca2+ channel antagonist, NC-1100, on cerebral blood flow (CBF) and metabolism in spontaneously hypertensive rats. The rats received a bolus injection of 0.2 or 1.0 mg/kg NC-1100 i.v. and 1-h cerebral ischemia was then induced by bilateral carotid artery occlusion (group 1). The rats in group 2 were continuously infused with NC-1100 0.03 or 0.1 mg/kg per min, starting immediately after bilateral carotid artery occlusion, for the 1 h of ischemia and following 3-h recirculation. Group 1: during ischemia, CBF in all rats decreased to 6-8% of the resting values. At 1 h cerebral ischemia, brain tissue lactate increased 11.5-, 10.1- and 9.8-fold of the normal control given vehicle or NC-1100, 0.2 and 1.0 mg/kg, respectively. The ATP levels were better preserved by NC-1100 administration; 0.61 +/- 0.04 (mean +/- S.E.M.), 0.80 +/- 0.09 and 0.97 +/- 0.14 mmol/kg (P < 0.05 vs. vehicle), respectively. Group 2: during recirculation, CBF in NC-1100-treated rats returned to 83-90% of the resting values, but to only 65% in the vehicle group. Postischemic brain lactate at 3 h was less well preserved and ATP was dose dependently better preserved in NC-1100- than vehicle-treated rats. It is considered that pre- as well as postischemic administration of a Ca2+ channel antagonist, NC-1100, is beneficial to attenuate and also ameliorate the metabolic and circulatory derangement in the ischemic brain.


Protective effect of vinconate on ischemia-induced neuronal damage in the rat hippocampus.

The protective effect of vinconate, a vinca alkaloid derivative, on ischemia-induced neuronal damage was investigated using a model of rat forebrain ischemia caused by occlusion of four vessels. Hippocampal cell loss was observed histologically and neurochemically 5 days after 10 min of ischemia. Treatment with vinconate (50 and 200 mg/kg i.p.) before cerebral ischemia significantly suppressed neuronal cell loss in the hippocampal CA1 region and the decrease in the content of neuroactive amino acids in the hippocampus. The release of neuroactive amino acids in the hippocampus was significantly increased by cerebral ischemia. Pretreatment with vinconate (50 and 200 mg/kg i.p.) significantly attenuated the increased release of glutamic acid and aspartic acid, but not the release of gamma-aminobutyric acid (GABA), taurine and glycine. This suppressive effect of vinconate was antagonized by scopolamine (10(-5) M). The addition of vinconate (10(-11)-10(-4) M) had no effect on the binding of [3H]MK-801. These results indicate that pretreatment with vinconate attenuates the ischemia-induced release of excitatory amino acids into the extracellular space of the hippocampus via the stimulation of presynaptic muscarinic acetylcholine receptors. The present results also suggest that this suppressive effect of vinconate on the release of excitatory amino acids (glutamic acid and aspartic acid) may play a crucial role in the protective action of this agent against ischemia-induced neuronal damage in the hippocampus.


Discharge dependencies of amygdala central nucleus neurons to the cardiac and respiratory cycle following local cocaine administration.

We examined dependencies of amygdala central nucleus neuronal discharge to the cardiac and respiratory cycles in freely behaving cats following local microinjection of cocaine (100 micrograms/0.2 microliter). Cross-correlation histograms showed cycle-by-cycle dependencies between neuronal discharge and the cardiac and respiratory cycles in 10 of 30 cells and 7 of 30 cells, respectively, during baseline periods. After cocaine delivery, the discharge rate of half of the central nucleus of the amygdala cells (16/30, 53%) were partly or completely inhibited in a reversible manner. Excluding cardiac- and respiratory-dependent neurons which ceased firing after cocaine, more than half (5/8) of the remaining cardiac and two-thirds (4/6) of respiratory-dependent neurons altered discharge dependencies following cocaine administration. Of the cells that did not exhibit cardiac and respiratory dependencies pre-cocaine, 2 of 20 developed cardiac correlations and 3 of 23 developed respiratory correlations following cocaine administration. We speculate that a portion of the cardiac and respiratory responses induced by cocaine may be mediated through the central nucleus of the amygdala.


Aggregation of human polymorphonuclear leukocytes by endothelin: role of platelet-activating factor.

The mechanisms by which endothelin-1 (ET-1) acts on polymorphonuclear leukocytes (PMN) are insufficiently known. In this study, we assessed the hypotheses that ET-1 is a PMN-aggregating agent, and that platelet-activating factor (PAF) is the principal mediator of ET-1-induced PMN aggregation. ET-1 induced dose-related PMN aggregation, which started 1 min after ET-1 exposure. Two different specific PAF receptor antagonists blocked the effect of ET-1 on PMN aggregation. In addition, ET-1 induced a significant increase in the production of PAF by PMN after 2 to 5 min of ET-1 incubation. ET-1 induced PAF release from PMN rather than accumulation. This PAF production was dependent on intra- and extracellular Ca2+. In this regard, the PAF receptor antagonists significantly blunted the ET-1-induced peak in cytosolic free Ca2+ ([Ca2+]i). Our results, therefore, indicate that ET-1 is effective in causing aggregation of human PMN and that its action appears to be mediated by PAF production via a Ca(2+)-dependent mechanism.


NNC-711, a novel potent and selective gamma-aminobutyric acid uptake inhibitor: pharmacological characterization.

NNC-711 (1-(2-(((diphenylmethylene)amino)oxy)ethyl)-1,2,5,6-tetrahydro-3- pyridinecarboxylic acid hydrochloride) is a novel, potent and selective gamma-aminobutyric acid (GABA) uptake inhibitor. NNC-711 inhibited synaptosomal (IC50 = 47 nM), neuronal (IC50 = 1238 nM) and glial (IC50 = 636 nM) GABA uptake in vitro NNC-711 lacked affinity for other neurotransmitter receptor binding sites, uptake sites and ion channels examined in vitro. In vivo, NNC-711 was a potent anticonvulsant compound against rodent seizures induced by methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) (ED50 (clonic) = 1.2 mg/kg i.p.), pentylenetetrazole (PTZ) (ED50 (tonic) = 0.72 mg/kg i.p., mouse; and ED50 (tonic) = 1.7 mg/kg, rat), or audiogenic (ED50 (clonic and tonic) = 0.23 mg/kg i.p.). At higher doses NNC-711 produced behavioral side effects characterized by inhibition of traction (ED50 = 23 mg/kg i.p.), rotarod (ED50 = 10 mg/kg i.p.) and exploratory locomotor activity (ED50 = 45 mg/kg i.p.) in the mouse. Following acute (3-h) in vivo pretreatment with NNC-711, behavioral tolerance developed to its motor impairing side effects (inhibition of traction, rotarod or exploratory locomotor activity) without corresponding tolerance to the anticonvulsant effects. These data suggest that NNC-711 will be useful for future in vitro and in vivo experiments to elucidate the role of the GABA uptake carrier in the central nervous system.


Regulation of the cellular transport and compactation of thyroglobulin.

The cellular transport ot thyroglobulin in thyrocytes is a TSH-regulated process and characterized by a bidirectional export-, storage- and recapture pathway. The regulation of single steps in this pathway is unclear, but a possible basis for this transport appeared to be the presence of the lysosomal targetting, signal mannose-6-phosphate (M6P), on porcine thyroglobulin and the detection of the cation-dependent M6P-receptor on the apical cell surface and the Golgi-complex in thyrocytes. The question arose, why thyroglobulin is not directed on an intracellular pathway to lysosomes thereby following the route characteristic of lysosomal enzymes. Recent observations have shown that the affinity of thyroglobulin to the M6P-receptor is very low and the receptor is presumably not effective in thyrocytes to direct thyroglobulin to lysosomes. Instead, a low affinity binding site for thyroglobulin has been found to operate on the surface of thyrocytes. This receptor is specific for thyroglobulin and is saturable at 8-13 mg thyroglobulin per ml (5-9 microM). The highest affinity of thyroglobulin was that to itself which is the basis for the positive cooperation observed during binding of thyroglobulin to thyrocyte membranes. The receptor has a relative molecular mass of 46 kDa but is not identical with the cation-dependent M6P-receptor. Because of the high-thyroglobulin-concentration in the follicle lumen (100-400 mg/ml) it has always been argued that there is no need for the presence of a thyroglobulin receptor.(ABSTRACT TRUNCATED AT 250 WORDS)


G-protein mutations in thyroid tumors.

Tissue specific G-protein mutations have been found in differentiated thyroid cancer with a higher prevalence in tumors from German than from American patients. Since activating mutations of Gs are principally comparable to chronic TSH stimulation of these tumor-thyrocytes such mutations may predict changes in tumor development and patient prognosis. The fundamental background and recent results of studies on mutational activation of G-proteins are presented.


