The antifertility agent, gossypol, changes several mitochondrial functions in the presence of Mg2+. 1. The effect of gossypol in the presence of K+ or Mg2+, or both, was studied on ATPase activity and respiration of rat liver mitochondria. 2. Respiration was uncoupled in the presence of gossypol, Mg2+, and K+, whereas in the presence of gossypol and Mg2+ a partial inhibition was observed. 3. Gossypol stimulated ATPase activity in the presence of K+ or Mg2+, but maximal activity was observed when both cations were in the incubation medium. 4. Stimulation of ATPase activity in the presence of Mg2+ was dose related. 5. EDTA reverted the stimulation produced by gossypol on ATPase activity. 6. Gossypol had no effect on the ATPase activity of submitochondrial particles, which suggests an indirect action of gossypol on the enzyme. 7. Mitochondrial membrane potential showed a higher collapse in the presence of gossypol and 1 mM MgCl2. 8. The observed effects of gossypol could be explained by the collapse of the mitochondrial membrane potential.


Effects of hydroquinones on intact Trypanosoma cruzi epimastigotes. 1. Hydroquinones inhibited the culture growth of Trypanosoma cruzi epimastigotes at concentrations lower than 1 mM. 2. Hydroquinones inhibited the oxygen consumption on the intact Trypanosoma cruzi cells. I50 values for hydroquinone, terbutylhydroquinone and 2,5-di-t-butylhydroquinone were 24.87 mM, 0.88 mM and 0.26 mM, respectively. t-Butylhydroquinone and 2,5-di-t-butylhydroquinone had a Michaelian type kinetic inhibition; hydroquinone also showed a Michaelian type kinetic inhibition at low concentrations but at higher concentrations it showed a positive cooperativity. 3. These hydroquinones changed the NAD(P) redox-state to a more reduced state and that of cytochrome b to a more oxidized state. The magnitude of the redox-state change was dependent of the hydrophobicity of the derivates. 4. These results suggest that the growth and oxygen uptake inhibition by the hydroquinones is due to a blockage of the mitochondrial electron transport chain before cytochrome b.


The allometric approach for interspecies scaling of pharmacokinetic parameters. A long standing problem in pharmacokinetics and toxicology is the extrapolation and correlation between results obtained in different animal species and man. Animal data may be scaled-up to predict PPs in man using the allometric approach. The allometric approach is empirical, but easy, and is based on the fact that the underlying physiological processes such as blood flow, heartbeat duration, breath duration etc. are essentially physical and related to B. This approach is generally applicable to compounds that are essentially renally excreted. For substances that are highly extracted by the liver, Cltot is a function of the LBF among various species. Based on the concept of neoteny, use of brain weight affords a more correct approach to the scaling of Cl(int) of low extraction ratio drugs. By using the invariant pharmacokinetic time, the superficial differences in concentration-time profiles due to chronological time among different species are removed. Finally, as Boxenbaum (1984) has said "parameters to be scaled, independent variables, and the mathematical relationships used in the scaling process are all at the discretion of the investigator. There are no proper or improper approaches; the only limitations are those imposed by the investigator."


Effects of copper and cadmium on growth, superoxide dismutase and catalase activities in different yeast strains. 1. Three strains of Saccharomyces cerevisiae have been adapted in vitro upon treatment with copper or cadmium. Growth rate, cellular size, metal uptake, superoxide dismutase and catalase activities were measured. 2. Growth rate and metal uptake are quite different among the yeast strains and also for copper and cadmium treatment. At the employed concentrations, only cadmium chiefly affects the cellular volume. 3. Cu, ZnSOD activity is stimulated in the presence of copper, while it is lightly inhibited in the presence of cadmium. Catalase level remains almost unchanged in the conditions tested. This lack of correlation is then discussed.


Sphingosine inhibits muscarinic cholinergic receptor binding in rat parotid acinar cells. 1. Sphingosine inhibited the binding of [3H]quinuclidinyl benzilate (QNB), a potent and specific muscarinic antagonist, in dispersed rat parotid acinar cells. 2. The inhibition of [3H]QNB binding was expressed as decrease in affinity without significant change of a number of membrane sites. 3. The effect of sphingosine on the binding was not affected by the chelation of extracellular Ca2+. 4. H-7, an inhibitor of protein kinase C, failed to decrease [3H]QNB binding. 5. Stearylamine, an analogue of sphingosine, was as effective as sphingosine in inhibiting [3H]QNB binding. 6. These results suggest that sphingosine inhibits muscarinic cholinergic receptor binding by a mechanism that is independent on extracellular Ca2+ and protein kinase C.


Equithesin: a hibernation-inducing drug? 1. Hibernating insectivore species (hedgehogs) and non-hibernating rodents (guinea pig and rat) were anaesthetized with 'equithesin' (a mixture of chloral hydrate, magnesium sulphate, and pentobarbitone sodium). 2. The physiological responses shown by the hedgehogs were similar to those observed in hedgehogs during a natural or cold-induced hibernation. 3. These responses included a strong reduction in body temperature, heart rate, respiration and oxygen consumption, and brain activity. 4. Such responses to equithesin were not observed in the non-hibernating rodent species. 5. These results suggest that equithesin is a potential tool for hibernation research.


