Intracellular Ca2+ dynamics in response to Ca2+ influx and Ca2+ release in autonomic neurones. Spatial and temporal changes in the intracellular free Ca2+ concentration in response to Ca2+ influx at the cell membrane and to Ca2+ release from intracellular organelles were studied by recording fluorescence of Ca(2+)-sensitive probes, fura 2 or indo 1, with conventional epifluorescence or confocal laser-scanning microscopy combined with recordings of Ca(2+)-dependent membrane responses in bullfrog sympathetic ganglion cells. It was found that an increase in the intracellular Ca2+ induced by (an) action potential(s) in freshly ganglion cells bathed in Ringer's solution was solely a result of Ca2+ influx, while a rise in the intracellular Ca2+ by Ca2+ current in voltage-clamped cultured neurones was caused by not only Ca2+ influx but also Ca2+ release. This Ca2+ release was suggested to occur by a voltage-dependent (and graded) mode of activation of a Ca(2+)-induced Ca2+ release mechanism, explaining the lack of Ca2+ release by action potentials (because of their short-lasting depolarization) in freshly isolated neurons. In both cases, there was an inward spread of an increase in intracellular Ca2+. On the other hand, all or nothing activation of Ca(2+)-induced Ca2+ release occurred in the presence of caffeine, leading to the oscillation of Ca2+ in the cells. Characteristics of this mode of Ca2+ release and unique properties of drugs to block Ca2+ release were described. Finally, the physiological significance of different types of Ca2+ release was discussed.


The calyx-type synapse of the chick ciliary ganglion as a model of fast cholinergic transmission. The calyx-type synapse of the embryonic chick ciliary ganglion is reviewed as a model of transmitter release from a vertebrate presynaptic nerve terminal. This nerve terminal is extensive in area, enabling the penetration with microelectrodes and the application of patch-clamp techniques. In other respects the calyx synapse is a typical fast-transmitting cholinergic nerve terminal. This synapse has been used to obtain the first recordings of action potentials and calcium currents in a vertebrate presynaptic nerve terminal and is the only preparation in which it has proved possible to record single calcium channels directly from the transmitter release sites. The calyx remains a powerful experimental preparation for the further analysis of the mechanism and control of neurotransmitter release in fast-transmitting nerve terminals.


Adrenergic receptors (alpha 1 and alpha 2) modulate different potassium conductances in sympathetic preganglionic neurons. Intracellular recordings were made from 168 sympathetic preganglionic neurons in the slice of the second or third thoracic spinal-cord segment of the adult cat to study the actions of noradrenaline on these neurons. Noradrenaline, applied by superfusion (0.5-50 microM), produced membrane depolarization in 73 neurons and membrane hyperpolarization in 39 neurons. In 26 neurons noradrenaline produced a biphasic response (depolarization-hyperpolarization or vice versa). The depolarization was blocked by prazosin, while the hyperpolarization was blocked by yohimbine. The noradrenaline-induced depolarization was associated with an increase in neuron input resistance, while the noradrenaline-induced hyperpolarization was associated with a decrease in neuron input resistance. Both responses decreased in amplitude with membrane hyperpolarization and were nullified at around the potassium equilibrium potential EK. The null potential of both responses became more and less negative with a decrease and an increase, respectively, in the extracellular potassium concentration. When the membrane potential was made more negative than EK, the noradrenaline-induced hyperpolarization reversed to depolarization in all cases, whereas in only 4 of 12 cases did the noradrenaline-induced depolarization reverse to hyperpolarization. These data suggest that the noradrenaline-induced depolarization is a result of a decrease, while the noradrenaline-induced hyperpolarization is a result of an increase in K+ conductance. Cobalt (2 mM), low calcium--high magnesium, and intracellular EGTA markedly reduced or abolished the noradrenaline-induced depolarization but had no significant effect on the noradrenaline-induced hyperpolarization. Barium (2 mM) depressed both responses. Tetraethylammonium (10-30 mM), 4-aminopyridine (3 mM), and cesium (2 mM) had no effect on either response.(ABSTRACT TRUNCATED AT 250 WORDS)


Changes in intracellular cAMP level and activities of adenylcyclase and phosphodiesterase during meiosis of lily microsporocytes. In the yeasts, Saccharomyces cerevisiae and Schyzosaccharomyces pombe, reduction of intracellular cyclic adenosine monophosphate (cAMP) is known to trigger the sporulation processes by activating various meiosis specific genes. In order to ascertain whether a similar mechanism is operative in higher plants, we carried out preliminary studies on lily microsporocytes. Measurement of cAMP levels as well as the activities of adenyl cyclase and phosphodiesterase in somatic cells and different stages of meiosis, and arrest of its in protoplasts cultured under conditions of high cAMP provided direct evidence that similar phenomena occur in plant meiocytes as earlier documented in yeasts.


Requirement for specific protein kinase activities during the rapid redistribution of F-actin that precedes the outgrowth of neurites in PC12D cells. Rapid changes in morphology of PC12D cells, a subline of PC12 cells, in response to various agents were studied in relation to the subsequent outgrowth of neurites. A few minutes after addition of NGF or of dbcAMP, staining of F-actin with rhodamine phalloidin revealed the formation of ruffles around the periphery of cells. Simultaneous relocalization of F-actin to the area of ruffles occurred in response to NGF. A moderate relocalization of F-actin occurred in dbcAMP-treated cells. Other neurite-promoting agents on PC12D cells, such as bFGF, EGF and PMA, also caused ruffling and an identical redistribution of F-actin. The actin bundles then condensed into several dot-like aggregates that subsequently became the growth cones of neurites. When an inhibitor of protein kinase, K-252a, was added, only the NGF-induced morphological change was selectively decreased. By contrast, an inhibitor of protein kinase A, H-89, selectively blocked the dbcAMP-induced change. These are analogous to the effects of those inhibitors on the outgrowth of neurites. These observations indicate that the formation of ruffles with the redistribution of F-actin might be one of the earliest steps in the neurite outgrowth and that the morphological changes might be triggered by the activation of specific protein kinases. Neither cytochalasin B nor colchicine prevented the series of morphological changes. However, processes formed in the presence of cytochalasin B had no filopodium and protrusions formed in the presence of colchicine were shaped like large filopodia. It appears that microtubules and microfilaments may not be absolutely required for the initiation of the rapid morphological changes, but that complete neurites might be formed with contribution by microtubules and by microfilaments.


Phenotypic conversion of SV40-immortalized human diploid fibroblasts to senescing cells by introduction of an antisense gene for SV40-T antigen. Normal human lung fibroblast diploid cells, WI-38, become senescent after a definite number of divisions. VA-13 is a line of immortalized cells established by transformation of WI-38 cells by SV40 virus. To determine whether SV40 large T (SV40-T) antigen is essential for this immortalization of WI-38 cells we introduced an antisense gene for T antigen into VA-13. Two morphologically different types of antisense transformant (VA-AS5-8 and VA-AS37-8) were obtained. In both antisense transformants the expression of T antigen was reduced by more than 70% as compared to that in the parent cells. The morphology of the antisense transformants indicated a partial conversion to the senescent phenotype of WI-38. The relative number of cells in the S phase of the antisense transformants was decreased as compared to that in cultures of VA-13 and about 50% of cells were at G1/0. The doubling time of the transformants was prolonged to close to the doubling time of WI-38. The level of expression of retinoblastoma protein (pRB) complexed with SV40-T antigen of the antisense transformants was significantly decreased although the level of total pRB was much higher than that in VA-13. The pRB was present exclusively in the underphosphorylated form. Thus, the decreased level of formation of the complex between SV40-T and pRB or the underphosphorylation of pRB may explain the suppression of growth of antisense transformants. Together, these results show that an antisense gene for SV40-T antigen can efficiently block the cell proliferation and the cell immortalization of VA-13 cells.


Action of diuretics at the cellular level. Classification of diuretics is based on their site and mechanism of action in the nephron. The most frequently used substances comprise 1. the mostly proximally acting carbonic anhydrase inhibitors (CAI); 2. the loop diuretics (LD); 3. the early distally acting thiazides (TZ); and 4. the K+ sparing diuretics (KS) acting in the distal tubule. CAI such as acetazolamide inhibit the dehydration of H2CO3 at the luminal membrane, the hydration of CO2 within the proximal tubule cell and the exit of HCO-3 out of the cell. As a result of this proximal reabsorption of HCO-3 is reduced, a slight diuresis and saluresis is induced. The enhanced urinary excretion of HCO-3 will cause a metabolic acidosis. LD inhibit the Na+2Cl-K+ carrier in the thick ascending limb of the loop of Henle (TAL). This produces a marked diuresis and saluresis which is accompanied by enhanced Ca2+, Mg2+, K+ and acid excretion. TZ inhibit the Na+Cl- cotransporter in the early distal tubule. The diuresis is less marked than that induced by LD but the renal losses of K+ are comparable. KS inhibit Na+ channels present in the luminal membrane of the cortical collecting tubule. This leads to a very limited diuresis, but a marked attentuation of renal K+ losses. All diuretics act by inhibiting the admission of Na+ (LD, TZ, KS) or HCO-3 (CAI) into the cell. Their organotropy is merely due to the fact that they are concentrated in tubule fluid by volume reabsorption and by proximal tubule secretion.


Pharmacological characterization of alpha 2-adrenoceptors in isolated jejunum of rabbits. In the isolated jejunum of rabbits, norepinephrine (NE) lowered the muscle tone in a dose-dependent manner which was potentiated by yohimbine, an antagonist of alpha 2-adrenoceptors. Guanethidine and/or bretylium, the adrenergic neuron blockers, attenuated this action of yohimbine, indicating the participation of neuronal alpha 2-adrenoceptors. In the presence of guanethidine and atropine, clonidine and guanabenz reduced the relative responses to forskolin, but they did not modify the responses to dibutyryl cAMP. The inhibition mediated by these alpha 2-adrenergic agonists was abolished by pertussis toxin, an inhibitor of Gi protein. The actions of clonidine and guanabenz were also blocked by yohimbine and/or rauwolscine. Thus, the Gi protein mediated inhibition of adenylate cyclase by postsynaptic alpha 2-adrenoceptors in muscle cells can be considered. The obtained data indicate that alpha 2-adrenoceptors are presented in adrenergic nerve terminals and smooth muscle of jejunum of rabbits to function as the feed-back autoreceptors in autonomic neurotransmission.