TSH-receptor and adhesion molecules in autoimmune thyroid disease.

Using a human TSH receptor (TSH-R) cDNA probe we investigated TSH-R expression levels in 20 human thyroid fragments by Northern blot analysis: 14 Graves' disease, 2 Hashimoto's disease, 3 endemic goiter and 1 healthy thyroid gland. TSH-R expression was low in those thyroids where expression of the major histocompatibility complex class I (MHC I), class II (MHC II) and intercellular adhesion molecule 1 (ICAM-1) was high, whereas expression of the endothelial leukocyte adhesion molecule 1 (ELAM-1) bore no relation to TSH-R expression. In situ hybridization showed that next to lymphocytes (MHC II), thyroid cells (MHC II, ICAM-1) and endothelial cells (ICAM-1, ELAM-1) were the source of transcripts of these T-cell activating antigens. We conclude that next to MHC I and II, the expression of additional T-cell activating antigens such as ICAM-1 and ELAM-1 play a role in the initiation of auto-immune thyroid disease.


Thyroid growth in vitro.

Whereas there is no doubt about the role of the growth factors EGF (epidermal growth factor) and IGF I (insulin like growth factor I) on thyroid cell proliferation, controverse results have been obtained in different in vitro studies with TSH (thyrotropin). Whereas in nearly all cell culture systems using thyroid cell monolayers TSH enhances cell proliferation, TSH inhibits cell growth induced by EGF in intact thyroid follicles. These differences are not due to species specificity, because i.e. growth of porcine thyroid cells or of human thyroid cells in monolayer cultures is stimulated by TSH, but growth of human or porcine thyroid follicles is not stimulated by TSH. In addition there is some evidence that IGF I is necessary for the growth stimulating effect of TSH and IGF I seems to be an autocrine growth factor, which is under the control of TSH and/or iodine deficiency within the thyroid cell.


Antagonists of the human TSH receptor: in vitro and in vivo studies of their functional and immunological effects.

We have developed and characterized a prototype of a TSH receptor antagonist derived from the hCG molecule. This may be used to block human TSH receptor both functionally and immunologically, particularly in the study of Graves' disease. Our hCG derived TSH receptor blocker compares favorably with other substances (e.g. deglycosylated forms of TSH, synthetic peptides of the alpha or beta subunit of TSH) that hare been reported to inhibit bTSH binding or bTSH-stimulated cAMP response (Joshi et al., 1981; Morris et al., 1988). It has a much higher affinity for human TSH receptor than the TSH subunit peptides and it is the only substance an efficacy of which has been proven in vivo so far. Recent progress in the synthesis of recombinant glycoprotein hormones should permit to biosynthetically produce this or a similar TSH receptor antagonist. With respect to Graves' disease, the data suggest that TSH receptor, in addition to its role in maintaining thyroid hyperfunction, plays also a role in propagating the thyroid autoimmune disease itself. Stimulation of TSH receptor by bTSH as well as TSAb enhances the expression of HLA class II antigens on the surface of thyrocytes, and a blockade of TSH receptor results in a substantial inhibition of this immunological key event. This could possibly explain why suppression of TSH by administering levothyroxine was found in a recent study by Hashizume and coworkers (1991) to decrease TSAb titers and to reduce relapse rate in patients with Graves' hyperthyroidism.(ABSTRACT TRUNCATED AT 250 WORDS)


In vivo effects of TSH receptor antibodies in xenotransplanted human thyroid tissue.

From the data presented it can be concluded that the xenotransplantation model is well suited for studies concerning pathogenesis, diagnosis and therapy of AITD. Human thyroid transplants of Graves' disease tissue, depending upon extrinsic stimulators, lose all signs of hyperfunction after transplantation into athymic nude mice, but hyperfunction can again be induced in the transplants by injections of sera or immunoglobulin fractions from patients with active Graves' disease, containing stimulating TSH receptor antibodies. Our data demonstrate that thyroid function and growth are independent phenomena and can be differentiated by means of the xenotransplantation model. Furthermore the presented results indicate that TSH receptor antibodies are able to alter the expression of gene products in human thyroid transplants of Graves' disease, emphasising their important role in inducing/perpetuating immune phenomena related to AITD.


Selective decontamination of the digestive tract in intensive care.

Nosocomial infection in intensive care unit (ICU) practice is a common problem and is associated with abnormal carriage of Gram-negative aerobic bacilli in the gastrointestinal tract, resulting in endogenous infections. Selective decontamination of the digestive tract (SDD) is a regimen aimed at preventing or eradicating this abnormal carriage. A large number of trials examining SDD in ICU practice have been published, the vast majority showing a significant reduction in the incidence of nosocomial, Gram-negative infection. However, the impact on morbidity and mortality is much less certain. A recent meta-analysis has suggested a 10-20% reduction in mortality (3-6% absolute difference) with SDD. A discussion of these results is presented together with potential criticisms of SDD.


Selective decontamination in bone marrow transplant recipients.

Patients undergoing bone marrow transplantation become immunocompromised for various reasons. Deep granulocytopenia, induced by conditioning (chemotherapy and total body irradiation), renders the patient at risk for serious bacterial and fungal infections. Our strategy for prevention of these infections by selective decontamination (SD) is the result of more than 15 years of clinical experience and research. The combination of antibiotics, used as standard SD (neomycin, polymyxin B, pipemidic acid and amphotericin B), with the application of local antimicrobial agents eliminates aerobic Gram-negative rods, Staphylococcus aureus and Candida spp. from the mucosal surfaces of the digestive tract, while the majority of the anaerobic flora persist and support colonization resistance (CR). The antibiotics used either are not resorbed or do not yield therapeutic serum concentrations. Antibiotics which induce therapeutic serum concentrations, such as ciprofloxacin and cotrimoxazole, are only used for SD on a limited scale. When Gram-negative rods persist despite intake of the standard regimen, ciprofloxacin is given until these persisting rods are eliminated. If the patients cannot swallow the oral regimen, i.v. cotrimoxazole is given temporarily. Streptococcal infections are prevented by the i.v. administration of penicillin for 14 days starting on the first day after cytotoxic treatment (conditioning for bone marrow transplantation). The combination of SD and systemic prophylaxis has been shown to be adequate; the major problem then remaining is a relatively mild catheter-associated infection with coagulase-negative staphylococci.


The relationship between infecting dose and severity of disease in reported outbreaks of Salmonella infections.

The relationship between size of the infecting dose and severity of the resulting disease has been investigated for salmonella infections by reanalysis of data within epidemics for 32 outbreaks, and comparing data between outbreaks for 68 typhoid epidemics and 49 food-poisoning outbreaks due to salmonellas. Attack rate, incubation period, amount of infected food consumed and type of vehicle are used as proxy measures of infecting dose, while case fatality rates for typhoid and case hospitalization rates for food poisoning salmonellas were used to assess severity. Limitations of the data are discussed. Both unweighted and logit analysis models are used. There is no evidence for a dose-severity relationship for Salmonella typhi, but evidence of a correlation between dose and severity is available from within-epidemic or between-epidemic analysis, or both, for Salmonella typhimurium, S. enteritidis, S. infantis, S. newport, and S. thompson. The presence of such a relationship affects the way in which control interventions should be assessed.


An outbreak of Salmonella dublin infection in England and Wales associated with a soft unpasteurized cows' milk cheese.

An outbreak of Salmonella dublin infection occurred in England and Wales in October to December 1989. Forty-two people were affected, mainly adults, and most lived in south-east England. Microbiological and epidemiological investigations implicated an imported Irish soft unpasteurized cows' milk cheese as the vehicle of infection. A case-control study showed a statistically significant association between infection and consumption of the suspect cheese (p = 0.001). Salmonella dublin was subsequently isolated from cheeses obtained from the manufacturer's premises. Initial control measures included the withdrawal of the cheese from retail sale and a Food Hazard Warning to Environmental Health Departments, as well as a press release, from the Department of Health. Subsequently, a decision was taken by the manufacturer to pasteurize milk used in the production of cheese for the UK market and importation of the cheese resumed in June 1990.


An investigation into an outbreak of Salmonella enteritidis phage-type 4 infection and the consumption of custard slices and trifles.

Epidemiological investigation into an outbreak of food poisoning in 17 patients caused by Salmonella enteritidis phage-type 4 demonstrated a highly significant association with consumption of custard, retailed in custard slices and trifles from a bakery on one day. The bakery had changed their recipe for custard 2 weeks earlier to include fresh shell eggs and had not followed earlier national advice on cooking eggs for human consumption. The case-control study supports earlier work associating Salmonella enteritidis phage-type 4 infection with consumption of uncooked or lightly cooked shell eggs.


The identification of Salmonella enteritidis-infected poultry flocks associated with an outbreak of human salmonellosis.