Effects of cholinergic and adrenergic agonists on the secretion of fluid and protein by submandibular glands of the hamster and the rat. 1. Significant differences were observed between the hamster and the rat in terms of the secretion of fluid and protein from submandibular glands in response to pilocarpine, phenylephrine and isoproterenol. 2. In both the rat and the hamster the secretory responses induced by pilocarpine, phenylephrine and isoproterenol were inhibited by pretreatment with 4-DAMP, prazosin and metoprolol, respectively. 3. These results suggest that the submandibular glands of the hamster and the rat have M3-cholinoreceptors, as well as alpha 1- and beta 1-adrenoceptors, and that these receptors play different roles in the secretion of fluid and protein from hamster and rat submandibular glands.


Postnatal changes in subcellular distribution of copper, zinc and metallothionein in the liver of bank vole (Clethrionomys glareolus): a possible involvement of metallothionein and copper in cell proliferation. 1. Dramatic interdependent changes in the intracellular concentrations of copper (Cu), zinc (Zn) and metallothionein (MT) in the liver of bank voles during the first 30 days of their life were observed. 2. The post-mitochondrial Cu, Zn and MT (Zn-MT) abruptly decreased between 1 and 3 days following birth but the nuclear MT (Cu-MT) and Cu increased at the same time, suggesting that Cu displaced Zn already bound to MT in the cytoplasm and subsequently the complex Cu-MT was translocated to the nuclei. 3. The nuclear Cu concentration reached the highest level (62-71% of the total tissue Cu) in the period from day 3 to day 20 post-partum, just prior to and during a rapidly growing liver. 4. The data indicate that MT and Cu may be involved in the hepatocyte proliferation.


Electrophysiological effects of a neurotoxin extracted from the skin of the Australian frog Pseudophryne coriacea. 1. The electrophysiological effects of a pumiliotoxin-B-like alkaloid extracted from the skin of the Australian frog Pseudophryne coriacea (PsC) have been studied in rat superior cervical ganglia at 37 degrees C. 2. PsC (50 mg/ml) elicits a broadening of the evoked compound action potential and, at rest, the appearance of spontaneous spike discharge at 10-20 Hz. Action potentials presumably originate far away from the soma, which is invaded in a typical IS-SD sequence. 3. The toxin effect is not related to any direct action on the preganglionic fibers of the sympathetic trunk, and does not involve synaptic mechanisms. 4. Two-electrode voltage-clamp experiments showed that the main properties of the major voltage-dependent ionic currents are apparently unaffected by the toxin, while the cell input resistance is considerably reduced. 5. The data are consistent with the hypothesis that PsC elicits a cationic permeability increase generating a pacemaker current in a region close to the cell soma.


Comparative study of the enzymatic, hemorrhagic, procoagulant and anticoagulant activities of some animal venoms. 1. The enzymatic, hemorrhagic, procoagulant and anticoagulant activities of venoms of some animals including snakes, lizards, toads, scorpions, spider, wasps, bees and ants were compared. 2. Snake venom was the richest source of enzymes among the animal venoms. Most other animal venoms were devoid of phosphodiesterase, L-amino acid oxidase, alkaline phosphomonoesterase and acetylcholinesterase activities and only a few exhibited arginine ester hydrolase activity. These venoms, however, exhibited wide ranges of protease, 5'-nucleotidase and hyaluronidase activities. Most of the animal venoms examined exhibited some phospholipase A activity. 3. Other than snake venoms, only venoms of the toad Bufo calamita and the lizards were hemorrhagic, and only venoms of the social wasps, social bees and harvester ant exhibited strong anticoagulant activity. Procoagulant activity occurs only in snake venoms.


Effects of amiodarone on triggered activity induced by overdrive stimulation in Ca2+ overloaded ventricular muscle from guinea pig. 1. The acute effects of amiodarone, a powerful antiarrhythmic drug, on transient depolarizations (TDs) and/or triggered activity (TA) induced by an overdrive stimulation in the condition of low potassium (2.7 mM) and high calcium (5.4 mM) solution were evaluated on isolated ventricular papillary muscles from guinea pig, by means of conventional microelectrode techniques. 2. The amplitude of the induced TDs was enhanced by increase in stimulus number and frequency during overdrive stimulation, and the coupling interval of TDs was shortened. 3. Amiodarone (4.4 x 10(-5) M) significantly decreased the amplitude of TDs, and prolonged the coupling interval. 4. On the other hand, superfusion with a higher concentration (4.4 x 10(-4) M) of amiodarone tended to induce automatic activity. 5. Possible implications with respect to the antiarrhythmic activity of amiodarone are discussed.


Effects of FMRFamide-related peptides and morphine on the isolated foregut of the locust Schistocerca gregaria. 1. Morphine and YAGFMamide were the most effective potentiators of 5-hydroxytryptamine (5-HT)-induced relaxation of the isolated foregut. 2. Morphine had no effect on proctolin-induced tissue contraction which was inhibited by YGGFMamide and YFMRFamide. 3. The differing potency of FaRPs and morphine to potentiate 5-HT effects and reduce proctolin responses suggests that there are two separate FaRP receptor sub types. 4. This proposal is supported by the observation that, while naloxone (10(-5) M) is a relatively potent antagonist of FaRP induced inhibition of proctolin contraction, it has less effect on FaRP-induced potentiation of 5-HT-induced relaxation.