Characterization of 3H-serotonin (5-HT) binding and effects on the phosphoinositides (PI) turnover in cultured C6 glioma and N2 neuroblastoma cells from rodents. 3H-5-HT (serotonin) binding and its displacement by various specific subtype ligands and effects on the phosphoinositides (PI) turnover were studied in cultured C6 glioma and N2 neuroblastoma cells from rodents. Saturation analysis of 3H-5-HT binding to C6 cells revealed that its Kd and Bmax were 3.0 nM and 18.0 pmole/mg protein respectively. DOI.HCl (5-HT2 agonist) and ketanserin (5-HT2 antagonist) had the highest affinities in the drug-displacement of 3H-5-HT binding to C6 cells studied. The IC50 values for DOI-HCl and ketanserin were 7.5 x 10(-7) and 3.5 x 10(-8) M respectively. 5-HT also induced 3H-PI breakdown and generated 3H-IP. The EC50 values for 5-HT for this event were in the dose range between 0.5 to 1.5 microM, and this 5-HT-induced response could be blocked by 5-HT2 antagonist ketanserin more effectively than the 5-HT1 antagonist or 5-HT3 antagonist studied. 3H-5-HT binding to N2 cells revealed that its Kd and Bmax were 4.0 nM and 80 pmole/mg protein respectively in the saturation analysis study. The drug-displacement of this binding revealed that MDL 72222 (5-HT3 antagonist) had a higher affinity than ketanserin. The IC50 values for MDL 72222 and ketanserin were 10 nM and 10 microM respectively, when 3 nM of 3H-5-HT was used. Our results indicate that the predominant receptor subtype of 5-HT in C6 and N2 cells are 5-HT2, and 5-HT3 respectively, and that the PI turnover is linked to 5-HT2, but not 5-HT3 receptor activation.


Sodium transport in hypertension.

Sodium transport in hypertension. Although most workers in the field agree that salt causes hypertension, but there is no well-established mechanism(s) explaining how salt works. Vast literature exists on abnormal sodium (Na+) transport processes in human and experimental hypertension from various cell systems. We examined the recent developments in the investigation of three important sodium transport pathways: Na+/K+ pump, Na+/Li+ countertransport and Na+/K+ cotransport in hypertension. The activities of these transporters may affect vascular reactivity or may serve as genetic marker for essential hypertension. Selected reports on the abnormalities of these transporters were summarized, potential problems in the interpretation of these data were discussed, and the current view about the physiological or pathophysiological meaning of these studies were presented. The multiple sites of defects and sometime conflicting reports on Na+ transport in hypertension also lead to alternative hypothesis associating membrane abnormalities with hypertension. Two models, considering membrane lipid bilayers and cellular calcium handling as primary sites of defect and the related evidence, were introduced. Finally, the role of natriuretic hormone (or endogenous Na+ pump inhibitor) in influencing Na+ transport was briefly discussed.


Synthesis and antitumour activities of quinolone antineoplastic agents. DNA topoisomerases, found in all prokaryotic and eukaryotic cells, play a key role in controlling the topological state of DNA. Quinolone antibacterial agents have been shown to be inhibitors of DNA gyrase, a bacterial topoisomerase II enzyme. The eukaryotic topoisomerase II is the target of various cytotoxic agents such as adriamycin and etoposide. Recently, several quinolones having C-8 fluoro and C-8 chloro substituents have been found to have cytotoxic activities and to interact with mammalian topoisomerase II. In searching for an antitumour agent of the quinolone class, we identified several quinolones having excellent in vitro cytotoxic activity. A-74932 also possesses good activity in vivo against both systemic tumour and subcutaneously implanted murine solid tumours as well as human tumour xenografts. The chemical synthesis as well as biological properties of A-74932 are described.


In vitro and in vivo evaluation of BMY 45243, a new 5-amino-naphthyridone derivative. BMY 45243 is the first representative of 5-amino naphthyridone derivatives prepared following a new methodology developed by the authors. BMY 45243 is a highly lipophilic compound, very active in vitro and in vivo on S. aureus and was found to be as potent as ciprofloxacin on Gram-negative organisms with identical in vivo activity against Pseudomonas aeruginosa. Pharmacokinetics in mice showed better AUC and Cmax and longer t1/2 for BMY 45243 than for sparfloxacin and ciprofloxacin.


Reorganisation of the microtubular cytoskeleton by embryonic microtubule-associated protein 2 (MAP2c). Microtubule-associated protein 2c (MAP2c) is one of a set of embryonic MAP forms that are expressed during neuronal differentiation in the developing nervous system. We have investigated its mode of action by expressing recombinant protein in non-neuronal cell lines using cell cDNA transfection techniques. At every level of expression, all the MAP2c was bound to cellular microtubules. At low MAP2c levels, the microtubules retained their normal arrangement, radiating from the centrosomal microtubule-organising centre (MTOC) but at higher levels an increasing proportion of microtubules occurred independently of the MTOC. In most cells, radially oriented microtubules still attached to the MTOC co-existed with detached microtubules, suggesting that the primary effect of MAP2 is to increase the probability that tubulin polymerisation will occur independently of the MTOC. The MTOC-independent microtubules formed bundles whose distribution depended on their length in relation to the diameter of the transfected cell. Short bundles were attached to the cell cortex at one end and followed a straight course through the cytoplasm, whereas longer bundles followed a curved path around the periphery of the cell. By comparing these patterns to those produced by two chemical agents that stabilise microtubules, taxol and dimethyl sulphoxide, we conclude that effects of MAP2c arise from two sources. It stabilises microtubules without providing assembly initiation sites and as a result produces relatively few, long microtubule bundles. These bend only when they encounter the restraining influence of the cortical cytoskeleton of the cell, indicating that MAP2c also imparts stiffness to them. By conferring these properties of stability and stiffness to neuronal microtubules MAP2c contributes to supporting the structure of developing neurites.


Drosophila homologs of two mammalian intracellular Ca(2+)-release channels: identification and expression patterns of the inositol 1,4,5-triphosphate and the ryanodine receptor genes. We have identified and cloned portions of two Drosophila genes homologous to two classes of mammalian intracellular Ca(2+)-release channels, the ryanodine receptor and the inositol 1,4,5-triphosphate (IP3) receptor. The Drosophila ryanodine receptor gene (dry) encodes an approx. 15 kb mRNA. It is expressed in the mesoderm of early stage-9 embryos and subsequently in somatic muscles and their precursor cells. In adults, dry mRNA was detected in tubular muscles and at a lower level in neuronal tissues. Embryonic expression of the Drosophila IP3 receptor gene (dip) appears more dynamic and is associated with developing anterior sense organs. In adults, dip expression occurs in several tissues, and relatively high levels of dip mRNA in adult antennae suggest a role for this gene product during olfactory transduction.


Induction of a RAR beta 2-lacZ transgene by retinoic acid reflects the neuromeric organization of the central nervous system. The hormone retinoic acid (RA) has been implicated in the organization of the anteroposterior (AP) body axis. In this paper, we describe the effects of RA on the activity of the RA-inducible retinoic acid receptor-beta 2 (RAR beta 2) promoter. When transgenic embryos carrying a RAR beta 2-lacZ reporter gene were exposed to a single dose of RA between gestational days 8.5 to 10.5, lacZ expression was induced in the anterior central nervous system (CNS). Strikingly, the transgene was expressed in a segmented pattern reminiscent of that of Drosophila 'pair-rule' genes. RA treatment of midgastrulation embryos at day 7.5 disturbed the segmentation and produced severe craniofacial defects. We discuss the possibility that the entire anterior CNS is segmented and that this segmentation is reflected by the RAR beta 2-lacZ induction pattern.


Chromosomal gene transfer elements of the Bacteroides group. Many human colonic Bacteroides strains carry large ( > 60 kbp) chromosomal elements that can transfer themselves from the chromosome of the donor to the chromosome of the recipient. Most of these elements carry a tetracycline resistance gene (tetQ) and many also carry an erythromycin resistance gene (ermF), but at least one cryptic member of the family has been identified. Molecular analysis of excision and integration events has shown that the self-transmissible Bacteroides elements are not transposons but may represent a new class of integrating elements. The Bacteroides elements are most similar to the streptococcal conjugative transposons, such as Tn916. The Bacteroides Tcr/TcrEmr elements can mobilize DNA that is not contained within the elements themselves. They not only mobilize co-resident plasmids but also cause the excision, circularization and mobilization of discrete unlinked 10-11 kbp segments of chromosomal DNA. Self-transfer and other activities of the Tcr/TcrEmr elements are regulated by tetracycline. Thus, tetracycline not only selects for acquisition of an element but also stimulates element transfer in the first place.


Effect of beta-lactamase inhibitors on beta-lactamases from anaerobic bacteria. Three beta-lactamase inhibitors in clinical use--clavulanic acid, sulbactam and tazobactam--were investigated for their activity on beta-lactamases from Bacteroides uniformis, Clostridium butyricum and Fusobacterium nucleatum. Purification of the beta-lactamases was carried out by anion-exchange chromatography, gel filtration and FPLC. The inactivation of beta-lactamase activity was determined spectrophotometrically with nitrocefin as substrate. Various concentrations of the inhibitors were preincubated at 30 degrees C together with the enzyme for different periods of time before determination of the beta-lactamase activity. The three beta-lactamases tested were more susceptible to tazobactam than to clavulanic acid and sulbactam. Clavulanic acid and sulbactam reduced the enzyme activity of the Bacteroides uniformis beta-lactamase more effectively than the Clostridium butyricum and Fusobacterium nucleatum beta-lactamases.


A retrospective view of obesity.

A retrospective view of obesity. To gain a perspective on 'obesity in the nineties' requires an understanding of where the field came from during the 1980s. Three main themes seem to characterize much of the research during the 1980s. First there was completion of development of multi-disciplinary treatment programmes utilizing the best behavioural modification, nutrition education, exercise and on occasion pharmacological adjuncts. Most characteristic of all was the widespread use of very low calorie diets which first appeared at the beginning of the decade and peaked at its end. The second major development of the 1980s was the recognition of the importance of fat distribution in addition to total fat as a risk factor for the consequences of obesity. Finally, the 1980s saw the development of serotonin agents and the initial studies with beta adrenergic agonists for the treatment of obesity based on the concepts of nutrient partitioning and reduced sympathetic activity. As we enter the 1990s, three additional themes appear to be in the ascendancy. The first will be the emphasis on molecular mechanisms as an explanation for the genetic diversity of obesity, including fat distribution. The second will be the focus on strategies for prevention of obesity. The third will be the development of newer and more innovative techniques for treating that obesity which we cannot prevent.


Strategies to minimize weight gain after smoking cessation: psychological and pharmacological intervention with specific reference to dexfenfluramine. Cigarette smoking suppresses body weight, discouraging many smokers from trying to quit. Behavioural therapies have so far proved unsuccessful in preventing post-cessation weight gain, and have in fact tended to undercut abstinence from smoking. The mental demands of implementing behavioural weight management strategies may compete with the concentration needed to maintain abstinence from smoking. Consequently, a pharmacological approach offers potential treatment advantages by minimizing the effort needed to achieve weight control. Of the agents found effective in minimizing weight gain, serotoninergic drugs, particularly dexfenfluramine, show special promise because they prevent an increase in caloric intake but do not decrease energy intake below pre-cessation levels.