In the summer of 1991 a human outbreak of Salmonella enteritidis infection occurred following a barbecue in which about 100 persons were involved. Eggs, supplied by one or more of 10 different layer farms, were the most probable source of the infection. To identify the S. enteritidis-positive flocks, an immunoassay was used to detect salmonella serogroup D-specific antibodies in the yolk of hens eggs. Antibody titres in the eggs from two layer farms, farm A and B, clearly exceeded the titres found in randomly collected eggs. Further investigation on farm A and B yielded high antibody titres in the eggs from flocks A1, A2 and B2, and low titres in the eggs from flock B1. S. enteritidis was isolated from the faecal samples of flocks A1, A2 and B2, whereas no salmonella was detected in the faecal samples of flock B1. The flocks present on both farms originated from the same breeder flock.


Occurrence of quinolone resistance in Staphylococcus aureus from nosocomial infection.

Among 63 Staphylococcus aureus isolates (one isolate per one patient) counted from infections (from August to November 1991) in hospital T., eight exhibited resistance to fluoroquinolones. Seven of these quinolone-resistant isolates were multiply- and methicillin-resistant S. aureus (QR-MRSA). The results of phage-, plasmid- and genotyping (pulsed field electrophoresis) revealed that six different strain-clones of these MRSA were spread in the hospital. In vitro spontaneous mutants resistant to fluoroquinolones are 10-100-fold more frequent in MRSA than in other S. aureus when selected on isosensitest-agar containing 1 microgram/ml of ciprofloxacin. However, the same mutant frequencies were found in strain 8325-4 with and without the mecA-determinant. The resistance phenotype was stable over 30 generations of subculture in nutrient broth as well in natural quinolone resistant MRSA as in mutants of other types of S. aureus selected in vitro. The phenotypic association of quinolone resistance and MRSA is rather likely due to a higher frequency of spontaneous resistant mutants which are present in natural populations of MRSA. Data of chemotherapy prior to the isolation of S. aureus show that three of seven patients from whom QR-MRSA were isolated were treated with a quinolone. In eight cases of infections with non-MRSA and quinolone treatment the isolated S. aureus strains were in vitro sensitive to quinolones.


A study of phenotypic variation of Staphylococcus epidermidis using Congo red agar.

This study examines a series of phenotypic variants of Staphylococcus epidermidis that were generated from a pair of parent variants, isolated from valvular tissue of a patient with prosthetic valve endocarditis. The variants were initially classified by examining their colonial morphology on Congo red agar. In addition to differences in Congo red binding and colonial morphology, they differed in the expression of several surface components and enzymes. Despite these phenotypic differences, all variants had the same restriction endonuclease profile of plasmid DNA. Examination of a collection of clinical isolates demonstrated that phenotypic variation is a common property of S. epidermidis. The ability to express different combinations of surface components and enzymes could contribute to the virulence of S. epidermidis strains by enabling these organisms to colonize a range of diverse environments.


A longitudinal study of Staphylococcus hyicus colonization of vagina of gilts and transmission to piglets.

High Staphylococcus hyicus colonization rates were found in vaginal samples of healthy breeding sows and in skin samples of their offspring. Twenty-two different phage types were identified among the 720 isolates of S. hyicus examined. Two to 13 different phage types were isolated per herd. Phage typing, as well as characterization of about 10% of the isolates by plasmid profiles and antibiogram patterns, showed that, several different clones of S. hyicus could be present simultaneously in vagina of gilts and also on skin of piglets. Generally isolates from the vagina of one animal were identical as regards to phage types, plasmid profiles, and antibiogram patterns during the entire investigation period. Isolates from the skin of piglets were of the same type as their mothers, indicating that vertical transmission had taken place. S. hyicus strains isolated from the skin of piglets within 24 h after birth were identical to strains isolated 3 weeks after birth from the same litter, indicating that the vaginal strains became part of a stable skin flora.


Secretor status and humoral immune responses to Neisseria lactamica and Neisseria meningitidis.

Non-secretors of ABO blood group antigens are over-represented among patients with meningococcal diseases. Lower levels of secretory IgA reported for non-secretors have been suggested to compromise mucosal defences. Total serum and salivary IgG, IgA and IgM and levels of these isotypes specific for Neisseria lactamica and five isolates of meningococci were determined by ELISA for 357 pupils and staff of a secondary school in which an outbreak of meningitis occurred. There were no differences in total or specific levels of serum IgG, IgA or IgM or salivary IgG or IgA of secretors compared with non-secretors. Non-secretors had significantly lower levels of salivary IgM (P = 0.022). A similar pattern was observed for levels of IgM specific for N. lactamica and five meningococcal isolates. The significance of these results is discussed with reference to the role of secretory IgM in protection of mucosal surfaces in infants.


High level resistance to trimethoprim, cotrimoxazole and other antimicrobial agents among clinical isolates of Shigella species in Ontario, Canada--an update.

A total of 598 isolates of Shigella species (24 S. dysenteriae, 254 S. flexneri, 30 S. boydii, 290 S. sonnei) submitted to the Ontario Public Health Laboratories in 1990 were tested for their susceptibility to 14 antimicrobial agents by the agar dilution method. Overall 79.6% of isolates were resistant to one or more antimicrobial agents and 52.0% were resistant to four or more. Trimethoprim resistance ranged from 26.7% among isolates of S. boydii to 39.4% among S. flexneri strains. The majority of the 224 TMP resistant isolates (88.8%) demonstrated high level resistance (MIC > 1000 mg/l) to trimethoprim. Resistance to cotrimoxazole increased from 3% in 1978 to between 26.7 and 37.6% in 1990. MICs for 90% of isolates (MIC90s) for ampicillin, ticarcillin and piperacillin were 128 to > 256 mg/l, > 256 for tetracycline and chloramphenicol, and > 2.0/38.0 for cotrimoxazole. These results from the Canadian Province of Ontario emphasize the need for prudent use of antimicrobial agents in the treatment of shigellosis.


Transferable high-level trimethoprim resistance among isolates of Escherichia coli from urinary tract infections in Ontario, Canada.

Of 1171 isolates of Escherichia coli isolated from urine samples at the Public Health Laboratory, Toronto, Ontario, Canada, between May 1990 and December 1991, 120 (10.3%) were resistant to trimethoprim (TMP), cotrimoxazole (TMP/SMX), sulfamethoxazole (SMX) and other antimicrobial agents; 110 of the 120 isolates (91.7%) were resistant to four or more agents. The majority of resistant isolates (91.7%) exhibited high-level resistance (MIC > 1000 mg/L) to TMP. The MIC of TMP/SMX for all 120 isolates was > 2.0/38.0 mg/L and for SMX > 1024 mg/L. High-level resistances were also present among the beta-lactam antimicrobials with MICs ranging from 16- > 256 mg/L. Forty-three of 120 TMP-resistant (35.8%) isolates conjugally transferred TMP-resistance to E. coli K-12. Co-transfer of several other resistances was observed. SMX cotransferred from 86% of the 43 donors and beta-lactams together with SMX cotransferred from 70%. Nalidixic acid resistance was present among 22 (18.3%) of the 120 resistant isolates, however, nalidixic acid resistance was not transferred to E. coli K-12.


Cockroaches as carriers of bacteria in multi-family dwellings.

The potential risk of bacterial dissemination due to the presence of cockroaches (Blattella germanica, Blattellidae) in low-income flats was investigated. Cockroaches can carry a great variety of bacterial species; we identified 30 different species from 52 different flats. Klebsiella oxycytoca, K. pneumoniae and Enterobacter cloacae were the most frequently found. Pathogenic and potentially pathogenic bacteria represented 54% of all the bacterial identifications. Bacteria were carried either on the cuticle or in the gut. Contamination through external contact is sufficient to insure bacterial diffusion. There was a very low level of overlap estimated by Pianka's index (a) between the bacterial flora of neighbouring blocks of flats, and (b) between bacterial flora of different flats in the same block.


Destruction of bacteria on fresh meat by hot water.

Strains of different bacterial genera; Escherichia coli (7), Salmonella serotypes (7), Enteropathogenic E. coli O 157 (2), Aeromonas hydrophila (4), Yersinia enterocolytica (1), Pseudomonas fragi (5), and Listeria monocytogenes (5), inoculated onto the surface tissues of pieces of fresh beef were found to be susceptible to the lethal action of hot water. On average, more than 3 log10 (99.9%) of these organisms were destroyed when water at 80 degrees C was applied for 10 or 20 s. Therefore, a hot water decontamination cabinet would be effective for destroying in situ any viable cells of these bacteria which might be present on the surface tissues of fresh beef carcasses.


Circulating antibodies against faecal bacteria assessed by immunomorphometry: combining quantitative immunofluorescence and image analysis.