The effect of high concentrations of alpha 2-adrenoceptor antagonists on the lipolytic responsiveness of isolated human adipocytes. 1. Non-specific effects of alpha 2-adrenergic antagonists on human adipocyte lipolysis are reported, and revealed that yohimbine and RX-821002 (10(-4) M) decreased the maximum lipolytic response and increased the ED50 towards norepinephrine and isoproterenol. 2. These effects were not mediated by adenosine receptors, alpha 2-adrenoceptors or Gi proteins. 3. Whereas alpha 2-antagonists, even as low as 10(-8) M, decreased the binding affinity of [3H]CGP-12177, levels as high as 10(-3) M of these antagonists were needed to cause 10-20% displacement of the beta-selective antagonist. 4. We propose that the antilipolytic effects of alpha 2-antagonists can only partly be explained by non-specific binding to beta-adrenoceptors, and that some other mechanism is also involved.


The nature of Cl- secretion, induced by carbaryl, across the isolated skin of Rana esculenta. 1. The pesticide carbaryl induces Cl- secretion through the isolated frog skin. 2. This effect is due to the activation of both processes responsible for this phenomenon: (a) Na+/K+/2Cl- cotransport on the serosal membrane; (b) Cl- selective channels on the external membrane. 3. Cl- outflux is inhibited by bumetanide (10(-5) M) on the serosal side and by diphenylamine-2-carboxylic acid (DPC) (10(-3) M) on the external side. 4. The DPC action is not mimicked by Naproxen, a specific inhibitor of cyclooxygenase. 5. A comparison with isoprenaline, demonstrates that the carbaryl action is, paradoxically, more selective than that of isoprenaline. 6. This selectivity of carbaryl action on Cl- permeability is confirmed by the fact that, unlike isoprenaline, carbaryl does not affect the permeability of Na+ and thiourea.


Effects of a high molecular weight toxin from the sea anemone Condylactis gigantea on cholinergic responses. 1. The effects of a high molecular weight toxin isolated from the sea anemone Condylactis gigantea (Condytoxina 2) on the cholinergic responses were studied in two different preparations: identified cells of a land snail and enzymatically dissociated mice sensory neurons. These neurons were studied using intracellular recording and concentration clamp techniques respectively. 2. The toxin produces a concentration-dependent dual effect on the cholinergic responses in both preparations. Thus the application of the toxin at concentrations up to 25 nmol/l produces a reversible block of the response whereas higher doses potentiates it. 3. These results suggest that Condytoxina 2 contains an active compound(s) with the capacity to bind to the nicotinic acetylcholine receptor of excitable cells in both snail and mice neurons. During this action complex allosteric interactions among the binding sites could occur.


Adrenergic stimulation of bovine non-pigmented ciliary epithelial cells raises cAMP but has no effect on K+ or Cl- currents. Freshly isolated bovine nonpigmented ciliary epithelial cells were studied using the whole-cell voltage-clamp and permeabilized patch techniques. In a study of 148 cells three classes of cell were found; those containing inward currents alone, 31%; those containing outward currents alone, 37% and those containing both inward and outward currents, 32%. The outward currents exhibited slow, delayed activation, were blocked by tetraethylammonium (TEA+) but were not effected by changes in [Cai] suggesting they are K(+)-currents similar to IK(V), the delayed rectifier. The inward currents were reduced by TEA+ and blocked by Cs+ suggesting they are inward rectifier K(+)-currents. Intracellular cAMP levels were increased by isoprenaline with a Ka of 20 nM. However, the resting membrane potential recorded from whole ciliary processes in intracellular microelectrode studies was not effected by adrenergic stimulation, neither were the leak current, the outward current nor the sustained inward current significantly effected by isoprenaline, noradrenaline or dibutyryl-cAMP in whole-cell and permeabilized patch clamp studies.


Pharmacological evidence for heterogeneity of ocular alpha 2 adrenoceptors. Previous studies have shown that ocular alpha 2 adrenoceptors are located prejunctionally on sympathetic neurons and postjunctionally on cells in the iris/ciliary body. While the activation of alpha 2 adrenoceptors at each site has been postulated to alter aqueous humor dynamics, little is known about the pharmacological characteristics of these receptors or their role in the modulation of anterior segment function. The purpose of the current study was to determine the possible heterogeneity of ocular alpha 2 adrenoceptors using relatively selective alpha 2 adrenoceptor agonists and antagonists to examine ocular pre- and postjunctional alpha 2 adrenoceptors. Prejunctional alpha 2 effects were evaluated by means of the cat nictitating membrane (CNM) preparation. Postjunctional alpha 2 effects were evaluated by means of the cAMP assay in rabbit iris root/ciliary body. In the CNM, the administration of UK-14, 304 (UK) produced a dose-related inhibition of neuronally mediated contractions. Pretreatment with the alpha 2 antagonist rauwolscine caused a 1 to 2 log unit right shift in the dose-response curve of UK in the CNM. However, pretreatment with alpha 2 antagonist SKF 104078 had no demonstrable effect on UK-induced inhibition of neuronally mediated contractions of the CNM. In the rabbit iris root/ciliary body, UK produced a concentration-dependent inhibition of cAMP accumulation on isoproterenol- and VIP-induced cAMP production. Pretreatment of iris root/ciliary bodies with SKF 104078 or rauwolscine reversed the inhibitory effect of UK on isoproterenol- and VIP-induced accumulation of cAMP. These data provide the first evidence that the pre- and postjunctional alpha 2 adrenoceptors represent pharmacologically distinct subpopulations of receptors in the eye.(ABSTRACT TRUNCATED AT 250 WORDS)