Body weight evolution during dexfenfluramine treatment after initial weight control. While very low calorie diets (VLCD) can lead to rapid weight loss, even in patients who have often failed with conventional diets, maintaining this loss remains a clinical challenge. Since dietary re-education or more formal behaviour modification techniques are not easily applicable to large numbers of patients and are not always successful, drugs to maintain and improve upon initial VLCD success would be of clinical benefit. Pharmacological treatment of obesity has evolved in recent years with the development and licensing of potent serotonin agonists, such as dexfenfluramine (D-F). VLCD followed by D-F has been shown to be effective. A double-blind trial randomized 45 patients who had successfully completed eight weeks' VLCD treatment, to either placebo or D-F 15 mg b.d. for 26 weeks. Patients continued on a diet giving 60-75% of daily energy needs. Patients treated with D-F had lost 14.9 +/- 0.9 kg on the Cambridge diet and a further 5.8 +/- 1.8 kg during the drug treatment. In contrast, patients who received placebo regained 2.9 +/- 1.3 kg of the 13.5 +/- 1.0 kg they had lost during VLCD. The total weight loss after 34 weeks in total was thus 21.3 +/- 2.6 versus 11.3 +/- 1.9 kg. Patients were offered the option of continuing on D-F, or switching from placebo, in an open continuation of the trial. Ten patients, regaining weight on placebo, stabilized their weight without further regain over the next 24 weeks when switched to D-F.(ABSTRACT TRUNCATED AT 250 WORDS)


The metabolic signal of hunger and satiety, and its pharmacological manipulation. Hunger is elicited by the depletion of available macronutrients to the cells. The question is how the depletion is sensed and transduced into a biologically meaningful signal of hunger. Accumulating data show that the signal comes not from depletion of carbohydrates alone or from one of the other major macronutrients. Instead, the signal is generated by the overall cellular power production that induces hunger when it decreases and satiety when it increases. As for satiation, i.e. the state that causes the termination of a meal before nutrients cross the intestinal barrier, it is induced by early metabolic events elicited reflexly from orogastric afferences and tuning the preprandial metabolism towards the level of the postprandial metabolism. Neuronal responses from the medial hypothalamus suggest that they may integrate information on the degree of utilization of glucose and of lipids. Some of the pharmacological agents that reduce feeding seem to operate by increasing the metabolic rate in a way similar to that induced by a meal. These pharmacological agents are active because they mobilize endogenous metabolites, such as lipids, so inducing a sort of 'autocanibalic' meal.


Progress report on the anorectic effects of dexfenfluramine, fluoxetine and sertraline. This progress report on the anorectic effect of serotoninergic indirect antagonists compares the action of D-fenfluramine, fluoxetine and sertraline and their N-dealkylated metabolites. Brain levels of drugs and their metabolites were measured after equi-active anorectic doses. Fluoxetine and sertraline inhibit 5-HT uptake in vitro with a potency which is at least one order of magnitude higher than for D-fenfluramine while all three drugs release 5-HT from synaptosomes and the active concentrations are closer to the brain concentrations reached after anorectic doses. However, a number of differences have been observed between D-fenfluramine and fluoxetine regarding the mechanisms of 5-HT release. Furthermore fluoxetine affected storage of 5-HT in vesicles much more than D-fenfluramine did. The anorectic effect induced by fluoxetine was not antagonized by antiserotoninergic drugs. No evidence of an involvement of CCK in the anorectic effect of D-fenfluramine was found when food intake was determined in rats previously submitted to food deprivation.


Dexfenfluramine and appetite in humans.

Dexfenfluramine and appetite in humans. Dexfenfluramine has been demonstrated to produce decreases in daily energy intake varying between 13 and 25% depending on the time for which the drug has been administered. This reduction in energy intake is achieved by a decrease in the size of meals (11-40% depending on the dose) and by a decrease in the frequency of snacks consumed between meals. These objective adjustments are accompanied by a decrease in the level of perceived hunger, particularly apparent in the postprandial period. Taken together these changes can be interpreted as an action of dexfenfluramine to intensify the satiating power of food. This effect is consistent with the proposed role of serotonin systems in the mediation of satiety. The effect of dexfenfluramine on eating parameters leads to an overall modulation of the pattern of eating and the profile of motivation to eat. The reduction in energy intake achieved by this modulation is consistent with the weight losses recorded following several months of treatment. This restraint over the expression of appetite exerted by dexfenfluramine appears to be present even after 12 months of continuous treatment. These demonstrated actions should help overweight people to achieve a better management of their appetite in order to assist weight loss and to prevent weight regain.


Effect of dexfenfluramine on energy expenditure in man. A short review has been made of the experimental studies performed in man, in which the effect of dexfenfluramine (D-F) on resting energy expenditure has been explored. It appears that the extent to which D-F possesses thermogenic properties (in addition to its anorectic effect) still remains controversial. Some investigators found either no significant increase in energy expenditure in response to the drug or a moderate effect in post-absorptive and/or postprandial state. It may be reasonable to assume that the supplementary weight loss observed with D-F as compared to a placebo can be primarily attributed to its anorectic effect rather than to its putative thermogenic effect.


Thermogenesis, brown adipose tissue and dexfenfluramine in animal studies. D-Fenfluramine is a potent stimulator of thermogenesis in the rat, an action which may contribute to its effects on body weight. The actions of D-fenfluramine appear to control release of endogenous 5-HT which subsequently induces release of eicosanoids and corticotrophin-releasing factor. Peripheral heat production results mainly from sympathetic activation of thermogenesis.


Neuroendocrine regulation and obesity.

Neuroendocrine regulation and obesity. The combined syndrome of android (upper body) obesity, diabetes, hyperlipidaemia and hypertension is discussed in terms of a deranged endocrine regulation of metabolism. The syndrome is characterized by insulin insensitivity and an increased control of metabolism by cortisol. The antagonism between the two hormones appears to be partly responsible for the hyperglycaemia, hypertriglyceridaemia and hypercholesterolaemia. The synergism between insulin and cortisol in stimulating energy deposition, associated with a decreased effect of corticotropin-releasing factor in stimulating energy expenditure, is likely to contribute to the development of obesity. The efficacy of D-fenfluramine in treating the obese-diabetic-hyperlipidaemic-hypertensive syndrome probably depends on its actions on the serotoninergic system in the hypothalamus which both decreases food consumption and tends to normalize hormonal balance through the hypothalamic-pituitary-adrenal axis.


Treatment of obesity.

Treatment of obesity. Current treatment of obesity seems to be focused mainly on the success of losing body weight, which can be achieved, in order of increasingly drastic manoeuvres: by simple nutritional advice; professional follow-up of a negative energy balance; drugs with effects on appetite regulation, energy absorption or expenditure; total seclusion with control of every administered calorie; surgical intervention; or even jaw-wiring. The only treatment of this sort that has been convincingly shown to have long-lasting effects is surgical intervention in the gastrointestinal tract, but this can only be accepted for use in severe cases. Thus, the problem of treatment of moderate obesity is to find an effective therapeutic modality which iss efficient in maintaining a reduced weight. Obesity treatment also seems to have focused too much on the mass of excess body fat, which is not necessarily an indicator of the medical hazards of the condition. It is important to realize that the risk factor clusters following obesity are often efficiently treated by successful reduction of the obese condition. Instead of specific treatment of each of these complications by, for example, multi-pharmacological therapy, a sufficiently efficient obesity treatment would be a preferable substitute. This goal may, if necessary, be achieved by treatment with a single drug with a useful therapeutic profile, including efficiency in the long-term to prevent relapse. Chronic treatment might then be considered acceptable in the same way as chronic pharmacological treatment of hypertension and hyperlipidemia, for example. No drug has as yet proven to have these characteristics.(ABSTRACT TRUNCATED AT 250 WORDS)


Purification and characterization of a possible protooncogene fyn product, p59fyn, from a rat brain particulate fraction. Four tyrosine-protein kinases that reacted with antibodies specific to p62c-yes, p60c-src, p60c-src+, and p59fyn, respectively, were solubilized from a rat brain particulate fraction and separated by casein-Toyopearl column chromatography. Possible p59fyn, with a pI of 6.5, was purified 490-fold as a single 59-kDa protein band on SDS-PAGE. The purified enzyme contained almost no phosphotyrosine residues but was autophosphorylated with Mg2+. ATP exclusively at tyrosine residues, with a concomitant increase in the kinase activity toward tyrosine-glutamate (1:4) copolymers. The rate of the copolymer phosphorylation was proportional to the square of the enzyme concentration, suggesting activation through intermolecular catalysis. In the presence of Mn2+, however, the reaction showed a first-order dependence on the enzyme concentration.


Purification and characterization of a 7Fe ferredoxin from a thermophilic hydrogen-oxidizing bacterium, Bacillus schlegelii. A ferredoxin (Fd) was purified from a thermophilic hydrogen-oxidizing bacterium, Bacillus schlegelii. This ferredoxin was a monomer with apparent molecular weight of 13,000 and contained 7 mol Fe/mol ferredoxin. The oxidized ferredoxin showed the characteristic EPR spectrum for [3Fe-4S]1+ (1.2 spin/mol Fd). This signal disappeared upon reduction with dithionite and new signals due to [3Fe-4S]0 and [4Fe-4S]1+ (0.7 spin/mol Fd) appeared. The quantitation of EPR signals and the iron content reveal that B. schlegelii ferredoxin contains one [3Fe-4S]1+/0 and one [4Fe-4S]2+/1+ cluster. The ferredoxin has the characteristic distribution of cysteines (-Cys8-X7-Cys16-X3-Cys20-Pro-) for 7Fe ferredoxins in the N-terminus.