A new technique to study the prevalence of circulating antibodies directed against different morphological groups ('morphotypes') of bacteria in fresh faeces is presented. The technique combines quantitative indirect immunofluorescence with digital image analysis. Plasma antibody titres and patterns of IgA, IgG and IgM isotype against morphotypes of faecal bacteria were determined in ten healthy individuals.


Factors influencing oral carriage of yeasts among individuals with diabetes mellitus.

A total of 439 individuals with diabetes mellitus were examined for carriage of yeasts by the oral rinse and palatal swab techniques. Eighteen genetic or environment variables were assessed for their contribution to carriage of yeasts. The factor contributing to palatal and oral carriage of yeasts among individuals with insulin dependent diabetes mellitus (IDDM) was age (P < 0.01). The factor contributing to palatal carriage of yeasts among individuals with non-insulin dependent diabetes mellitus (NIDDM) was poor glycaemic control (glycosuria P < 0.01); carriage in the oral cavity as a whole was influenced additionally by non-secretion of ABH blood group antigens (P < 0.05). Introduction of a denture altered the above risk factors. For individuals with IDDM, oral carriage was associated with the presence of retinopathy (P < 0.05); palatal carriage was influenced by poor glycaemic control (HbA1P < 0.01, plasma glucose levels P < 0.05) and age (P < 0.05). For those with NIDDM, palatal carriage was associated with continuous presence of the denture in the mouth (P < 0.01); oral carriage was associated with plasma glucose levels (P < 0.05).


Epidemiology of enteric adenovirus infection in prospectively monitored Argentine families.

To examine the role of enteric adenoviruses (EAV) in an urban area of Buenos Aires (Argentina), we prospectively studied faecal samples from 49 families of newborns. These were monitored weekly for diarrhoea for 2 years. A total of 180 samples from cases of diarrhoea and 766 samples obtained during diarrhoea-free periods were studied by dot-blot hybridization with an EAV-specific DNA probe. EAV were found in 6/180 (3.3%) cases of diarrhoea and 6/766 (0.8%) asymptomatic samples (P < 0.015). Incidence of EAV was 3.9 cases per 100 person-years in children < 60 months old. EAV-related diarrhoeas were slight and of short duration. In addition, 129 faeces from hospital out-patients, 1-30 months old, were also studied. EAV was identified in 7/129 cases (5.4%). These cases were 9.5 +/- 3.5 months old and the diarrhoea was mild or severe, of 3 +/- 1.5 days of duration. We suggest that EAV are low-risk causes of diarrhoea under natural conditions, although a few children may develop more severe diarrhoea. The diagnosis of EAV needs to be considered in these patients.


Comparison of the HA genes of type B influenza viruses in herald waves and later epidemic seasons.

From January 1985 to May 1991, herald strains of influenza B virus were isolated in 1987 and 1989 in Japan. In both cases, influenza epidemics caused by the same type followed in the next winter season. The HA gene sequences of the influenza B viruses isolated in Japan from 1987-91, which covers two herald waves of influenza B viruses, were analysed and located on the phylogenetic tree for influenza B viruses after the B/Singapore/64 strain. Co-circulation of at least two evolutionary lineages of the HA genes existed for influenza B viruses in Japan during the period of this study. The herald viruses in one wave (1987) were genetically close to the winter isolates and were considered to be the parental viruses for the following influenza season, while in the other wave (1989) winter isolates belonged to another lineage on which one of the herald viruses was located, but they were genetically and antigenically different from the herald viruses.


Use of etoposide in combination with cyclosporin for purging multidrug-resistant leukemic cells from bone marrow in a mouse model.

Cyclosporin (CsA) is a potent modulator of multidrug resistance (MDR) and has been combined with etoposide (VP-16) to purge MDR leukemic cells from human bone marrow (BM) in vitro. We studied the feasibility of this approach in an in vivo model for autologous BM transplantation using the murine leukemia cell line P388 and its MDR variant P388/ADR. Colony-forming assays with 2-h drug exposure revealed a tumor selectivity of VP-16 for P388 cells compared to normal murine marrow granulocyte-macrophage colony-forming units (CFU-GM), whereas P388/ADR cells were resistant to VP-16. Simultaneous incubation with CsA restored sensitivity in these cells. Almost 4 logs of cell kill were achieved by treating P388/ADR cells with 60 microM VP-16 plus 2.5 microM CsA (combination A) or 40 microM VP-16 plus 10 microM CsA (combination B), whereas there was a 2.5-log reduction of CFU-GM at these doses. Even though the myelotoxicity of VP-16 was increased by the addition of CsA, this effect was nonspecific as shown by a similar chemosensitization in sensitive P388 as well as in P388/VP 2.5 cells, an atypical MDR variant lacking P-glycoprotein. In vivo experiments addressed the ability of BM treated with VP-16 and CsA to rescue lethally irradiated mice and to purge leukemic cells. In total, 1/14 lethally irradiated mice died due to sepsis within 10 days after receiving 15 x 10(6) BM cells treated ex vivo with combination A in contrast to 1/4 for combination B. All 16 surviving animals demonstrated long-term engraftment. When simulated remission marrow contaminated with 0.1% P388/ADR was purged with VP-16 (60 microM) or CsA (2.5 microM) alone, all mice died from leukemia before day 16 after transplantation (median 14.3 and 12.2 days). In contrast, nine of ten animals receiving similar marrow purged with combination A survived > 60 days without any evidence of disease (p < 0.01). We conclude that combining VP-16 and CsA was effective in purging MDR leukemia cells from transplanted BM in this murine model.


Methylation status of c-myc oncogene in leukemic cells: hypomethylation in acute leukemia derived from myelodysplastic syndromes.

DNA methylation plays an important role in gene regulation. We have analyzed the methylation status of CCGG sites in and around the human proto-oncogene c-myc in blood cells from patients with acute and chronic leukemias and with myelodysplastic syndromes using restriction endonucleases. The 5' region of c-myc was unequivocally hypomethylated in all the 58 specimens studied, including 10 from normal bone marrow and 1 from human placenta. In contrast, the 3' region was hypermethylated in a great majority of cases. However, this region was hypomethylated in 1 of 12 patients with de novo acute myeloid leukemia, 1 of 6 patients with chronic myeloid leukemia, and 4 of 5 patients with acute myeloid leukemia preceded by a documented stage of myelodysplastic syndromes. One possible mechanism for the 3' region of c-myc to have remained hypomethylated may be a "delayed methylation" during transforming events toward a more aggressive stage of the disease, but the precise mechanism is unknown.


Composition and functional integrity of the in vitro hemopoietic microenvironment in acute myelogenous leukemia: effect of macrophage colony-stimulating factor.

In the present work, we have investigated the composition and hemopoietic supportive capacity of eleven normal and six acute myelogenous leukemia (AML) marrow-derived stromal adherent layers, established in the absence or in the presence of recombinant human colony-stimulating factor 1 (rhCSF-1, macrophage colony-stimulating factor). Two of six AML adherent layers were deficient in composition (i.e., no confluency, reduced numbers of macrophages and fibroblastic progenitors, and no fat cell formation), resulting in reduced CSF-1 production and a poor hemopoietic supportive capacity (assessed by the ability of an irradiated stroma to sustain the growth of myeloid, erythroid, and multipotential progenitors derived from a second innoculum of normal bone marrow). Four out of six AML adherent layers showed levels of macrophages, fibroblastic progenitors, fat cells, and CSF-1 similar to those observed in adherent layers from normal bone marrow; however, their capacity to sustain normal hemopoiesis was still significantly reduced. The deficient hemopoietic supportive capacity of all AML adherent layers correlated with the presence of a soluble activity in the culture supernatant that inhibited hemopoietic colony formation. Addition of rhCSF-1 during the establishment of AML adherent layers significantly increased their hemopoietic supportive capacity. In contrast, the hemopoietic supportive capacity of normal adherent layers was reduced by rhCSF-1. The opposite effects of rhCSF-1 on the hemopoietic supportive capacity of normal and AML adherent layers correlated with the levels of the soluble inhibitory activity, that is, increased levels in cultures containing rhCSF-1-treated normal adherent layers, and slightly reduced levels in cultures of rhCSF-1-treated AML layers. These results indicate that, despite a morphologically normal composition in most cases (four out of six), the hemopoietic microenvironment developed in long-term marrow culture (LTMC) from all AML marrows analyzed has a deficient hemopoietic supportive capacity, due, at least in part, to the production of hemopoietic inhibitor(s). Such a deficiency can be partially overcome by establishing the stroma layers in the presence of rhCSF-1.


Botrocetin agglutination of rat megakaryocytes: a rapid method for megakaryocyte isolation.