The effect of exogenous dopamine on ileal smooth muscle of guinea-pigs. In the isolated ileum of guinea-pig, treatment with dopamine produced a lowering of muscle tone in a dose-dependent manner. Dopamine-induced relaxation at low concentration was reversed by haloperidol, the specific antagonist of dopamine receptors. The relaxations by dopamine induced at concentration of 1 microM or higher were abolished by haloperidol with propranolol. At the concentration sufficient to block beta-adrenoceptors, propranolol attenuated this relaxation by dopamine at high concentrations. Similar results were also observed in tissues concerning the dopamine-stimulated formation of cyclic AMP. Mediation of dopamine receptor and beta-adrenoceptor in this cyclic AMP-related relaxation can thus be considered. Failure of sulpiride, an antagonist of dopamine DA-2 receptors, to reverse the actions of dopamine ruled out the participation of DA-2 receptor. Otherwise, SCH23390, the blocker of dopamine DA-1 receptors, reversed the responses to dopamine at low concentration only. Actions of dopamine induced at high concentration were disappeared in the presence of SCH23390 with propranolol. Thus, the obtained data suggest that dopamine induced relaxation of ileal smooth muscle through an activation of dopamine DA-1 receptors and/or a stimulation of beta-adrenoceptors at the concentrations over 1 microM to result in an increase of cyclic AMP in guinea-pigs.


Evaluation of a method for detection of cells with reduced drug retention in solid tumours. A method for detection of cells with reduced drug retention was evaluated in solid tumours. After a 1 h incubation with daunorubicin (DNR), the right angle scatter (RAS), forward angle scatter (FAS), and specific fluorescence (Fluo) were measured in sensitive and resistant cells; only Fluo was related qualitatively, but not quantitatively, to resistance. Various incubation conditions were examined. When the pH of the incubation medium increased, the DNR retention increased in sensitive and resistant cells. In contrast, when the cell concentration increased, the DNR retention decreased. Using sensitive and resistant cell lines, a proportion of resistant cells lower than 10% can be detected in a mixture. To analyse cells from solid tumours, the cells were dissociated by repeated fine needle aspirations. Tumours from 22 patients have been processed with this technique; 8 samples were classified as S (sensitive); 2 as R (resistant); and 12 as I (intermediate). Further experiments were run to study and improve the method. Another method of detection of dead cells was tested. The intra-assay variability of the technique was found to be less than 10%. When the study was performed with different fragments of the same tumour, the variation, corresponding to the tumour heterogeneity, rose to 21 to 36%. The inter-assay reproducibility was too bad, so a variant of this technique has been adapted, using verapamil or cyclosporin A, which is able to block DNR efflux; this new method allows tumour cells to be used as their own controls.


The role of the teashirt gene in trunk segmental identity in Drosophila. The phenotypes of different mutant combinations of teashirt (tsh) and homeotic genes together with their regulatory interactions are described in order to gain insight into tsh gene function. We show that when tsh, Scr, Antp and BX-C genes are missing, the ventral part of the trunk (or thorax and abdomen) is transformed to anterior head identity showing that tsh is a homeotic gene. These genes act synergistically to suppress the expression of the procephalic gene labial (lab) in subsets of cells in each segment of the trunk. Transcripts from the tsh gene always accumulate in segments destined to acquire trunk identities. tsh gene activity is required for the normal function of the Antp and BX-C genes, which modulate in part the expression of tsh. As a whole, our results suggest that tsh plays an essential dual role, during embryogenesis, for determining segmental identity of the trunk. First, tsh is required critically for the identity of the anterior prothorax. Second, tsh is required globally for segmental identity throughout the entire trunk whereas the "classical" homeotic genes have more specific roles. Our results are consistent with the idea that tsh is defining the ground state of the Drosophila trunk region seen in the absence of the Antp and BX-C genes.


MHox: a mesodermally restricted homeodomain protein that binds an essential site in the muscle creatine kinase enhancer. Myogenic helix-loop-helix (HLH) proteins, such as myogenin and MyoD, can activate muscle-specific transcription when introduced into a variety of nonmuscle cell types. Whereas cells of mesodermal origin are especially permissive to the actions of these myogenic regulators, many other cell types are refractory to myogenic conversion by them. Here we describe a novel homeodomain protein, MHox, that binds an A+T-rich element in the muscle creatine kinase (MCK) enhancer that is essential for muscle-specific transcription and trans-activation by myogenic HLH proteins. MHox is completely restricted to mesodermally derived cell types during embryogenesis and to established cell lines of mesodermal origin. In contrast to most other homeobox genes, MHox expression is excluded from the nervous system, with the highest levels observed in limb bud and visceral arches. In adult mice, MHox is expressed at high levels in skeletal muscle, heart and uterus. The DNA-binding properties and pattern of MHox expression are unique among homeobox genes and suggest a role for MHox as a transcriptional regulator that participates in the establishment of diverse mesodermal cell types.