Thiobacillus ferrooxidans cytochrome c oxidase: purification, and molecular and enzymatic features. Cytochrome c oxidase from Thiobacillus ferrooxidans was purified to homogeneity and some of its properties were studied. The oxidase was solubilized with n-octyl-beta-D-thioglucoside (OTG) under acidic conditions (pH 4.0) and purified by one step of ion-exchange chromatography with a CM-Toyopearl column. The absorption spectrum of the oxidase showed peaks at 420 and 595 nm in the oxidized form and at 440 and 595 nm in the reduced form. Its CO compound showed a novel absorption spectrum; a double-peaked gamma band appeared at 429 and 438 nm. The oxidase seemed to have CuA-like copper atom from its ESR and near-infrared spectra. The oxidase molecule consisted of three polypeptides with molecular weights of 53,000, 22,000, and 17,000, respectively, as estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The molecular weight of the enzyme in a solution containing detergents was estimated to be 169,000 on the basis of the results obtained by gel filtration, while the molecular weight per heme alpha was estimated to be 83,700. The copper content of the oxidase was 1.01 g atom per mol of heme alpha. Therefore, the cytochrome seemed to contain one molecule of heme alpha and one atom of copper in the minimal structural unit consisting of one molecule each of the three subunits, and to occur as a dimer of the unit in the solution. The oxidase oxidized ferrocytochrome c-552 of the bacterium, and the optimal pH of the reaction was 3.5.(ABSTRACT TRUNCATED AT 250 WORDS)


Reactive oxygen species and human spermatozoa: analysis of the cellular mechanisms involved in luminol- and lucigenin-dependent chemiluminescence. We have shown that human spermatozoa generate and release reactive oxygen species that can be detected by chemiluminescence techniques. Analysis of the cellular mechanisms responsible for this activity suggests that the probe, luminol, undergoes an intracellular dioxygenation reaction mediated by hydrogen peroxide and a sperm peroxidase located within the acrosome. Support for this model included the following observations: (1) the luminol-dependent signal could be suppressed with peroxidase inhibitors, phenylhydrazine and sodium azide; (2) this suppression could be reversed by the addition of an azide-insensitive peroxidase, horse radish peroxidase (HRP); (3) inhibition of intracellular superoxide dismutase (SOD) with potassium cyanide (KCN) suppressed the luminol signal; (4) peroxidase activity could be detected in purified populations of human spermatozoa with 3,3',5,5' tetramethylbenzidine (TMB); (5) this peroxidase was active at the pH prevailing within the acrosomal vesicle; and (6) peroxidase activity and luminol-dependent chemiluminescence were minimal in spermatozoa exhibiting a congenital absence of acrosomes. Human spermatozoa could also generate lucigenin-dependent chemiluminescent signals that could neither be suppressed with peroxidase inhibitors nor enhanced by the addition of peroxidase. However, these signals could be enhanced by suppression of intracellular SOD with KCN or inhibited by exogenous SOD, suggesting that lucigenin was responding to superoxide anion released into the extracellular space. The ability of chemiluminescent techniques to detect and discriminate the production of superoxide and hydrogen peroxide by spermatozoa should facilitate the further analysis of reactive oxygen species as mediators of normal and abnormal human sperm function.


Effect of camptothecin on mitogenic stimulation of human lymphocytes: involvement of DNA topoisomerase I in cell transition from G0 to G1 phase of the cell cycle and in DNA replication. The possible involvement of DNA topoisomerase I in cell transition from G0 to G1 and in progression through the cell cycle was studied by estimating the ability of human peripheral blood lymphocytes to undergo mitogenic stimulation in the presence of the topoisomerase I inhibitor camptothecin (CAM). Exposure of quiescent G0 lymphocytes to up to 3 microM CAM for 24 h had no significant effect on their ability to subsequently undergo mitogenic stimulation in the presence of phytohemagglutinin (PHA); higher doses of CAM, although not immediately cytotoxic, impaired the mitogenic response. Stimulation of lymphocytes with PHA in the presence of < or = 1.5 microM CAM resulted in unperturbed transition of these cells from G0 to G1 characterized as an increase in cellular rRNA content, appearance of interleukin-2 receptor, and, after removal of CAM, response to interleukin-2 by entering S phase of the cell cycle. However, lymphocytes were prevented from entering S phase in the presence of CAM at a concentration of > or = 30 nM, and their rate of progression through S was minimal even at CAM concentration as low as 3 nM. When cycling lymphocytes (48 h after stimulation by PHA) were treated with CAM, the cell progression through S and G2 was also very sensitive to the inhibitor: the cells were "frozen" in S and G2 at > or = 6 nM CAM. These cells died within 24 h; their selective loss from the cultures (with only G0/G1 cells remaining) coincided with the appearance of cells with fractional DNA content, typical of apoptotic cells. Human lymphocytic leukemic MOLT-4 cells were arrested in S and G2 at > or = 7.5 nM CAM. Thus, progressions through S and G2 of both normal and leukemic lymphocytes were perturbed at approximately two orders of magnitude lower CAM concentration than the G0 to G1 transition. These data suggest that DNA replication and chromosomal events during G2 are more sensitive to inhibition of DNA topoisomerase I, compared with the early events of lymphocyte stimulation, which involve activation and transcription of numerous genes associated with the G0 to G1 transition. The antitumor properties of CAM may be related to its high cytostatic/cytotoxic activity toward cycling cells and relative resistance of cells in G0 or undergoing transition from G0 to G1.


Elevated cAMP is required for stimulation of eicosanoid synthesis by interleukin 1 and bradykinin in BALB/c 3T3 fibroblasts. In Swiss 3T3 murine fibroblasts, interleukin 1 (IL-1) and bradykinin stimulate prostaglandin E2 (PGE2) synthesis. However, in the present study, we found that neither agonist stimulated PGE2 synthesis in BALB/c 3T3 murine fibroblasts, this in spite of expression of similar numbers of receptors for each agonist compared to Swiss 3T3 cells. When BALB/c 3T3 cells were preincubated with cAMP analogs, both IL-1 and bradykinin stimulated PGE2 synthesis to levels similar to those observed in Swiss 3T3 cells. Similarly, when the cells were preincubated with forskolin, which activates the catalytic subunit of adenylate cyclase directly, or NECA, which stimulates cellular cAMP accumulation by activating adenosine receptors, IL-1 and bradykinin stimulated PGE2 synthesis. Rp-cAMPS, an inhibitor of cAMP-dependent protein kinase, blocked the ability of cAMP or NECA to render cells responsive to IL-1 and bradykinin. In basal BALB/c 3T3 cells, bradykinin and IL-1 stimulated arachidonate release in the absence of cAMP, but little conversion of released arachidonate to PGE2 occurred. cAMP, forskolin, and NECA all increased cyclooxygenase activity in the cells. SV-T2 is a clonal line originating from BALB/c 3T3 transformed with SV-40. In these cells, IL-1 and bradykinin stimulated PGE2 synthesis despite basal intracellular cAMP concentrations similar to BALB/c, and cAMP only modestly potentiated the response. In summary, cyclooxygenase expression appears to be regulated by cAMP in BALB/c 3T3 cells, and SV-40 transformation results in increased cyclooxygenase expression, apparently independent of cAMP.


Adenosine receptors, cyclic AMP accumulation, and DNA-synthesis in aortic smooth muscle cell cultures of adult and neonatal rats. The effects of two adenosine analogs on cyclic AMP (cAMP) accumulation and DNA synthesis were studied in cultured smooth muscle cells (SMCs) isolated from adult and neonatal rat arteries. N-ethylcarboxamido adenosine (NECA) dose-dependently increased intracellular cAMP levels and appeared to be more potent in adult than in neonatal SMCs. R-phenylisopropyl adenosine (R-PIA), in nanomolar concentrations, counteracted the increase in cAMP evoked by 10 microM forskolin in adult but not in neonatal SMCs, indicating that the enhanced "A2" response seen in adult SMCs was not due to a lack of "A1" receptors in these cultures. Binding experiments performed using the adenosine antagonist XAC did not reveal any differences in the number or affinity of the adenosine receptors between neonatal and adult SMCs. This indicates effects presumably on the G-protein level. A high capacity to spontaneously synthesize DNA and a weak response to platelet-derived growth factor (PDGF) were seen in the neonatal SMCs. Furthermore, NECA had no effect on PDGF-induced DNA synthesis in these cells. In contrast, adult SMCs presented a low rate of spontaneous DNA synthesis and a marked proliferative response to PDGF, which was inhibited by NECA. This inhibition paralleled the increase in cAMP elicited by NECA. Our findings suggest that neonatal and adult SMCs differ both in their response to growth factors and growth inhibitors.


An ultrastructural study of thrombomodulin endocytosis: internalization occurs via clathrin-coated and non-coated pits. The regulation of thrombomodulin (TM) expression has been reported to occur by several mechanisms. We have examined constitutive internalization of TM using immunofluorescent and electron microscopic (EM) methods. A cell model was developed to study this process by introducing TM DNA into COS-7 cells for expression. The recombinant TM was determined to behave similarly to native TM from human umbilical vein endothelial cells (HUVEC) with respect to M(r) and cell surface functional activity. The transfected cells expressed 8-100-fold more functional TM per cell than HUVEC. Immunofluorescent studies on these cells indicated that anti-TM antibody-TM complex was internalized in a time- and temperature-dependent manner, with internalization detectable within 10 minutes. When the cells were incubated at 4 degrees C with gold-labelled anti-TM antibody, most of the gold particles were surface bound and detected by EM as individual particles or clusters of 2 or 3 particles. Initiation of endocytosis for 10 to 60 minutes resulted in a redistribution of gold particles into small clusters predominantly in non-coated pits and rarely in clathrin-coated pits, subsequently in early endosomes, multivesicular bodies, and lysosomes. Similar studies were performed with gold-conjugated thrombin, demonstrating a similar route of intracellular processing. These studies provide ultrastructural evidence that the process of endocytosis of TM involves the participation of both clathrin-coated and non-coated pits and vesicles, but that the latter process predominates. Further structure/function studies are indicated using our cell model, since defects in the endocytic pathway of this important anticoagulant receptor may contribute to the development of thromboembolic disease.


A high level of cell surface phosphatidylinositol-specific phospholipase C activity is characteristic of growth-arrested 3T3 fibroblasts but not of transformed variants. Confluent monolayers of four contact-inhibited mouse fibroblast lines (Swiss 3T3, Balb/c 3T3, NIH 3T3, and C3H10T1/2) were found to have substantial levels of a cell surface phosphatidylinositol-specific phospholipase C (ecto-PLC). In contrast, confluent cultures of virally, chemically, or spontaneously transformed variants derived from these cell lines expressed undetectable or negligible levels of this enzyme activity. A simple and rapid assay, using lysophosphatidylinositol radio-labeled in the inositol group ([3H]-lysoPI) as the substrate was developed to provide a quantitative measure of the phospholipase C activity present at the external cell surface. For cells testing positive for ecto-PLC activity, rapid uptake of [3H]-lysoPI is accompanied by the simultaneous appearance of [3H]-inositol phosphate in the external medium. Confluent monolayers of the four mouse fibroblast lines exhibiting density-dependent growth inhibition had levels of ecto-PLC activity in the range of 50-800 pmol/min/10(6) cells (i.e., about 20-50 times greater than the activity observed for the transformed variants). The expression of ecto-PLC activity at the cell surface of the Swiss or Balb/c cells was dependent on the state of cell proliferation. Cultures which had become quiescent through attainment of confluence displayed a tenfold increased activity over that of subconfluent, growing cultures of these cells. Similarly, subconfluent Swiss 3T3 cells which had become quiescent following exposure to low serum conditions also showed increased activity. These results indicate that there may exist a correlation between the control of cell proliferation in contact-inhibited mouse fibroblasts and the expression of inositol phospholipid-specific phospholipase C activity at the external cell surface.