Botrocetin isolated from the venom of Bothrops jaracara has been shown by others to induce binding of von Willebrand Factor to glycoprotein Ib and thereby produce platelet agglutination in a wide range of animal species. We have found that botrocetin also facilitates the agglutination of megakaryocytes and have used this property to develop a method to isolate megakaryocytes from rat bone marrow. When botrocetin is added to a mixture of rat bone marrow, rat platelets, and rat plasma, coagglutination of megakaryocytes and platelets occurs. The agglutinated complexes, containing > 95% of the megakaryocytes, may then be separated from the remaining marrow cells by filtration. Megakaryocytes account for 39% (range 30%-48%) of the isolated cells and 83% (range 77%-88%) of the isolated cell mass. This method allows the virtually complete removal of megakaryocytes from bone marrow as well as their isolation to a high degree of purity. It should provide a useful, inexpensive, general method for the rapid isolation of megakaryocytes from multiple, small marrow samples from a wide range of species.


Cell cycle status of murine megakaryocyte and granulocyte-macrophage colony-forming cells in bone marrow and spleen.

The cell cycle status of megakaryocyte colony-forming cells (Meg-CFC) and granulocyte-macrophage colony-forming cells (GM-CFC) from the spleen and bone marrow of C57BL mice was evaluated by determining the effects of hydroxyurea (OHU) or cytosine arabinoside (Ara-C), both in vivo and in vitro, upon colony-forming cells (CFC). The concentrations of cells in culture (2 x 10(6) to 4 x 10(6)/ml for spleen and 0.25 x 10(5) to 1.0 x 10(5)/ml for bone marrow) did not alter cell cycle status of either Meg-CFC or GM-CFC. Determination of cell cycle status following in vivo administration of OHU indicated that 25.2% of Meg-CFC and 28.1% of GM-CFC in the spleen, and 26.0% of Meg-CFC and 29.5% of GM-CFC in the bone marrow, were in cycle. In vitro incubation of CFC with OHU showed that in the spleen 25.1% of Meg-CFC and 24.2% of GM-CFC were engaged in DNA synthesis, whereas in bone marrow 28.5% of Meg-CFC and 29.2% of GM-CFC were synthesizing DNA. Incubation with Ara-C, in vitro, gave similar results, with 26.0% of Meg-CFC and 26.2% of GM-CFC in the spleen, and 27.1% of Meg-CFC and 31.4% of GM-CFC in the bone marrow, in cycle. In summary, significant differences were not observed between the cell cycle status of Meg-CFC and GM-CFC, whether derived from spleen or bone marrow. In vitro and in vivo measurements (with OHU) and in vitro measurements with two cytotoxic drugs (OHU versus Ara-C) also provided similar results. The data suggest that the regulation of DNA synthesis in both Meg-CFC and GM-CFC in the murine spleen and bone marrow is similar.


Expression of stem cell inhibitor (SCI) gene in patients with bone marrow failure.

Stem cell inhibitor (SCI) has been shown to inhibit the proliferation of primitive progenitors. The inhibitor, a product of bone marrow macrophages, activated lymphocytes, and monocytes, is identical to macrophage inflammatory protein (MIP-1 alpha). We report homologous (SCI/hMIP-1 alpha) sequences in freshly isolated lymphocytes, monocytes, and granulocytes and have found that SCI mRNA can be induced in monocytes by lipopolysaccharide (LPS) and interleukins 1, 2, and 6. In contrast, interferon gamma (IFN-gamma) decreases the expression of SCI/hMIP-1 alpha. Although only a low level expression of SCI/hMIP-1 alpha mRNA can be detected in normal human bone marrow nucleated cells (NCBM), very significant increases in the levels of SCI/hMIP-1 alpha RNA transcripts are observed in NCBM from patients with aplastic anemia (AA) and myelodysplastic syndrome (MDS). These data suggest that the expression of SCI/hMIP-1 alpha in bone marrow may reflect dysregulated cytokine production and activation of the immune system that may possibly contribute to disease progression.


Production of interleukin 3 and granulocyte-macrophage colony-stimulating factor from stimulated blood mononuclear cells in patients with aplastic anemia.

Blood cells from patients with aplastic anemia (AA) were evaluated for the ability to produce interleukin 3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) on stimulation with phytohemagglutinin (PHA) or antilymphocyte globulin (ALG) by the use of an IL-3-dependent cell-line, TF-1, and the GM-CSF-IRMA kit. The IL-3 levels in patients with active AA were significantly lower, both in PHA-stimulated conditioned medium (CM) and in ALG-CM, than those of normal healthy donors (HD; p < 0.01). The degree of reduced IL-3 production in AA patients correlated well with the severity of neutropenia; the level of IL-3 returned to normal after successful treatment with ALG plus methylprednisolone (ALG therapy). On the other hand, GM-CSF production in AA patients varied widely and was only significant in remission patients in PHA-CM; in this case production was higher than that in active AA patients (p < 0.05) or in HD (p < 0.01). Sensitivity to PHA or ALG stimulation was evaluated by the ratio of IL-3 concentrations in ALG-CM versus PHA-CM (ALG/PHA index). The index varied widely from < 0.1 to > 10 in AA patients, contrasting to the clustered values in HD. Seven of the eight patients who had an ALG/PHA index of > 1.0 showed a good clinical response to ALG therapy. However, 12 of the 14 patients who had a lower index (< 1.0) failed to respond. The ALG/PHA index might have an ability to predict the response to ALG therapy.


Adult and neonatal patterns of human globin gene expression are recapitulated in liquid cultures.

We have recently described a two-step liquid culture system that supports the proliferation and maturation of human erythroid progenitors. Several days after the addition of erythropoietin, the cultures undergo erythroid differentiation in a synchronized fashion. The purpose of the present study was to determine detailed kinetics of globin gene expression at the mRNA level in adult and newborn erythroid cells. Our results show that in cultures derived from normal adult peripheral blood, the mRNA levels of alpha- and beta-globin genes increased throughout most of the culture period, whereas gamma-globin mRNA remained at a low level. In contrast, high expression of all three globin genes, alpha, beta, and gamma, was observed in cultures derived from cord blood. The results demonstrate that the populations of erythroid progenitors in cord blood and in adult peripheral blood are fundamentally different, suggesting that this culture system recapitulates the normal pattern of globin gene expression, providing a valuable tool in the investigation of the regulation of the switch from fetal to adult hemoglobin.


Triggering of stem cell (CFU-S) proliferation after transplantation into irradiated hosts.

The paper demonstrates the beginning of increased proliferation of the bone marrow clonogenic cells, detected by the spleen colony technique, taken from normal mice and transplanted into syngeneic recipients, lethally irradiated 24 h previously. A lag period of about 10 h preceded the beginning of increased proliferation that was determined according to the sensitivity of transplanted cells to the lethal action of hydroxyurea.


A DNA fragment homologous to F1-ATPase beta subunit was amplified from genomic DNA of Methanosarcina barkeri. Indication of an archaebacterial F-type ATPase.

A 490 bp DNA fragment was amplified from Methanosarcina barkeri genomic DNA by the polymerase chain reaction (PCR) using oligonucleotide primers designed based on conserved amino acid sequences of the F1-ATPase beta subunits. The amino acid sequence deduced from the DNA sequence of this fragment was highly homologous to a portion of the F1-ATPase beta subunit. This indicates that this archaebacterium has a gene of F-type ATPase in addition to a gene of V-type ATPase.


Nucleotide sequence of the genes for ribosomal proteins HS15 and HSH from Haloarcula marismortui: an archaeon-specific gene cluster.

The nucleotide sequences of the genes for two ribosomal proteins, HS15 and HSH, from the archaeon Haloarcula marismortui, have been determined. The genes were found in a cluster together with another open reading frame with a probable regulatory function. HS15 and HSH have counterparts in eucarya. HS15 is significantly homologous to S19 from frog (Xenopus laevis). HSH is related to S37 from yeast (Saccharomyces cerevisiae) and S27 from fly (Drosophila melanogaster), as well as to other members of the S27 family. Eubacterial counterparts were not found, suggesting that these proteins are 'extra proteins' that are absent in eubacterial ribosomes.


Molecular cloning of G protein alpha subunits from the central nervous system of the mollusc Lymnaea stagnalis.

The central nervous system of the pond snail, Lymnaea stagnalis, contains many large, identified neurons which can be easily manipulated making it an advantageous model system to elucidate in vivo the architecture of neuronal signal transduction pathways. We have isolated three cDNA clones encoding G protein alpha subunits that are expressed in the Lymnaea CNS, i.e. G alpha o, G alpha s and G alpha i. The deduced proteins exhibit a very high degree of sequence identity to their vertebrate and invertebrate counterparts. The strong conservation of G protein alpha subunits suggests that functional insights into G protein-mediated signalling routes obtained through the experimental amenability of the Lymnaea CNS will have relevance for similar pathways in the mammalian brain.