Relationship between Wnt-1 and En-2 expression domains during early development of normal and ectopic met-mesencephalon. Grafting a met-mesencephalic portion of neural tube from a 9.5-day mouse embryo into the prosencephalon of a 2-day chick embryo results in the induction of chick En-2 (ChickEn) expression in cells in contact with the graft (Martinez et al., 1991). In this paper we investigate the possibility of Wnt-1 being one of the factors involved in En-2 induction. Since Wnt-1 and En-2 expression patterns have been described as diverging during development of the met-mesencephalic region, we first compared Wnt-1 and En-2 expression in this domain by in situ hybridization in mouse embryos after embryonic day 8.5. A ring of Wnt-1-expressing cells is detected encircling the neural tube in the met-mesencephalic region at least until day 12.5. This ring consistently overlapped with the En-2 expression domain, and corresponds to the position of this latter gene's maximal expression. We subsequently studied ChickEn ectopic induction in chick embryos grafted with various portions of met-mesencephalon. When the graft originated from the level of the Wnt-1-positive ring, ChickEn induction was observed in 71% of embryos, and in these cases correlated with Wnt-1 expression in the grafted tissue. In contrast, this percentage dropped significantly when the graft was taken from more rostral or caudal parts of the mesencephalic vesicle. Taken together, these results are compatible with a prolonged role of Wnt-1 in the specification and/or development of the met-mesencephalic region, and show that Wnt-1 could be directly or indirectly involved in the regulation of En-2 expression around the Wnt-1-positive ring during this time. We also provide data on the position of the Wnt-1-positive ring relative to anatomical boundaries in the neural tube, which suggest a more general role for the Wnt-1 protein as a positional signal involved in organizing the met-mesencephalic domain.


Kindled rats are more sensitive than non-kindled rats to the behavioural effects of combined treatment with MK-801 and valproate. The effects of combined treatment with low doses (0.025-0.05 mg/kg i.p.) of the non-competitive NMDA receptor antagonist, MK-801 (dizocilpine), and the antiepileptic drug, valproate, were studied in amygdala-kindled and non-kindled rats. MK-801, 0.05 mg/kg, did not exert anticonvulsant effects in fully kindled rats but increased the anticonvulsant potency of valproate, 100 mg/kg i.p. However, the increase in anticonvulsant activity was paralleled by a marked increase in adverse effects such as motor impairment and hyperactivity, resulting in a considerable reduction of the therapeutic index of the combined treatment compared to valproate alone. Furthermore, MK-801 potentiated the adverse effects but not the anticonvulsant activity of 50 mg/kg valproate. Combined treatment with MK-801 and valproate induced much less marked adverse effects in non-kindled rats than in kindled rats. The competitive NMDA receptor antagonist, CGP 37849 1 mg/kg i.p., did not alter the effects of valproate in kindled rats. The data on combined treatment with MK-801 and valproate substantiate the conclusion that kindling alters the susceptibility to manipulations of NMDA receptor-mediated events.


Quantitative in vitro and ex vivo autoradiography of the alpha 2-adrenoceptor antagonist [3H]atipamezole. Atipamezole is an alpha-adrenoceptor antagonist with high affinity and selectivity for the alpha 2-receptor. In vitro autoradiography of [3H]atipamezole on rat and human brain sections revealed a pattern virtually identical to the one observed using 3H-labeled antagonists such as idazoxan or methoxyidazoxan or agonists such as p-aminoclonidine and 5-bromo-6-(2-imidazolin-2-yl-amino-quinoxaline (UK-14304). In vivo studies of [3H]atipamezole demonstrated good penetration into the brain (0.3-1.8% injected dose/g at 5 min, depending upon brain region). In addition, [3H]atipamezole displayed rapid in vivo clearance of nonspecific binding such that at 1 h following an i.v. injection of the drug (100 microCi/animal, rat tail vein administration) the pattern of radioactivity in the brain correlated very well with the receptor distribution as seen by in vitro autoradiography. Atipamezole binding in vivo was displaceable by idazoxan. These results demonstrate the potential of suitably labeled atipamezole for regional studies of brain alpha 2-adrenoceptors in vitro and in vivo, in experimental animals as well as in human positron emission tomography (PET) studies.


Evidence for a noradrenergic component in the antinociceptive effect of the analgesic agent tramadol in an animal model of clinical pain, the arthritic rat. The analgesic agent tramadol has a potent antinociceptive effect in arthritic rats. In the present study, the actions of the selective alpha 2-adrenoceptor antagonists yohimbine and idazoxan on this antinociceptive effect were tested in arthritic rats, using vocalization thresholds to paw pressure as a nociceptive test. The antagonists were administered 30 min before tramadol, at doses (0.5 and 1 mg/kg i.v.) without action per se, but which prevented the antinociceptive action of the prototypic alpha 2-adrenoceptor agonist clonidine (0.1 mg/kg i.v.) in these animals. The potent antinociceptive effect of tramadol (1 mg/kg i.v.) was significantly decreased (mean total effect reduced about 2-fold) by yohimbine and idazoxan. In alpha 2-adrenoceptor antagonists-pretreated arthritic rats, the effect of tramadol was almost abolished when tramadol was coinjected with the opioid antagonist naloxone. In addition to the involvement of opioid receptors, these results provide evidence for a noradrenergic component to the antinociceptive action of tramadol in this model of clinical pain.