Biochemical events accompanying macrophage activation and the inhibition of colony-stimulating factor-1-induced macrophage proliferation by tumor necrosis factor-alpha, interferon-gamma, and lipopolysaccharide. Agents that can arrest cellular proliferation are now providing insights into mechanisms of growth factor action and how this action may be controlled. It is shown here that the macrophage activating agents tumor necrosis factor-alpha (TNF alpha), interferon-gamma (IFN gamma), and lipopolysaccharide (LPS) can maximally inhibit colony stimulating factor-1 (CSF-1)-induced, murine bone marrow-derived macrophage (BMM) DNA synthesis even when added 8-12 h after the growth factor, a period coinciding with the G1/S-phase border of the BMM cell cycle. This inhibition was independent of autocrine PGE2 production or increased cAMP levels. In order to compare the mode of action of these agents, their effects on a number of other BMM responses in the absence or presence of CSF-1 were examined. All three agents stimulated BMM protein synthesis; TNF alpha and LPS, but not IFN gamma, stimulated BMM Na+/H+ exchange and Na+,K(+)-ATPase activities, as well as c-fos mRNA levels. IFN gamma did not inhibit the CSF-1-induced Na+,K(+)-ATPase activity. TNF alpha and LPS inhibited both CSF-1-stimulated urokinase-type plasminogen activator (u-PA) mRNA levels and u-PA activity in BMM, whereas IFN gamma lowered only the u-PA activity. In contrast, LPS and IFN gamma, but not TNF alpha, inhibited CSF-1-induced BMM c-myc mRNA levels, the lack of effect of TNF alpha dissociating the inhibition of DNA synthesis and decreased c-myc mRNA expression for this cytokine. These results indicate that certain biochemical responses are common to both growth factors and inhibitors of BMM DNA synthesis and that TNF alpha, IFN gamma, and LPS, even though they all have a common action in suppressing DNA synthesis, activate multiple signaling pathways in BMM, only some of which overlap or converge.


Estimation of the infectivity of herpes simplex virus type I passage III on two different tissue culture. This technique has been developed for the estimation of the infectivity of herpes simplex virus type I passage III on a tissue culture of Vero and HBK21. The plaque assay method was performed and the number of plaques on the two culture media was counted per each dilution from (10(-5) to 10(-8)). It was found that the number and the size of plaques in 10(-5) dilution are the best in the two types of tissue cultures. The number indicates the number of virus particle (per each dilution point), which can be used for infection of experimental animals to demonstrate its infectivity character.


Fiber intake of older adults: relationship to mineral intakes. This study evaluated reported dietary fiber and mineral intakes of 45 healthy adults, 60 years and over, using two sets of three-day dietary food records. The average age of the participants was 73 years, with an age range of 60 to 87 years. Mean reported dietary fiber intake was 18.3 g per day. Percentage of dietary fiber from different food sources was; breads and cereals 33%, vegetables 22%, fruits 21%, beans and legumes 7%, nuts and seeds 3%, combination foods 7% and others 7%. Significant positive correlations were observed between dietary fiber intake and copper, magnesium, selenium, potassium, iron and zinc intakes. Those participants with dietary fiber intakes of 20 g/day or more had significantly higher intakes of copper, magnesium, selenium, potassium and iron compared to those who had dietary fiber intakes less than 20 g/day, although mean kilocalorie intakes were not significantly different. Thus, increasing grain products, vegetables and fruits to increase dietary fiber intake, also may improve the mineral intakes of older adults.


[Primary malignant fibrous histiocytoma of the lung]. On the basis of one personal case, the various clinicopathological features of primary malignant fibrous histiocytoma of the lung are summed up. This tumor, which has a nonspecific radiological appearance, most often appears as a large peripheral pulmonary opacity, for which the prognosis is guarded and the positive diagnosis based on surgical exeresis or biopsy.


[Evaluation of fibrotic effects from dusts originating from KWK "Brzeszcze" on lung tissue]. In order to determine biological aggressiveness of settled dusts and dusts collected using the gravimetric method, experimental studies were carried out, including: 1) evaluation of the physicochemical parameters (size of dust particles, content of silica, metals and other chemical compounds); 2) evaluation of the haemolytic activity; 3) experimental of evaluation fibrogenic potentials by means of: a) intraperitoneal test--to identify morphological type of reactive changes in peritoneum and; b) intratracheal test--to determine the level of hydroxyproline (collagen) in lungs and the morphological type of reactive changes. Albino rats were used for the experiment. The animals were divided into ten groups which received a single intratracheal injection of 50 mg of mining dust in 0.9% NaCl suspension. Comparative evaluation of biological aggressiveness of mining dusts was conducted basing on findings of collagen levels in lungs. After the end of the experimental period (3 and 6 months) histopathological examination of the lungs and mediastinal lymph nodes was made and the collagen levels in the pulmonary tissue (following Stegeman) were determined. As evidenced by the results of the pathomorphological examination and statistical analysis: 1) after intratracheal injection the mining dusts induced changes within the respiratory system e.g. inflammatory process and emphysema.(ABSTRACT TRUNCATED AT 250 WORDS)


[The behavior of serum angiotensin-converting enzyme in normotensive subjects, in subjects with essential hypertension and in subjects with primary hyperaldosteronism on the orthostatic test]. The results of this study demonstrate that only in healthy normotensive subjects during extended orthostatism that the renin-angiotensin-aldosterone system remains integral and is characterized by a significant increase in serum angiotensin-converting enzyme (SACE), plasma renin activity (PRA) and plasma aldosterone (PA). SACE modification do not seems to directly follow that of PRA (as shown by the absence of a direct correlation between SACE and PRA). In essential hypertension, the behavior of SACE seems to change, without demonstrating a significant increase in the mean levels of this enzyme. Finally in hypertension of known origin, such as primary hyperaldosteronism, the low levels of SACE in the recumbent position (not stimulated by orthostatism) seem to depend (by mechanism of negative biofeedback) on the increased serum levels of PA, which is moreover verified in the same group for PRA.


Heparin inhibition of human vascular smooth muscle cell hyperplasia. Vascular smooth muscle cell (VSMC) growth is responsible for intimal hyperplasia, a major cause of failure after vascular surgery and angioplasty. Heparin is the first described inhibitor of VSMC growth, but has not proved effective in the prevention of human intimal hyperplasia. Heparin is a heterogeneous substance, which may contain a mixture of components which differ in antiproliferative activity. Isolation of an active component may favourably influence its therapeutic profile. Growth of human VSMC cultured from operative specimens, assessed by cell counting and labelled thymidine incorporation, was used as a model of VSMC proliferation in intimal hyperplasia. Unfractionated (UFH) and low molecular weight (LMWH) heparins inhibit cell growth and thymidine uptake by human VSMCs in response to 15% foetal calf serum. UFHs are more active than LMWHs and this difference increases with increasing heparin dose. To confirm this effect, size-based fractions of heparin were prepared by gel permeation chromatography, and characterised by high performance liquid chromatography. High molecular weight fractions (MW > 21000) have higher activity than fractions of medium (MW 12000-21000) or low molecular weight (MW < 12000). These differences become more pronounced at higher dose, and are statistically significant at 100 micrograms/ml (Mann-Whitney, p < 0.05). The antiproliferative activity of heparin appears to be maximal in its high molecular weight component.


Neuromuscular blocking effect and placental gradient of pipecuronium bromide in elective caesarean section. We studied the suitability of pipecuronium for maintenance of neuromuscular block during 20 elective Caesarean sections under light general anaesthesia with a rapid-sequence technique facilitated by i.v. suxamethonium 0.6-0.8 mg kg-1. An initial dose of 0.035 mg kg-1 or 0.05 mg kg-1 of pipecuronium provided satisfactory relaxation of similar speed of onset (3.1 +/- 0.3 vs 3.8 +/- 0.6 min, p = 0.25); however the higher dose had a significantly longer duration of action than the lower dose (from injection to recovery to 20% of baseline 13.1 +/- 1.8 vs 39.3 +/- 6.9 min, p < 0.05). A spontaneous recovery of the T1 twitch of the train-of-four to 25% of baseline (residual block 75%) assured rapid antagonism of the residual block with either edrophonium 0.5 mg kg-1 or neostigmine 0.04 mg kg-1. A residual block greater than 80% depression of T1 markedly prolonged the time taken to achieve a satisfactory reversal. Pipecuronium had negligible cardiovascular effects and placental transfer. It had no observable effect on the newborn. In spite of some advantages, we conclude that pipecuronium is a suitable alternative during Caesarean section only when used judiciously in small doses for maintenance of neuromuscular block and recommend that the initial dose be no greater than 0.035 mg kg-1.


[Viral hepatitis sequelae].

[Viral hepatitis sequelae]. The viral hepatitis is a serious public health problem worldwide. Some problem is hepatitis B, particularly superinfection HBV-HDV and least hepatitis C (HCV), because they are transmitted via parenteral routes. About 20% of patients becomes a chronic carrier. Some chronic carriers are healthy: and they have no functional deficiencies. Others however, chronic active hepatitis develops and can lead to cirrhosis of the liver and finally to hepatocellular carcinoma, that is one of the major cancers of the world today. The immunocomplexes play a role in pathogenesis of several syndromes, such as: polyarthritis nodosa, glomerulonephritis, acrodermatitis. In the study based on questionnaires mailed 645 persons after acute viral hepatitis they were observed: cholecystitis--13.9%, stomach and/or duodenum ulcer--11.5%, and cholelithiasis--8.1%. An important results of the investigation is the conclusion that hepatitis caused distinct decrease of the health condition and change of the lifestyle. After the viral hepatitis 9% of patients shifted to a lighter job for a time, 3.8% for good and 5.6% patients after hepatitis B were receiving disability payment. In the light of the problems discussed here the vaccination would prevent not only the acute liver illness but also the sequelae of the disease.