The amino acid sequence of a type I copper protein with an unusual serine- and hydroxyproline-rich C-terminal domain isolated from cucumber peelings.

We have determined the amino acid sequence of a small copper protein isolated from cucumber peelings. This cupredoxin contains 137 amino acids including a pyroglutamate as the first residue. The N-terminal 110 amino acid-long domain shows 30-37% identity to 2 other cupredoxins, stellacyanin and cucumber basic blue protein. A unique feature of this protein is a 27 amino acid-long C-terminal domain rich in 4-hydroxyproline and serine and resembling certain plant cell wall proteins. The prolines in this domain are hydroxylated to a different extent depending on the surrounding sequence.


Proteolytic processing of human intestinal lactase-phlorizin hydrolase precursor is not a prerequisite for correct sorting in Madin Darby canine kidney (MDCK) cells.

Maturation of lactase-phlorizin hydrolase (LPH) (EC 3.2.1.23-62) requires proteolytic processing of precursor (pro-LPH) to mature microvillus membrane enzyme (m-LPH). Subcellular site and function of this processing are unknown. We studied the processing and sorting of human LPH expressed permanently in MDCK cells. LPH was inserted into the apical membrane and small amounts were found basolateral. Of the LPH immunoprecipitated from the apical membrane, 42% was in the mature, i.e. proteoytically processed form; on the basolateral membrane it was 20%. Thus, LPH-processing occurs after sorting and is not necessary for surface expression.


Inhibition of passive cutaneous anaphylaxis by synthetic human immunoglobulin E peptide fragments.

In an attempt to determine the regions responsible for type I immediate hypersensitivity, a total of 42 peptide fragments, which cover the CH3-CH4 domains in human immunoglobulin E (IgE), were chemically synthesized. Several peptide fragments located in the amino acid sequences Ala329-Thr357 and Arg419-Ala463, inhibited passive cutaneous anaphylaxis (PCA) in vivo. In order to pinpoint the sites responsible for the inhibition of the PCA reaction, various fragment peptides in these two regions were synthesized. As a result, residues Pro343-Leu348, Pro426-Thr433, and Ser456-Thr461 were suggested to be involved in type I immediate hypersensitivity.


Processing of the chloroplast transit peptide of pea carbonic anhydrase in chloroplasts and in Escherichia coli. Identification of two cleavage sites.

The chloroplast transit peptide (cTP) of pea carbonic anhydrase was shown to be processed at two different sites, giving protein subunits of two sizes. The cleavage sites were identified and found to be localized immediately before and after a highly charged part, containing 8 acidic and 6 basic residues, of the cTP. Properties of pea carbonic anhydrase produced in Escherichia coli show that folding, oligomerization and catalytic activity do not depend on the presence of the acidic part or the rest of the cTP. The pattern of processing of the cTP in E. coli indicates that cleavage at site I is specific for a chloroplastic stromal peptidase and that cleavage at site I prevents processing at site II.


DNA-specific antiidiotypic antibodies in the sera of patients with autoimmune diseases.

Blood sera of patients with autoimmune diseases scleroderma (Scl), systemic lupus erythematosus (SLE), and rheumatoid arthritis (RA) have been shown to yield a specific immune response to topoisomerase I, the product of expression of a cDNA fragment cloned into lambda gt11 and monoclonal antibodies (MAB) to the enzyme. The 'topoisomerase test' is not absolutely specific for Scl. The stable positive response of autoimmune sera to anti-topoisomerase monoclonal antibodies has a specific character and is associated with the interaction of the Fab fragment of MAB with the IgG fraction of autoimmune serum. The response observed indicates the induction of anti-idiotypic antibodies against topoisomerase. The anti-idiotype, isolated by HPLC and affinity chromatography demonstrated the following functional activities: (i) the immunological reaction against DNA; (ii) high-affinity DNA-binding with topoisomerase-specific consensus; (iii) ability to compete with the native enzyme for binding with DNA and MAB to topoisomerase; (iv) immunological reaction against MAB to topoisomerase.


Air saturation of the medium reduces the rate of phosphorylating oxidation of succinate in isolated mitochondria.

Rat brain mitochondria were isolated and their respiration was polarographically measured without contact with air oxygen. Gas-saturated experimental mixtures close to the in vivo partial oxygen pressure (normoxic) were compared with the air-saturated, i.e. hyperoxic, mixtures. The rate of phosphorylating oxidation of added succinate under normoxic conditions was found to be 70-100% higher compared with hyperoxic ones. The addition of succinate dehydrogenase activators results in a more than two-fold stronger stimulation of succinate oxidation under normoxia than under hyperoxia. Thiol group donors are shown to stimulate respiration under hyperoxia and not under normoxia. Hyperoxic conditions prevented oxidation of the low succinate concentrations corresponding to the physiological ones.


Investigation of the neighbour relationships between photosystem II polypeptides in the two types of isolated reaction centres (D1/D2/cytb559 and CP47/D1/D2/cyt b559 complexes).

The nearest neighbour relationships within the D1/D2/cyt b559 complex (PSIIRC) and the CP47/D1/D2/cyt b559 complex (RC-CP47) were investigated by using different length bifunctional crosslinking agents. The crosslinking products were identified by immunoblotting with polyclonal antibodies and by two-dimensional gel electrophoresis. Seven products (CP47/D2, D1/D2/alpha, D1/D2, D2/alpha, D1/alpha, alpha/alpha, alpha/beta) have been revealed in both complexes. The crosslinking of both complexes does not increase their photostability. The photocrosslinking products (D1/alpha and D2/alpha) appeared under illumination of complexes with light of high intensity.


Diversion of the sign of phototaxis in a Chlamydomonas reinhardtii mutant incorporated with retinal and its analogs.

The blind mutant FN68 of the unicellular flagellate green alga Chlamydomonas reinhardtii is negatively phototactic in the presence of the native chromophore all-trans retinal. In contrast, analog chromophores such as a ring-acyclic retinal and those in which trans/cis isomerization about the C11 = C12 double bond was blocked induced predominantly positive phototaxis in the same strain under the same experimental conditions. These observations can be interpreted by assuming that the negative and the positive phototaxis is mediated distinctively by two rhodopsin species which differ in their affinities with the exogenous chromophores. However, a more reasonable explanation, which requires fewer assumptions, is that the sign of phototaxis depends on a delay in intracellular photosignal transduction. This novel view was deduced directly from the widely accepted hypothesis [1980, Microbiol. Rev. 44, 572-630] on phototaxis mechanisms.


The murine NPY-1 receptor gene. Structure and delineation of tissue-specific expression.

The murine gene for the NPY-1 receptor subtype for neuropeptide Y was characterized by DNA sequencing and expression studies. It comprises three exons with a 6,400 bp 5'-untranslated and a 80 bp internal intronic sequence. The 5'-flanking region of this gene lacks TATA or CCAAT consensus sequences in the proximity to the multiple transcription initiation sites. A 1,300 bp genomic fragment of the 5'-flanking region drives the expression of the lacZ reporter gene in NG108-15 cells and primary cultured neurons but not in glial and human embryonic kidney cells. In addition, it contains consensus sequences for various transcription factors including cAMP- and glucocorticoid-responsive elements.


Inhibition of epidermal growth factor receptor functions by tyrosine kinase inhibitors in NIH3T3 cells.

Epidermal growth factor (EGF) induces transformed phenotypes in EGF receptor-overexpressing NIH3T3 (ER12) cells. Tyrosine kinase inhibitors such as erbstatin and its stable analogue methyl 2,5-dihydroxycinnamate inhibited the EGF-induced phenotypic changes in these cells; while 5'-O-methylerbstatin, an inactive analogue, did not. Methyl 2,5-dihydroxycinnamate inhibited intracellular tyrosine kinase activity in EGF-treated ER12 cells. Methyl 2,5-dihydroxycinnamate also delayed the EGF-induced DNA synthesis from the quiescent phase ER12 cells without showing irreversible cytotoxicity. It inhibited the DNA synthesis most efficiently at the early G1 phase. Thus, tyrosine kinase inhibitors may modify malignant phenotypes in EGF receptor-overexpressing neoplasms.


Resonance Raman studies of the carbonmonoxy form of catalase. Evidence for and effects of phenolate ligation.

Resonance Raman spectra are reported for the carbon monoxide (CO) adduct of catalase formed from the reaction of peracetic acid or hydrogen peroxide with the azide adduct of catalase in the presence of CO. The expected three normal vibrations of the FE-CO fragment are detected at 1,908,593 and 543 cm-1 for the nu(C-O), delta(Fe-C-O) and nu(Fe-CO), respectively. The expected coordination of the phenolate group in this adduct is confirmed by the enhancement of an internal vibration of phenolate, nu 19a at 1,515 cm-1, and an extraordinary intensity enhancement of the nu(Fe-CO) mode.