[Immunocytochemical studies on the pancreatic endocrine cells in the Japanese newt (Cynopus pyrrhogaster)]. Pancreatic endocrine cells were examined by light and electron microscopic immunocytochemistry to discuss the co-localization of peptides in one cell type. A cells were irregular in shape with an occasional long cytoplasmic process, and contained glucagon-immunoreactive granules with various contours. These granules were 160-300nm in diameter with various density, and also immunoreactive to anti-human pancreatic polypeptide (PP) serum. A part of them were further immunoreactive to anti-somatostatin serum. B cells were round to elliptical in shape, and often aggregated around the capillaries. Granules of B cells were round to irregular in shape, 270-410 nm in diameter, and immunoreactive to anti-insulin serum. D cells were irregular in shape with meager cytoplasm, and contained somatostatin-immunoreactive granules. These granules were ovoid or teardrop in shape, 140-250nm in longitudinal diameter, and immunoreactive to both anti-somatostatin and anti-human PP sera. PP cells were round to spindle-shaped, and contained human PP-immunoreactive round granules 150-35nm in diameter. These findings reveal the existence of at least 4 types of endocrine cells secreting glucagon, insulin, somatostatin, and PP, respectively, in the newt pancreas, and suggest the co-localization of some of these peptides in one cell type.


Heat-shock responsive elements in the induction of the multidrug resistance gene (MDR1). The MDR1 gene, considered to be involved in multidrug resistance of cancer cells, is expressed in liver, kidney, small intestine and the blood-brain barrier. We investigated MDR1 gene expression in the well-differentiated hepatoma cell line HepG2 after exposure to several stresses and found that sodium arsenite treatment increased MDR1 gene expression 2.6-fold. Deletion analysis of the MDR1 promoter indicated that the transcriptional activation after exposure to arsenite depends on a 60-bp region containing two heat-shock responsive elements.


The sequence of human caveolin reveals identity with VIP21, a component of transport vesicles. Caveolin is a protein present in membrane specializations termed caveolae where it may play a structural role. Previous studies on the cDNA sequence of chicken caveolin demonstrated that it is an integral membrane protein without significant homology to any known protein. The cDNA sequence of human lung caveolin is presented here. A striking sequence homology is observed with the chicken protein, as well as a very recently reported protein termed VIP21 [(1992) J. Cell Biol. 118, 1003-1014], a putative vesicle transport protein isolated from MDCK cells. Genomic Southern blots suggest that caveolin is present in a single copy, and Northern blots confirm that the caveolin mRNA is elevated in muscle.


Nitric oxide-releasing compounds inhibit Dictyostelium discoideum aggregation without altering cGMP production. The effects of nitric oxide-releasing compounds on Dictyostelium discoideum cell development and guanylyl cyclase activity were studied. The addition of SNP (sodium nitroprusside) or SIN-1 (3-morpholino-syndnonimine) to starved cells inhibited their differentiation and aggregation in a concentration-dependent manner. In contrast to mammalian systems, SNP did not significantly affect guanylyl cyclase activity in cell lysates of D. discoideum, nor did it stimulate cGMP production in intact cells. The results suggest that the inhibitory effects of NO on D. discoideum cell aggregation are through a mechanism independent of an effect on guanylyl cyclase activity.


The single-copy gene psbS codes for a phylogenetically intriguing 22 kDa polypeptide of photosystem II. Recombinant phages that encode the complete precursor polypeptide for the 22 kDa polypeptide associated with photosystem II have been serologically selected from two lambda gt11 expression libraries made from polyadenylated RNA of spinach seedlings. The cDNAs hybridize to a 1.3 kb RNA species. The precursor protein is comprised of 274 amino acid residues and carries an N-terminal transit peptide of probably 69 amino acid residues. The mature protein exhibits four predicted transmembrane segments and is shown to be an integral component of photosystem II originating in a single-copy gene. The unique characteristics of this protein are: (i) it is the result of a gene-internal duplication of an ancestor with two membrane spans, (ii) a striking resemblance to LHC I/II, CP24/CP29 apoproteins, and ELIPs, although it does not bind chlorophyll and is present in cyanobacteria, and, as these proteins, (iii) it integrates into the membrane with uncleaved routing signals that display remarkable resemblance to patterns found in bipartite transit peptides.


Regulation of rat erythrocyte L-glutamine, L-glutamate and L-lysine uptake by short term starvation. 1. The kinetic parameters (Km, Vmax and Kd) of L-glutamine, L-glutamate and L-lysine uptake by isolated red blood cells in fed and 24 hr starved rats have been determined. 2. L-Lysine and L-glutamine uptake was best fitted by a two transport component: a saturable component and a diffusion one. 3. Starvation brought about important decreases in the Km and Vmax for both L-lysine and L-glutamine uptake. 4. The Kd for L-glutamine showed a significant increase whereas that corresponding to L-lysine did not change by starvation. 5. L-Glutamate uptake adjusted to diffusion kinetics, with a Kd which did not change due to starvation. 6. It is concluded that the amino acid uptake showed specific regulation by starvation. 7. The mechanism involved is not dependent on protein synthesis--given the unnucleated nature of mammal red cells. 8. The magnitude of the changes observed in the uptake kinetic parameters may account for the extent of the blood amino acid pool changes as those produced in vivo over physiological limits.