Human placental gonadotrophin-releasing hormone (GnRH) binding sites: I. Characterization, properties and ligand specificity. Radioiodinated gonadotrophin-releasing hormone tracers were prepared from mammalian (m GnRH), salmon (s GnRH), lamprey (l GnRH) and the two forms of chicken GnRH (ch GnRH I and ch GnRH II), and also from the GnRH agonist (GnRHA) analogues, Buserelin ([D-Ser(tBu)6] 1-9 GnRH ethylamide) and Tryptorelin ([D-Trp6 GnRH] 1-9 ethylamide) and a GnRH antagonist (GnRHANT; [Ac 3,4-dehydro-Pro1, D-p-F-Phe2, D-Trp3,6] LHRH). Specific binding of hormone tracers was compared in homogenates and membrane fractions from human placenta and rat pituitary. GnRH agonist tracers bound readily to pituitary and placental binding sites. Binding of m GnRH to rat pituitary membranes was low compared to agonist binding, whereas other GnRH iso-forms were not bound. Binding of 125I-labelled m GnRH to human placental membranes was also low compared to that of Buserelin, and l GnRH and ch GnRH I tracers bound poorly. However, human placental membranes bound s GnRH and ch GnRH II to the same extent as GnRHA. Studies of the inactivation of GnRH tracers following incubation with rat pituitary and human placental membranes demonstrated that, although GnRH isoforms were degraded at different rates in these tissues, the differential ability of GnRH isoforms to bind to pituitary or placental binding sites was not related to differences in degradation of tracers, but rather to differences in ligand specificity. Specific binding of 125I-labelled GnRH agonists (GnRHA) and mammalian GnRH (m GnRH), s GnRH and ch GnRH II tracers to human placental membrane fractions increased linearly with increasing membrane protein at low concentrations. Binding was dependent on both the duration and temperature of incubation, and pH profiles of 125I-labelled GnRHA, s GnRh and ch GnRH II binding to placental membranes were similar. Once bound s GnRH formed a tighter complex with placental receptors than GnRHA, though 125I-labelled s GnRH was inactivated more rapidly than agonist tracer during incubation with placental membranes. Binding of GnRH tracers was specific for molecules with the GnRH structure. Deletions of amino acid residues at positions 1-3 and/or deamidation at Gly10 reduced binding potencies for both human placental and rat pituitary binding sites, indicating that both ends of the GnRH molecule were required for optimal binding. Modifications which conferred increased agonist activity led to markedly increased receptor binding potency in the rat pituitary, but only slightly increased potency for placental receptors. In contrast, GnRH antagonist analogues had increased potency towards pituitary receptors, but much reduced potency towards human placental binding sites.(ABSTRACT TRUNCATED AT 400 WORDS)


[Intravenous gamma globulins (i.v.IG) in the treatment and prevention of virus infections]. Active research is currently underway to determine the role of intravenous administration of IgG (i.v.Ig) in preventing and treating viral infections (CMV, HIV, RSV, EB, ECHO, adeno). These studies strongly suggest that immunoglobulin therapy may be beneficial in renal and bone marrow transplant patients to prevent or treat in CMV infections. Intravenous gamma globulins appears to be effective not only reducing the overall prevalence of coronary artery abnormalities in Kawasaki disease but, more important, in preventing the formation of giant aneurysms, the most serious form of coronary abnormality after this illness. However role of intravenous immunoglobulin in AIDS and in other viral diseases remains controversial. There are also dates that the high doses i.v.Ig in specific clinical situations may present certain adverse effects.


Human placental gonadotrophin-releasing hormone (GnRH) binding sites: II. Comparison of binding and inactivation of 125I-labelled GnRH agonist to subcellular fractions following density gradient centrifugation. Human placental homogenates and membrane fractions were centrifuged on continuous sucrose density gradients, with or without buoyant density perturbation of plasma-membranes by digitonin, and aliquots of each gradient fraction were assayed for a range of plasma-membrane and intracellular organelle markers, and for specific binding and inactivation of radiolabelled GnRH agonist (GnRHA), Buserelin ([D-Ser(tBu)6] GnRH 1-9 ethylamide). GnRH agonist (GnRHA) binding equilibrated in the same regions of control gradients as the plasma-membrane markers, EGF-receptor and alkaline phosphatase. Moreover, binding of 125I-labelled GnRHA and 125I-labelled chicken GnRH II (ch GnRH II) was enriched in the same regions of the gradients, indicating that both bound to the same membrane fractions. Digitonin pretreatment increased the buoyant density of all three placental plasma-membrane markers to a similar degree. Intracellular organelle markers (and hCG content) equilibrated in different regions of the gradient to placental surface-membranes, and were not perturbed appreciably by digitonin. In contrast, inactivation of 125I-labelled GnRHA was associated largely with the soluble (cytosol) fraction which failed to enter the gradient, and little tracer inactivation was observed in fractions enriched in GnRHA binding activity. Similar results were obtained with fractionated rat pituitary membranes. We conclude that: (a) placental GnRH binding sites do not represent binding of radiolabelled ligand to GnRH-degrading enzymes, (b) degradation of radiolabelled ligand is associated largely with placental cytosol fractions, and (c) GnRH binding activity appears to be associated largely with placental plasma-membranes.


[Should the extended evaluation of bronchial adenocarcinoma be different from that of non-small cell lung carcinoma?]. The records of 132 patients explored for initial evaluation of non-small cell lung cancer (NSCLC) were reviewed to find out whether the evaluation of extrathoracic extension could be influenced by anatomicopathological data. Brain, liver and bone metastases were found to be significantly more frequent in adenocarcinomas than in NSCLCs. This relative frequency was observed at all stages, including stages I and II as defined by computerized tomography of the chest, and in asymptomatic patients. We therefore recommend to evaluate fully the tumoral extension in patients with bronchial adenocarcinoma irrespective of its stage, and to do so even in the absence of clinical symptoms.


[Bronchopulmonary cancer and peripheral neuropathy: diagnostic difficulties. Apropos of a case]. The well-known neurotoxicity of cisplatin may be difficult to diagnose when the neuropathy it produces becomes worse during the weeks or months following the discontinuation of treatment or, exceptionally, appears at the time when cisplatin therapy is withdrawn. In addition, the drug-induced peripheral neuropathy must be distinguished from a paraneoplastic syndrome and in particular from epidural or radicular tumoral invasion. These different diagnoses are discussed in the light of the case reported here.


[Multiple trichoepithelioma, cylindroma, miliaria and carcinomatous transformation]. We report a new case of multiple trichoepitheliomas, milia and cylindromas syndrome. This new case differed from the others in that it was complicated by the occurrence of a basal cell carcinoma presenting as an ulcus rodens affecting the nasal pyramid. In such cases, the main differential diagnosis to be excluded is basal cell naevomatosis.


[Effect of the starch sources barley, corn, potatoes and their ration fractions on the nutrient digestibility and energy utilization in ruminants. 1. Comparative studies of nutrient digestibility in cattle and sheep]. Rations with energy parts of 50, 25 and 10% from barley, maize and potatoes were investigated comparatively by means of total metabolism experiments with oxen with a view to arriving at a more precise estimation equation of net energy fat for cattle. Parallel to the investigations with oxen the energy and nutrients digestibility of the rations in wethers was measured. The crude fibre content of the rations ranged from 166 to 271 g and the starch content from 69 to 330 g/kg DM. The daily starch intake of the oxen ranged from 575 to 2739 g on nutrition level (NL) 1.7 and from 365 to 1804 g on NL 1.1. The energy digestibility of the rations in oxen with energy parts of 50% barley, maize and potatoes was on average 73.5, 73.9 and 75.3%, of the rations with energy parts of 25% on average 72.2, 71.6 and 72.4% and of the rations with energy parts of 10% on average 68.8, 69.5 and 69.8%. The digestibility of energy and nutrients in cattle and sheep was in good agreement excepted crude protein and crude fat; these were digested 4-5%-units lower from cattle than from sheep. The increase of the nutrition level by one unit lowered the digestibility of rations with energy parts of 50 and 25% from concentrates in cattle about 3-6 units and of rations with energy parts of 10% from concentrates about one %-unit. Information about rumen physiological data is given comparatively between cattle and sheep.


Feeding value of dried sugar beet pulp from Egyptian production. Dried sugar beet pulp was fed to sheep in mixture with molasses (BPM), molasses plus urea (BPMU) or with hay (BPH) in comparison to whole diet of hay (H) in four metabolic trials. There were no significant differences among the four rations although BPMU reflected the highest digestibility of nutrients. The BPH had the highest feeding value (total digestible nutrients "TDN" and metabolizable energy) and caused the highest retention of nitrogen. Blood profile revealed that there were no significant effect of the rations on packed cell volume, haemoglobin, activity of glutamic pyruvic transaminase, Mg and Na. Whereas glucose, total N, Ca, P and K significantly affected. The BPMU and BPH were found to be the best mixtures of the four tested rations in respect to the digestibility, feeding value and blood picture. Thus, dried sugar beet pulp could be offered to animals in a mixture with molasses and urea or with hay according to the availability and price of the other ingredients than beet pulp, i.e. molasses and urea or hay.


Cytomegalovirus-associated neonatal hepatitis. Fifty-five patients with cytomegalovirus (CMV)-associated neonatal hepatitis (NH) were followed for 12 to 90 months. Six patients (10.9%) died from either a fulminant course or a chronic liver disease. Among the remaining 49 patients, whose liver function was completely recovered, there were eight with retardation of developmental or growth status, and two with hearing impairment. Overall, 20.4% of the survivors suffered from a long-term impact. The unfavorable outcome was related to several clinical and pathological parameters. These included persistence of clay-colored stool, presence of splenomegaly, ascites or anemia, high peak total and direct bilirubin, low nadir albumin levels, diffuse giant cell transformation and cirrhosis of the liver. The seropositivity of CMV infection did not significantly correlate with the outcome.


The effect of genetic variability (degree of homozygosity) on serum levels of the anterior pituitary hormones prolactin, corticotropin, and growth hormone in rats. Male and female wild Norway rats (Rattus norvegicus Erxleben) and males and female albino outbred rats (Ipf:RIZ) were crossbred. The resulting animals (F1 hybrids) were the control, noninbred group (0% inbred). By systematic full-sib mating, two experimental groups (50 and 91% of inbred) were produced. Half of each group (both males and females) was exposed to physical stress (3 days of starvation and 3 hr of swimming). The other half of each group was anesthetized using ether to collect blood. The anterior pituitary hormone concentrations of prolactin (PRL), corticotropin (ACTH), and growth hormone (rGH) in blood serum were determined by the radioimmunoassay method. Significant relationships between the PRL, ACTH, and rGH concentrations in blood serum and the inbreeding coefficient were observed: A significant PRL content decrease in blood serum occurred (linear function) and the rGH and ACTH content diminished significantly rapidly (quadratic function). These changes were affected by an increase in homozygosity. Stress significantly influenced PRL, ACTH, and rGH concentrations as well. The sex of rats significantly determined PRL and ACTH content only. Hormone levels were also influenced by interactions between the factors studied (inbred level, sex, stress).


Inhibitory effect of interleukin-3 on interleukin-2-induced cortisol release in the immunotherapy of cancer. The stimulatory effect of IL-2 on cortisol rise represents an undesirable biological event during IL-2 cancer immunotherapy. At present, no cytokine has been proven to be able to counteract IL-2-induced cortisol increase. This study was carried out to evaluate the influence of IL-3 on IL-2 stimulation of cortisol secretion. Five lung cancer patients were investigated after IL-2 (3 x 10(6) IU s.c.), after IL-3 (1 mcg/kg b.w. i.v.) and after IL-3 plus IL-2, by administering IL-3 two hours before IL-2 injection. IL-2 significantly stimulated cortisol secretion. IL-3 alone had no effect on cortisol levels. The pretreatment with IL-3 completely neutralizes IL-2-induced cortisol release. These preliminary results would suggest that IL-3 may be associated with IL-2 during cancer immunotherapy to modulate the effect of IL-2 on the endocrine system.