Interdependence of respiratory NO reduction and nitrite reduction revealed by mutagenesis of nirQ, a novel gene in the denitrification gene cluster of Pseudomonas stutzeri.

An open reading frame, designated nirQ, was identified upstream of nirS, the structural gene for the respiratory nitrite reductase of Pseudomonas stutzeri ZoBell. Its derived gene product (275 amino acids, M(r) = 30,554) shows similarity to the NtrC protein family of transcriptional activators. Deletion-replacement mutagenesis of the nirQ gene resulted in the simultaneous loss of nitrite reduction and NO reduction in vivo. However, both reductases were still synthesized, with only nitrite reductase being active in vitro. NO reductase was overproduced by a factor of about 2. Our results indicate that the systems for nitrite reduction and NO reduction are functionally coupled.


Role of ADP-ribosylation in endothelial signal transduction and prostacyclin production.

ADP-ribosylation of proteins by the enzymatic transfer of ADP-ribose from NAD has been implicated in a number of biological processes. We report that inhibitors of ADP-ribosylation, most notably the novel inhibitor of arginine specific cellular mono(ADP-ribosyl) transferase, meta-iodobenzylguanidine (MIBG) as well as nicotinamide, L-arginine methyl ester (LAME) and guanyltyramine, inhibit histamine-induced endothelial production of inositol phosphates, release of arachidonic acid and production of prostacyclin (PGI2). Those same responses were unaffected by MIBG when triggered by thrombin or leukotriene C4. These findings suggest that ADP-ribosylation serves a role in histamine-induced production of prostacyclin and imply differences in transduction pathways employed by the different agonists.


A novel nuclear 42-kDa casein kinase identified in Chironomus tentans.

We have purified and characterised an apparently novel nuclear 42-kDa casein kinase from epithelial cells of Chironomus tentans which comigrates with a phosphoprotein associated with transcriptionally active salivary gland genes. The protein kinase promotes phosphorylation of casein and phosvitin, using either ATP or GTP as phosphate donors, and undergoes autophosphorylation. The casein kinase activity of the 42-kDa protein is sensitive to heparin, 5,6-dichloro-1-beta-D-ribofuranosylbezimidazole (DRB), spermine and spermidine indicating that it is a novel enzyme with similar but not identical properties to casein kinase II or nuclear protein kinase NII.


A novel ubiquinone reductase activity in rat cytosol.

Ubiquinone (UQ) reductase activity which reduces UQ to ubiquinol (UQH2) in rat tissues was roughly proportional to the UQH2/total UQ ratio in respective tissues. The highest activity was found in the liver, showing the highest UQH2/total UQ ratio. A greater part of liver UQ reductase activity was located in the cytosol. Within a week, the liver UQ reductase activity decreased by 80% even at -20 degrees C. The DT-diaphorase activity was stable. UQ reductase required NADPH as the hydrogen donor and was not inhibited by a less than 1 microM concentration of dicoumarol. There was no stimulation of UQ reductase in the presence of bovine serum albumin nor in Triton X-100. Yet, both stimulated DT-diaphorase. As a result, UQ reductase appeared to be a novel NADPH-UQ oxidoreductase and responsible for the UQ redox state in liver.


Photoreaction of N560 intermediate in the photocycle of bacteriorhodopsin.

Sophisticated measurements were made on the nanosecond time-resolved absorbance change of the purple membrane of Halobacterium halobium under cw background light irradiation (440-800 nm, 11-441 mW/cm2). A red-shifted transient species R660 (KN, Q) was found in alkaline conditions (pH > 9.3). Background light intensity effect shows that (i) R660 is photochemically formed from N560 intermediate which is accumulated under background light irradiation because of the elongated lifetime in alkaline suspension, and that (ii) the slow decaying M412 is not photochemically formed from N560 but from bR568.


Preferential binding of alpha-actinin to actin bundles.

At 37 degrees C, the alpha-actin-F-actin binding isotherm is anomalous. In 6.7% polyethylene glycol 6000, concomitantly with the formation of actin bundles, the binding isotherm becomes hyperbolic (Kdiss. = 11.3 microM). alpha-Actinin increases the rigidity of the networks formed by actin bundles in polyethylene glycol and by paracrystalline actin in 16 mM MgCl2 but not by F-actin. It is proposed that in the cell alpha-actinin functions are mostly carried on by interaction with actin bundles.


Pulsed magnetic field effects on calcium signaling in lymphocytes: dependence on cell status and field intensity.

The effect of 3-Hz, monopolar, quasi-rectangular magnetic field pulses on 45Ca2+ uptake in resting and mitogen-treated rat thymic lymphocytes was evaluated. A 30-min, non-thermal exposure to the pulsed magnetic field (Bpeak = 6.5 mT, Emax = 0.69 mV/cm, Jmax = 2.6 microA/cm2) reduced Concanavalin A-induced 45Ca2+ uptake by 45%. It was observed that (i) the induction of the 3-Hz field response depended on Ca2+ signal transduction activation; (ii) the response direction (stimulation or inhibition) depended on the level of lymphocyte mitogen responsiveness, and (iii) the field response magnitude increased with increasing magnetic field flux densities (Bpeak = 0, 1.6, 6.5 and 28 mT). Our results demonstrate field effects at Bmax nearly 10(4) greater than that of the average human environment for low-frequency magnetic fields and they are consistent with the independent results from other 3-Hz pulsed magnetic field studies with lymphocytes.


Bone morphogenetic proteins (BMP-2 and BMP-3) induce the late phase expression of the proto-oncogene c-fos in murine osteoblastic MC3T3-E1 cells.

Here we report that bone morphogenetic proteins 2 and 3 (BMP-2 and BMP-3) induced marked expression of c-fos mRNA in a biphasic manner, i.e. the late phase (48 to 60 h) as well as the immediate-early phase (0.5 h), in murine osteoblastic MC3T3-E1 cells in vitro. The BMP-induced late phase c-fos gene expression was temporally associated with the onset of marked expression of the genes for osteocalcin and alkaline phosphatase, differentiation markers of mature osteoblasts. In contrast, none of TGF-beta 1, 10% FBS, IGF-I and IGF-II, which induced only the immediate-early c-fos mRNA expression, stimulated the expression of osteocalcin and alkaline phosphatase genes. These data suggest that in osteoblasts BMP-2 and BMP-3 induce the late phase expression of c-fos, which may play a role in transcriptional activation of the genes involved in differentiation of osteoblasts.


Brefeldin A inhibits protein synthesis in cultured cells.

The fungal metabolite brefeldin A (BFA) is known to disrupt the Golgi apparatus resulting in redistribution of Golgi proteins to the endoplasmic reticulum and inhibition of protein secretion. BFA was found to inhibit protein synthesis in rat glioma C6 cells by up to 70% between 0.1 and 1 microgram/ml. Inhibition was both time-dependent and reversible. BFA inhibited protein synthesis to varying degrees in a number of other cell lines but not in BFA-resistant marsupial kidney cells. The same concentrations of BFA which inhibited protein synthesis, also blocked the inhibitory effects of Pseudomonas exotoxin and ricin on BFA-sensitive cells. BFA, however, was unable to block the inhibition of protein synthesis by the toxins in the resistant marsupial kidney cells.


The organisation of the mouse secretogranin II gene.

We have characterized the gene which encodes mouse secretogranin II (previously also referred to as chromogranin C), a tyrosine-sulfated secretory protein belonging to the granin (chromogranin/secretogranin) family which is found in secretory granules of most endocrine cells and neurons. The secretogranin II gene was found to contain 2 exons. In contrast to chromogranin A and chromogranin B, the two previously characterized granin genes, the entire secretogranin II protein is encoded by a single exon, exon 2, with exon 1 containing only a 5'-untranslated sequence. Consistent with previous data on the expression of secretogranin II, the putative promoter region was found to contain a cAMP-responsive element and a potential AP-1 binding site.


Presence and coding properties of 2'-O-methyl-5-carbamoylmethyluridine (ncm5Um) in the wobble position of the anticodon of tRNA(Leu) (U*AA) from brewer's yeast.

The unknown modified nucleoside U* has been isolated by enzymatic and HPLC protocols from tRNA(Leu) (U*AA) recently discovered in brewer's yeast. The pure U* nucleoside has been characterized by electron impact mass spectroscopy, and comparison of its chromatographic and UV-absorption properties with those of appropriate synthetic compounds. The structure of U* was established as 2'-O-methyl-5-carbamoylmethyluridine (ncm5Um). The yeast tRNA(Leu) (U*AA) is the only tRNA so far sequenced which has been shown to contain ncm5Um. The location of such a modified uridine at the first position of the anticodon restricts the decoding property to A of the leucine UUA codon.