Home deliveries in The Netherlands--perinatal mortality and morbidity. The obstetrical organizational system in the Netherlands is based on the selection for risk factors. We conclude that: (i) The reporting of perinatal death is not complete. (ii) Perinatal mortality can be reduced. (iii) More iatrogenic interventions are present in low-risk deliveries in hospitals. (iv) Neurological behavior of low-risk babies born at home is equal to those born at the hospital, despite the worse maternal profile of the latter and the level of acidemia at birth in the first. Good data especially in referred cases are necessary before adopting a similar system.


Longitudinal study of serum thyroid hormone levels during normal pregnancy. We undertook a prospective longitudinal study of thyroid function in 60 normal pregnant women and measured serum concentrations of T4, triiodothyronine (T3), T-uptake, thyroxine binding globulin (TBG), free thyroxine index (FTI), free T4, albumin and thyrotropin (TSH). From these data we established reference ranges for each of these analytes for each trimester and examined the inter-relationships between laboratory measurements of thyroid function tests. We observed significant increases in serum concentrations of thyrotropin and decreases in free T4, assays commonly used as first line investigations of thyroid activity during pregnancy. However, the 95th centile intervals for both analytes remained within the reference range for nonpregnant women.


Perinatal outcome of very low birthweight infants by mode of delivery. In order to evaluate the influence of mode of delivery on perinatal morbidity and mortality in vertex infants weighing less than 1500 g (VLBW), we made a retrospective study of 152 singleton newborns, in vertex presentation, with a birthweight of less than 1500 g, delivered in the Cruces Hospital (Vizcaya, Spain), a major perinatal referral center, between 1 January 1987 and 31 December 1989. Twins and infants with lethal congenital anomalies or gross intrauterine growth deviations were excluded from the study (n = 71). Of the infants studied (n = 81), 37 were delivered by cesarean section (mean weight 1120 +/- 206 g, range: 680-1495 g) and 44 were delivered vaginally (mean weight 1029 +/- 283 g, range: 530-1475 g). The patients were divided into four groups: Group A: 500-749 g (n = 10); Group B: 750-999 g (n = 21); Group C: 1000-1249 g (n = 27); and Group D: 1250-1499 g (n = 23). The percentages of cesarean sections in each group were 10%, 42%, 66% and 39%, respectively. A comparison within each group of immediate perinatal outcome (Apgar score and umbilical vein cord pH), as well as mortality and sequelae up to 1 year of age did not yield any significant differences between cesarean and vaginal birth. We conclude that cesarean delivery does not appear to offer improved outcome over vaginal delivery in live births without congenital anomalies. For this reason, we believe that fetal weight should not be the only obstetrical variable considered when deciding whether or not to perform a cesarean section in these circumstances.


Interval to delivery in high-risk patients: do tocolytic agents really work? Some question whether tocolytic drugs reduce uterine activity and prolong gestation. The interval from discontinuance of tocolytics until spontaneous labor and delivery in patients (n = 69) with documented preterm labor (PTL) versus subjects receiving prophylactic tocolytic therapy (n = 41) was studied. Women with documented PTL delivered sooner after cessation of tocolytics (6.1 +/- 6.9 days) than control (C) patients (14.7 +/- 10.8 days, P less than 0.001). Also, 28 of the 69 (41%) patients in the PTL group delivered within 24 h of discontinuation of tocolysis compared to 4 (10%) in the C group (P less than 0.0004). We conclude that tocolytic therapy for documented preterm labor suppresses uterine activity and when these agents are discontinued, contractions return and labor ensues.


Maternal antihypertensive therapy with beta-blockers associated with poor outcome in very-low birthweight infants. The progress of 36 very-low birthweight (less than or equal to 1500 g) infants born to mothers with pregnancy-induced hypertonia or pre-eclampsia was studied. During the first year of life, 7 out of 19 infants died when the mothers' antihypertensive regimen included beta-blockers. Four of the deaths occurred within 15 days. There were no deaths in 16 infants whose mothers were treated with other antihypertensive treatment (P = 0.006). These results suggest that maternal beta-blocker therapy may have adverse effects on the very-low birthweight infants.


Infraumbilical-ring capillary abdominocentesis in late presentation ectopic pregnancy: a prospective study. Infraumbilical-ring capillary abdominocentesis was compared with culdocentesis in 75 cases of suspected ruptured ectopic pregnancy, with patients serving as their own controls. There were 43 proven ectopic cases and the diagnosis was known in the remaining 32. The accuracy of results was equivalent: P greater than 0.15 for false positives and P greater than 0.63 for false negatives. For combined testing the positive predictive value reached 97%. The new method was easier to perform and should be preferred.


A comparison of ovulation suppression and ovulation stimulation in the treatment of endometriosis-associated infertility. This study compares ovulation stimulation or suppression with the pregnancy rate among infertile couples with endometriosis. The design is a nonrandomized, prospective, multicentered cohort analytic study. Two hundred and ninety-seven couples with laparoscopy-proven endometriosis were analyzed: 68 received no therapy, 42 received clomiphene and 74 received danazol. Forty patients (22%) conceived and pregnancy rates were similar in each treatment group. The relative likelihood of pregnancy associated with clomiphene was 2.9 (95% confidence limits 1.2-7.1); the relative likelihood of pregnancy with danazol was 1.02 (95% confidence limits 0.5-2.3). This study suggests that pregnancy rates during clomiphene treatment could be superior to expectant therapy, while pregnancy rate after danazol is similar to no treatment.