Action site of the lethal Ay gene in the mouse embryo. We investigated the lethal effect of Ay gene in embryos at the preimplantation stage in vitro. First, the development until the blastocyst stage and the division of individual cells from 8-cell stage embryos were examined. No difference in development was detected between embryos from the experimental cross (Ay/a x Ay/a) and those from the control cross (a/a x a/a). Therefore, it seems that the abnormality of the Ay/Ay embryo does not appear until blastocyst formation in vitro. We subsequently examined the hatching from zona pellucida of the blastocysts. The hatching ratio of the embryos from the experimental cross was significantly lower than that of the control crosses (Ay/a x a/a, a/a x a/a: p < 0.05). Our observation indicates that deficiency of the Ay/Ay embryo can be detected in vitro at hatching. In order to elucidate the mechanism of the gene action of the Ay, we attempted to rescue the lethal embryos from decreased hatching ratio in vitro. When dbcAMP at the concentration of 1 mM was added to the culture medium, the hatching ratio of blastocysts from the experimental cross increased until the level of those from the control crosses. Since this result indicates that the cAMP content in Ay homozygote seemed to be lower than those in a/a and Ay/a, the cAMP content in individual blastocyst was quantified. It is found that Ay homozygosity was associated with lower level of cAMP. When adenylate cyclase was activated by forskolin and cholera toxin, the hatching ratio was increased. These results seem to suggest that Ay homozygote embryos possess a defect in signal transduction system mediated by adenylate cyclase during hatching.


[Brain metastases from Wilms' tumor without systemic involvement--a case report and review]. A 3-year-old boy presented with multiple brain metastases 21 months after the resection of stage II Wilms' tumor. Metastasis to other organs was not found. He was treated by total removal of a large metastatic tumor in the left temporal lobe and post-operative radiotherapy and chemotherapy. He has been in complete remission for 20 months after surgery. Cerebral metastasis from Wilms' tumor without systemic metastases is very rare. It is speculated that brain metastases occurred in this patient because most of the anticancer agents used in the primary treatment for Wilms' tumor were not able to cross the blood brain barrier.


A new pattern of neuronal firing in the suprachiasmatic nucleus in vitro and a possible mechanism which induces rhythmicity. Electrophysiological studies of neurons in the suprachiasmatic nucleus, of rat brain slices in vitro, reveal characteristic firing patterns which have been previously classified as regular, irregular and bursting. A new kind of neuronal firing is found, by means of an interspike interval distribution analysis. Based on these results a new classification scheme is suggested. A model to explain the presence of all the experimental patterns is delivered. The model not only suffices to infer the presence of regular oscillators, but also suggests interesting consequences concerning the components of the circadian system and their statistical behavior.


Growth modulatory effects of some 6-methylenic steroids on human and hamster pancreatic adenocarcinoma cells in vitro. Similarities between pancreatic, prostate and mammary tumors in possession of steroidal receptors and enzymes led to investigation of the responsiveness of pancreatic cancer to steroids with potential for tumor inhibition. The compounds were tested in vitro against human (HPAF and PANC-1) and hamster (HP-1) pancreatic ductal tumor cell lines using a colorimetric enzyme-based assay (MTT) to assess both cytotoxic and cytostatic effects and the 3H-thymidine uptake assay for cytostatic effects. Only certain 6-methylenic steroidal 3-ketones and the anti-estrogen tamoxifen citrate exerted appreciable anti-tumor effects. Marked cytotoxic and cytostatic activity was shown by some 6-methylenic congeners of progesterone, testosterone and its acetate, and 4-androstene-3,17-dione on both human and hamster pancreatic tumor cell lines. In contrast to prostate cancer, testosterone, but not 5 alpha-dihydrotestosterone, enhanced growth of the well differentiated HPAF cell line as well as the poorly differentiated PANC-1 cell line. It is therefore surprising that the 6-methylene derivative of testosterone acetate, which is both a potent androgen and 5 alpha-reductase inhibitor, is a very active tumor inhibitor in our assay.


Amygdalar catecholaminergic input to septal nuclei, relation to clomipramine actions on lateral septal neurons in the rat. Antidepressants exert mixed actions on serotonergic and catecholaminergic systems. However, it is unknown whether a catecholaminergic blockade impinge on the actions of a tricyclic with serotonergic agonist properties (clomipramine) in limbic structures. The aim of the present study is to explore the effects of a catecholaminergic lesion in the basolateral amygdala on the firing rate of lateral septal, and hippocampal neurons in rats treated with clomipramine. An amygdaline lesion with 6-OHDA resembled the actions of clomipramine on the firing rate in lateral septal neurons, i.e. an increased rate of firing. However, the lesion blocked further effects of clomipramine on septal firing. Clomipramine decreased the firing rate in hippocampal neurons; however, neither the 6-OHDA lesion nor the added treatment with clomipramine modified the firing rate. It is concluded that an intact catecholaminergic amygdaloid input to lateral septal nuclei is necessary for clomipramine actions; however, the initial action of the tricyclic may involve a catecholaminergic blockade.


Quantitative structure-activity relationship study of some benzodiazepine-receptor ligands having inverse agonist/antagonist and agonist actions. Quantitative structure-activity relationship (QSAR) analyses of three different series of benzodiazepine ligands, pyridodiindoles, and beta-carbolines, having inverse agonist/antagonist and agonist actions have been performed with a view to understanding their likely mode of action at the benzodiazepine-receptor (BzR). From this study, the in vitro radioligand displacement activities of substituted 7,12-dihydropyrido[3,2-b: 5,4-b']diindoles (Figure 1) were found to be significantly correlated with resonance effect of R-substituents, van der Waals volumes at positions-1 and -3, hydrophobic constant at position-2 and field effect at position-4 of X-substituents. For 3-substituted beta-carbolines, a similar analysis has established that R-substituents at position-3 of beta-carbolines (Figure 2) are involved in strong hydrophobic interaction with some hydrophobic pocket on the receptor. For the analogues of 6-(benzyloxy)-4-(methoxymethyl)-beta-carbolines-3-carboxylic acid ethyl ester (Figure 3), however, the combined hydrophobicities of 6- and 4-substituents were shown to be important in determining ligand binding behaviour. The significant correlations so obtained are in agreement with the proposed models (Figures 4 and 5) of pharmacophores of inverse agonist/antagonist and of agonist sites at the BzR.


Effective visualization of suppressed thyroid tissue by means of baseline 99mTc-methoxy isobutyl isonitrile in comparison with 99mTc-pertechnetate scintigraphy after TSH stimulation. Baseline 99mTc-MIBI thyroid scintigraphy was compared with 99mTc-pertechnetate scintigraphy after TSH stimulation in seven patients with suppressed thyroid tissue due to an autonomously functioning thyroid nodule (AFTN). In all patients the suppressed thyroid tissue was visualized by means of both baseline 99mTc-MIBI and post-TSH 99mTc-pertechnetate scintigraphy, and in some cases the former technique provided better visualization. In one patient presenting a "warm" nodule T3-suppression did not affect the nodular/extranodular uptake ratio of 99mTc-MIBI, whereas the 99mTc-pertechnetate uptake ratio increased significantly. This leads us to hypothesize that the thyroid uptake of 99mTc-MIBI is not related to TSH control, but rather to other mechanisms such as the blood flow. Since exogenous TSH is no longer available, 99mTc-MIBI scintigraphy can be successfully used in the place of repeated 99mTc-pertechnetate scintigraphy after TSH stimulation in the assessment of AFTN.


A rapid PCR dependent microtitre plate screening method for DNA sequences altered by site-directed mutagenesis. A simple and rapid method for identifying DNA sequences altered by site-directed mutagenesis is described. The procedure requires that a restriction endonuclease recognition sequence is either created or removed from the target DNA sequence by the site-directed mutagenesis reaction. In the screening method presented, transformant colonies or M13 plaques are directly subjected to PCR using universal primers that flank the region containing the site-directed changes. The double stranded DNA products generated are then digested, with no purification, by the restriction enzyme whose site is modified, allowing mutant clones to be differentiated from those carrying wild type sequences. The protocol also provides for recovery of the bacterial cultures harbouring the mutated plasmid or M13 for further characterisation. All the procedures are carried out in small volumes in microtitre plates, thereby lowering costs and enabling the investigation of large numbers of clones. The technique allows a considerable amount of effort to be saved compared to conventional screening practices by circumventing time consuming DNA template preparations.


[Hepatic hematomas due to Menghini liver biopsy]. Hepatic hematomas, after liver biopsy, are collections of blood within the hepatic parenchyma and/or the hepatic capsula. The frequency of hematomas is reported to vary from 0% to 23% as a consequence of the patient selection and/or of the different diagnostic techniques (angiography, isotope techniques, ultrasound and CT scan). To study prospectively, using the ultrasound scan, the incidence and the clinical significance of hematomas. 115 liver punctures were prospectively studied; before and 24 hours after the procedure the patients were submitted to liver US scan and CBC, transaminase and bilirubin were also checked. The procedure was unsuccessful in one patient and none had more than one needle pass; five patients had two biopsies in different sessions. The 24 hour post-biopsy liver US scan did not show any hepatic hematomas. No patient had a significant drop in hemoglobin or in red blood cells. Hepatic hematomas after liver biopsy are uncommon and of little clinical significance.


Sequencing and analysis of genomic fragments from the NF1 locus. The sequence of five non-contiguous genomic fragments encompassing 14.4 kilobases from the NF1 locus have been determined by fluorescence-based automated DNA sequence analysis. These fragments included one kilobase of the NF1 coding region, which resulted in the identification of the intron/exon boundaries of five exons. Based on these sequences, five new NF1 exon-PCR assays have been developed, that could be useful for detecting new NF1 mutations. The genomic sequences were analyzed for the presence of Alu repetitive elements and their classification is described. This analysis may provide some insight into the characterization of genetic rearrangements resulting in disruption of the NF1 gene.


Nucleotide sequence of PAT, a retroid element with unusual DR organization, isolated from Panagrellus redivivus. We have isolated several copies of the transposable element PAT of Panagrellus redivivus and sequenced one full length, presumably autonomous, 5514 bp entity. The terminal sequences are found repeated inside the element, probably representing the homologous of the long terminal repeats in common retroid elements. Two major open reading frames are present with features typical of GAG and Pol. Both the structural features and open reading frame characteristics assign PAT to the retroid family of transposable elements, and more precisely to the gypsy class of retroids when putative functional domains of Pol are compared to published sequences.


A specific assay for leukotriene B4 in human whole blood. Leukotrienes (LTs) are potent mediators of inflammatory and allergic responses, and are present in biological fluids in minute amounts, that is, in the picogram range. The aim of this study was to develop and validate a method for determination of LTB4 synthesized in vitro in human whole blood. Heparinized blood was stimulated with calcium-ionophore A23187 at 37 degrees C. After 30 min cells were separated by centrifugation. LTB4 was analyzed by radioimmunoassay (RIA). When sample preparation was restricted to protein precipitation with acetone, interference was demonstrated by lack of parallelism between standard and sample dilution curves. Purification was, therefore, extended by combinations of the following steps: 1) protein precipitation, 2) lipid extractions, and 3) high-performance liquid chromatography (HPLC). One of two commercially available LTB4 standards was found to contain multiple components, several of which were immunoreactive in RIA. Even for the standard containing pure LTB4, interference was demonstrated by lack of parallelism between sample and standard dilution curves. Testing eight combinations of varying purification steps, we found that only a three-step purification procedure, including 1) solid-phase extraction, 2) protein precipitation at -20 degrees C, and 3) HPLC, was able to eliminate interference in RIA. Using this procedure, the recovery was 78%. Stimulation of whole blood from normal subjects with calcium-ionophore showed optimal LTB4 production at 10 microM ionophore, yielding 6.6 ng LTB4/mL blood.