Human urokinase contains GalNAc beta (1-4)[Fuc alpha (1-3)]GlcNAc beta (1-2) as a novel terminal element in N-linked carbohydrate chains.

Structural analysis of enzymically released N-linked carbohydrate chains of human urokinase (urinary-type plasminogen activator) by 1H NMR spectroscopy and FAB-MS demonstrated that the N-linked oligosaccharides on the only N-glycosylation site contain diantennary structures with the novel GalNAc beta (1-4) [Fuc alpha (1-3)]GlcNAc beta (1-2) element in the upper or the lower branch.


Effect of phosphoramidon on big endothelin-2 conversion into endothelin-2 in human renal adenocarcinoma (ACHN) cells. Analysis of endothelin-2 biosynthetic pathway.

The biosynthetic pathway of endothelin (ET)-2 was analyzed in cultured ACHN cells. In the supernatant, we detected three ET-2-related peptides, ET-2, big ET-2(1-38) and big ET-2(22-38). Phosphoramidon decreased the amount of ET-2 and increased that of big ET-2(1-38) dose-dependently. The amount of big ET-2(1-37) did not significantly change. These results suggest that big ET-2 is composed of 38 and not 37 amino acid residues, and that a putative ET-2-converting enzyme (ECE-2) should be classified as a phosphoramidon-sensitive neutral metalloprotease, bearing a resemblance to the putative ET-1-converting enzyme (ECE-1) in endothelial cells.


Altered response to growth factors in rat epithelial liver cells overexpressing human c-Fos protein.

Human c-fos cDNA was transfected into normal rat liver epithelial (REL) cells to identify cellular modifications associated with high expression of c-Fos protein. Responses to EGF and TGF beta were examined in the different cell lines, under anchorage-dependent and -independent conditions. Sensitivity to both factors was modified in transfected cells. While parental cells in monolayer did not respond to EGF, c-fos containing cells growth was stimulated by this factor. Overexpression of c-Fos protein led to an enhanced TGF beta-induced growth inhibition under anchorage dependent conditions, and TGF beta abolished spontaneous growth in soft agar of the cell lines containing c-fos oncogene. The mechanisms underlying the increased sensitivity to TGF beta in c-fos transfected cells are still to be determined.


Verapamil reverses the ultrastructural alterations in the plasma membrane induced by drug resistance.

Two P388 cell sublines with different levels of resistance to daunomycin (DNM), P388/20 and P388/100 cells (approximately 20- and 100-fold resistance, respectively), undergo a significant (approximately 2-fold) increase in the number of intramembrane particles (IMPs) present at their plasma membrane, as compared to that exhibited by the parental, drug-sensitive P388 (P388/S) cell line. Regardless of the level of resistance, incubation of drug-resistant cells with verapamil, a well known reverting agent of anthracycline resistance, restores the morphology of the plasma membrane in these cells, yielding a pattern in which the number and size distribution of IMPs at both leaflets of the bilayer, become undistinguishable from those displayed by drug-sensitive cells. Furthermore, verapamil did not affect the ultrastructural organization of the plasma membrane of drug-sensitive cells. It is possible that the alterations in the structural organization of the plasma membrane of the antineoplastic-resistant tumor cells, might represent a reliable 'marker' for early diagnosis of drug resistance.


Enhanced acetylcholine secretion in neuroblastoma x glioma hybrid NG108-15 cells transfected with rat choline acetyltransferase cDNA.

Neuroblastoma x glioma hybrid NG108-15 cells and mouse neuroblastoma N18TG-2 and N1E-115 cells were transiently transfected with the sense cDNA coding for rat choline acetyltransferase (ChAT). All transfected cell lines showed a high level of ChAT activity. ACh secretion was monitored by recording miniature end-plate potentials (MEPPs) in striated muscle cells that had been co-cultured with transfected cells. The number of muscle cells with synaptic responses and the MEPP frequency were higher in co-culture with transfected NG108-15 cells than with control or mock cells. No synaptic response was detected in muscle cells co-cultured with transfected N18TG-2 or N1E-115 cells. The results show that ACh secretion into the synaptic cleft was enhanced due to ChAT overexpression in NG108-15 hybrid cells but not in neuroblastoma cells.


The thermal stability of the tryptic fragment of bovine microsomal cytochrome b5 and a variant containing six additional residues.

Thermally induced denaturation has been measured for both oxidised and reduced forms of the tryptic fragment of bovine microsomal cytochrome b5 using spectrophotometric methods. In the oxidised state, the tryptic fragment of cytochrome b5 (Ala7-Lys90) denatures in a single cooperative transition with a midpoint temperature (Tm) of approximately 67 degrees C (pH 7.0). The reduced form of the tryptic fragment of cytochrome b5 shows a higher transition temperature of approximately 73 degrees C at pH 7.0 and this is reflected in the values of delta Hm, delta Sm and delta(delta G) of approximately 310kJ.mol-1, 900J.mol-1.K-1 and 5 kJ.mol-1. Increased thermal stability is demonstrated for a variant protein that contains the first 90 amino acid residues of cytochrome b5. These novel increases in stability are observed in both redox states and result from the presence of six additional residues at the amino-terminus. The two forms of cytochrome b5 do not differ significantly in structure with the results suggesting that the reorganisation energy (lambda) of the variant protein, as measured indirectly from redox-linked differences in conformational stability, is small. Consequently the reported subtle differences in reactivity between variants of cytochrome b5 may result from the presence of additional N-terminal residues on the surface of the protein.


Nucleolar and cytoplasmic localization of annexin V.

The subcellular localization of annexin V in cultured human umbilical vein endothelial cells, epithelial cells and fibroblasts was examined. Indirect immunofluorescence and immunoblotting studies using affinity-purified anti-annexin V antibodies revealed that annexin V is located within the cytoplasm and nucleus of these cells. Further examination and direct binding studies showed that annexin V within the nucleus is associated with the nucleolus. These findings suggest that annexin V may play a role in a nucleolar function, such as ribosome assembly and transport.


Characterization of carbohydrate chains of C1-inhibitor and of desialylated C1-inhibitor.

Carbohydrate chains of C1-inhibitor were identified with a binding assay using different lectins. Lectins from Sambucus nigra (SNA) and Maackia amurensis (MAA) that are specific for sialic acids bound to C1-inhibitor. Lectin from Datura stramonium (DSA) reacted also with the inhibitor indicating complex and hybrid sugar structures. C1-inhibitor was enzymatically desialylated and reexamined for lectin binding. SNA and MAA did not react anymore, but in addition to DSA, peanut agglutinin, which can bind to carbohydrate chains after sialic acids are removed, bound to desialylated C1-inhibitor. C1-inhibitor contains about 30 sialic acid residues per molecule. SDS-polyacrylamide gel electrophoresis showed that desialylated C1-inhibitor had a faster mobility than native C1-inhibitor. The N-terminal sequence of desialylated C1-inhibitor was the same as of native C1-inhibitor and no change in the inhibition of human plasma kallikrein was observed.


Determination of the relative configuration of 5,6,7,8-tetrahydromethanopterin by two-dimensional NMR spectroscopy.

The relative configuration of the pterin moiety of 5,6,7,8-tetrahydromethanopterin 1, a coenzyme isolated from methanogenic archaea, has been determined by two-dimensional NMR spectroscopy of N5,N10-methenyl-5,6,7,8-tetrahydromethanopterin 2 to be rel-(6R; 7S; 11R). The complete proton resonance assignment of the pterin moiety of N5,N10-methylene-5,6,7,8-tetrahydromethanopterin 3 is described including the relative stereospecific assignment of the C(14a) methylene protons.


Soluble interferon-alpha receptor molecules are present in body fluids.

Soluble forms of the interferon-alpha receptor (sIFN-alpha R) were identified in human serum and urine by Western blotting with monoclonal antibodies (MAb) directed against IFN-alpha R, and by immunoprecipitation (Iptn) of a covalently cross-linked complex of IFN-alpha R and [125I]IFN-alpha with anti IFN-alpha MAb. Elevated levels of sIFN-alpha R were found in sera of hairy cell leukemia patients. The soluble receptor from serum migrated as a 55 kDa protein in SDS-PAGE, and, as expected, the cross-linked product migrated as a 75 kDa protein. The soluble receptor from urine was found to be a protein of mol. wt. 45 kDa and its cross-linked complex migrated as a 65 kDa protein. The calculated mol. wt. of the entire extracellular domain of the IFN-alpha R prior to post-translational modifications is 47,000. Since there are 12 potential glycosylation points in this extracellular domain, its actual mol. wt. may be as high as 70,000 Da. It is therefore concluded that sIFN-alpha R molecules, corresponding to truncated forms of the extracellular domain of the cell surface IFN-alpha R, are present in human serum and in normal human urine.