Tissue distribution, elimination, and metabolism of [3H]leukotriene C4 by the conscious marine toad, Bufo marinus. Tissue distribution, elimination, and metabolism of 3H-labelled leukotriene (LT) C4 were studied in ureter-catheterized conscious marine toads, Bufo marinus. Six and 24 h after injection, organs containing the highest percent of injected radioactivity were small intestine, liver, and kidney. Radioactivity declined in these organs at 24 h by approximately threefold. Peak elimination time for radioactivity in the urine was between 2 and 4 h after the injection. During the 24-h collection period, 55.2 +/- 0.2% of the injected radioactivity was eliminated in the urine. Polar metabolites represented 40.3 +/- 1.1, 57.3 +/- 5.6, and 62.8 +/- 1.6% of the radioactivity at 2, 4, and 6 h, respectively. The primary urinary polar metabolite was 20-carboxy-LTE4, with 18-carboxydinor-LTE4 and 20-hydroxy-LTE4 also present. [3H]LTE4 decreased from 37.2 +/- 1.8% at 2 h to 15.8 +/- 3.3 and 15.0 +/- 2.1% of the radioactivity at 4 and 6 h, respectively. Bile radioactivity was low. N-Acetyl-LTE4 was not detected in urine or bile samples. Radioactivity in the pan water was 14.3 +/- 2.4 and 15.8 +/- 2.5% of the injected radioactivity, at 6 and 24 h, respectively, suggesting that the skin was a route for excretion of leukotrienes. The marine toad is an interesting model demonstrating both similarities and differences from mammals in distribution, elimination, and metabolism of peptide leukotrienes.


The effect of nizatidine on neuromuscular transmission. The effect of the H2-receptor antagonist, nizatidine, on neuromuscular transmission was investigated using sciatic nerve-gastrocnemius muscle preparations of rat in vivo. Nizatidine, administered by i.v. injection, potentiates the neuromuscular blockade induced by d-tubocurarine, pancuronium and the aminoglycoside antibiotic, kanamycin. Moreover, the drug alone is capable of producing a blockade on preparations stimulated at high frequency. The neuromuscular blockade induced by nizatidine is reversed by 4-aminopyridine but not by dimaprit.


Basolateral membrane lipid dynamics alter Na-K ATPase activity in rabbit small intestine. The role of basolateral membrane fluidity in regulating Na-K ATPase activity along the crypt-villus axis in rabbit distal small intestine was assessed. Basolateral membranes were prepared from isolated villus and crypt enterocytes at 24- to 28-fold enhancement. Villus basolateral membranes were significantly (p < 0.001) more fluid than crypt basolateral membranes as measured by 1,6-diphenyl-1,3,5-hexatriene. No difference was seen between the two groups as measured by either 2-(9-anthroyloxy)-stearic fatty acid or 16-(9-anthroyloxy)-palmitic acid. Fluidity alterations were accompanied by an increased phospholipid content in villus membranes, which resulted in a decreased cholesterol:phospholipid ratio and an increased lipid:protein molar ratio. Na-K ATPase activity was significantly (p < 0.01) greater in villus basolateral membranes than in crypt membranes, and demonstrated a greater sensitivity to ouabain inhibition. Ouabain inhibition curves calculated from villus data fit well (p < 0.001) with a two binding site model, with a high affinity (Ki 16 nM) and a low affinity (Ki 4.2 microM) ouabain binding site. In crypt basolateral membranes, only a low affinity site was apparent (Ki 3.0 microM). Fluidizing crypt basolateral membranes in vitro with benzyl alcohol to levels seen in villus basolateral membranes resulted in the appearance of a high affinity ouabain binding site (Ki 110 nM) and an increased sensitivity of Na-K ATPase to ouabain inhibition. The fluidization of villus basolateral membranes eliminated the binding associated with the high affinity site. Treatment with methanol, as a control, did not alter Na-K ATPase activity.(ABSTRACT TRUNCATED AT 250 WORDS)


Characterization of [3H]desmethylimipramine binding in bovine adrenal medulla: interactions with sigma- and (or) phencyclidine-receptor ligands. High-affinity binding sites (apparent KD 2.87 nM) for [3H]desmethylimipramine ([3H]DMI), have been demonstrated and characterized in membrane preparations of bovine adrenal medulla. The binding of [3H]DMI improved upon pretreatment of the membrane with KCl and was saturable, sodium dependent, and potently inhibited by nisoxetine and imipramine. [3H]DMI binding was also inhibited by various phencyclidine (PCP)- and (or) sigma-receptor ligands, with the following order of potency: haloperidol > rimcazole > (-)-butaclamol > dextromethorphan > MK-801 > (+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine ((+)-3-PPP) > PCP > N-(2-thienyl)cyclohexyl-3,4-piperidine (TCP) > (+)-SKF-10047 > (-)-SKF-10047. The inhibition produced by sigma ligands was not attributed to stimulation of either sigma 1- or sigma 2-receptors, owing to inactivity of the selective sigma-receptor ligands (+)-pentazocine and 1,3-di(2-tolyl)guanidine (DTG). The inhibition of [3H]DMI binding by sigma- and PCP-receptor ligands was not attributed to PCP1- or PCP2-receptor stimulation, owing to the decreased potency (100-fold) of these ligands in [3H]DMI assays compared with the affinity for brain PCP1 sites, and the ineffectiveness of the PCP2-ligand N-(1-(2-benzo(b)thiophenyl)cyclohexyl)piperidine (BTCP). Scatchard analysis of the inhibition by the sigma-ligands haloperidol and (+)-3-PPP, as well as the PCP1 receptor ligand MK-801, demonstrated noncompetitive interaction with the site bound by [3H]DMI. These studies indicate that bovine adrenomedullary membranes possess a specific receptor for the noradrenaline uptake inhibitor [3H]DMI, which is sensitive to allosteric modulation produced by PCP and sigma-ligands.


Biological effects of rat iso-atrial natriuretic peptide and brain natriuretic peptide are indistinguishable from each other. Rat brain natriuretic peptide (rBNP) and iso-atrial natriuretic peptide (iso-rANP) were discovered independently by two research laboratories. They are considered to be members of the B-type natriuretic peptides. Except for the Gln/Leu substitution at position 44, the amino acid sequence of iso-rANP is identical with that of the C-terminal 45 amino acids of rat pro-BNP and with the 5-kDa cardiac peptide from rat atria. To determine whether this amino acid substitution can modify the known biological effects of rBNP and iso-rANP, the present investigation examined the cardiovascular and renal responses, vasorelaxant effect, receptor binding characteristics, and cyclic GMP production by the two peptides in relation to that of rat atrial natriuretic peptide (rANP). Results indicate that rBNP and iso-rANP are indistinguishable from each other in terms of these known biological activities of atrial natriuretic peptide. We therefore conclude that rBNP and iso-rANP are identical peptides and that the amino acid substitution at position 44 represents a polymorphic form of the rat B-type natriuretic peptide.


Respiratory chain defect of myocardial mitochondria in idiopathic dilated cardiomyopathy of Doberman pinscher dogs. Idiopathic dilated cardiomyopathy (IDCM) is a primary myocardial disease of unknown cause. We tested the hypothesis that IDCM was associated with a myocardial metabolic defect by determining a comprehensive biochemical profile of metabolite concentrations and enzyme activities for the major metabolic pathways of the myocardium. We used the Doberman pinscher breed as a naturally occurring canine model of IDCM and compared its myocardial profile with that of healthy adult mongrels. Compared with controls, myocardium in IDCM had markedly reduced mitochondrial electron transport activity and myoglobin concentration, in association with acidosis and energy depletion following anoxic challenge: 60% decreased NADH dehydrogenase and 50% decreased ATP synthetase activities; 90% decreased myoglobin concentration; and 30% reduced ATP and 50% increased lactate and proton concentrations. Sarcoplasmic reticulum Ca(2+)-transport ATPase was decreased by 42%. There was a 15% compensatory increase in fatty acid oxidation and Krebs cycle activity. Other biochemical changes were mild by comparison with the mitochondrial defects. We conclude that IDCM is associated with a marked impairment of mitochondrial production of ATP, arising from decreased activity of the mitochondrial electron transport system, including myoglobin. These changes may be secondary to an underlying genetic defect or may indicate a deficiency of the mitochondrial respiratory chain that predisposes this breed to heart failure.


[Cytochrome oxidase induction following oxidative stress produced by adriamycin in the heart of rats fed olive oil]. Cytochrome oxidase is extremely susceptible to modifications that could occur at membrane level such as, for example, the deep influence that cardiolipin has on its maximal activity. Moreover, even the different ratio of fatty acid species in cardiolipin molecule could affect cytochrome oxidase properties. The influence of dietary fat and of endogenous lipid peroxidation with regard to cytochrome c oxidase activity and cytochrome a+a3 concentrations in mitochondria from rat heart has been investigated. Whilst the endogenous oxidative stress led to increase the peroxidation products, detected by means of hydroperoxides and conjugated dienes determinations, the features of the lipid environment were not affected as shown by fluorescence polarization technique. However, the oxidative stress was able to induce a significant increase of cytochrome a+a3 level even if the phenomenon did not reach the same extent as in the case of previous investigations on rat liver. Moreover, the results vary with the different fat source used and this might indicate a possible involvement of different dietary fats in peroxidation mechanisms. Furthermore the data we have obtained enable us to confirm once more that an induction of the synthesis of cytochrome a+a3 might be related to an enhanced production of peroxides at membrane level.


[Compression neuropathies of the ulnar nerve in occupational diseases of the arms]. Features of forming and course of ulnar nerve compression neuropathy in the cubital and Guyin's canals were studied in 86 subjects, suffering from occupational diseases of the arms due to vibration and functional overstrain. Compression neuropathy of the ulnar nerve aggravates the vibration disease and arm disorders caused by functional overstrain--increases frequency, intensity and duration of paroxysmal paleness of the IV-V fingers, aggravates paresthesia, hypalgesia and hypothermia of the ulnar antebrachium, markedly decrease power of the hand, which must be taken into account when solving exert problems and carrying out treatment and rehabilitation.


[The clinical aspects of vibration disease in miners of the Karaganda coal basin]. Studies of 242 patients showed that vibration disease in miners depends on age, length of service and occupation. Initial forms of disease predominate, they are characterized by monosyndromal clinical manifestations and slowly progressing course. Additional informative methods are suggested to diagnose the distal neurovascular lesions caused by vibration disease in miners. Marked clinical signs evidence the severity of changes. The therapeutic effect depends on the age of disease and clinical manifestations.