Endocrinological profile of oligomenorrheic strenuously exercising adolescents. The endocrinological profile of 20 strenuously exercising oligomenorrheic adolescents divided into 2 groups (groups A and B), was correlated with that of 10 athletes (group C) with normal menstrual cycles and without strenuous exercise. Group A LH serum baseline values were found to be statistically significantly lower than those of group C (P less than 0.001). FSH/LH basic values were 1.9- and 2.9-times higher in group A athletes than those of group B or C (P less than 0.05 and P less than 0.001, respectively). 17 beta-estradiol (E2) and prolactin serum levels were found lower in group A and B athletes than those of group C (P less than 0.01-0.05). Dehydroepiandrosterone sulfate and delta 4 androstenedione serum levels were found lower in group A athletes than those of group C (P less than 0.001). The low LH and E2 values indicate the anovulatory status of group A and B cases which were also confirmed by ultrasound. It is concluded that no severe endocrinological changes exist in strenuously exercising oligomenorrheic athletes in relation to menarche.


Evaluation of regret after tubal sterilization. Many different criteria and profiles have been suggested for the possible cause of regret and requests for reversal after tubal sterilization. Evaluation of data obtained from 2253 women who had undergone tubal sterilization showed a strong correlation between regrets and youthful age and to changes in marital situation. Previously demonstrated risk factors, such as sterilization in connection with abortion or labor were not related to regret in this study material.


Evaluating the readability of informed consent forms used in contraceptive clinical trials. The readability level of informed consent forms used in clinical trials on contraceptives was determined. Three different formulas for measuring readability were used. Some forms received relatively high scores by all three methods. The most common problems associated with high readability scores were the use of 'unfamiliar' words, long words and long sentences. At present all forms used must be readable, using the SMOG formula, at a grade 6 level or less.


Pregnancy complicated by malaria, precipitate labor and uterine rupture. A case of Plasmodium falciparum malaria is reported in a 25-year-old pregnant woman with a history of three previous cesarean sections. She developed premature precipitate labor which was complicated by stillbirth, uterine rupture, bladder and vaginal tears necessitating hysterectomy.


Antibodies to glutamic acid decarboxylase are associated with HLA-DR genotypes in both Australians and Asians with type 1 (insulin-dependent) diabetes mellitus. Antibodies to glutamic acid decarboxylase, previously known as the 64 kD antigen, appear to be more predictive of Type 1 (insulin-dependent) diabetes mellitus in Caucasoids than other autoantibodies to islet cell antigens. However, seropositivity to glutamic acid decarboxylase is not universal at the onset of Type 1 diabetes and the prevalence in Asians is low compared to Caucasoid patients. This suggests the involvement of multiple pancreatic autoantigens in the Type 1 diabetes autoimmune process or, genetic differences within and between ethnic groups that contribute to the heterogeneous autoimmune response to glutamic acid decarboxylase or both. Alternatively some cases of Type 1 diabetes could have an aetiology unrelated to autoimmunity. This study examined the differential response to glutamic acid decarboxylase according to HLA-DR and -DQ genotypes, as determined by RFLP, in 49 white Australian and 44 Asian patients with Type 1 diabetes. Among Australians heterozygous for HLA-DR3, DR4, 85% were positive for antibodies to glutamic acid decarboxylase, significantly different (p = 0.039) from the prevalence of 48% in patients with at least one HLA-DR antigen other than DR3 or DR4. Also, among Australians, the presence of "low risk" HLA-DQ antigens, namely DQw5, DQw6 or DQw7, reduced the prevalence of antibodies to glutamic acid decarboxylase by 40% (p = 0.064). Among Asians with Type 1 diabetes and with antibodies to glutamic acid decarboxylase, HLA-DR9 was significantly (p = 0.037) increased in frequency, at 63% compared with 22% in those without glutamic acid decarboxylase antibodies, and the presence of a "low risk" HLA-DQ allele reduced the antibody rates by 87% (p = 0.003).(ABSTRACT TRUNCATED AT 250 WORDS)


Modulation of HOX2 gene expression following differentiation of neuronal cell lines. The expression of the genes in the human HOX2 locus has been studied during differentiation of two human neuroblastoma (SH-SY5Y and Kelly), a human glioblastoma (251-MG), and the murine F9 embryonal carcinoma cell lines. Cells were differentiated with retinoic acid (RA), or with RA together with dibutyral cyclic AMP (db-cAMP) and nerve growth factor (NGF) in order to assess the changes in the expression patterns of these homeobox genes during neuronal differentiation. We show that the genes of the HOX2 locus are expressed in a complex transcription pattern that varies with cell type. The two uninduced neuroblastoma cell lines show a similar pattern of expression for a number of HOX2 genes although the levels of expression are different for individual cell lines. The embryonal carcinoma cell line F9 expresses low levels of several HOX2 genes which is restricted to the 5' region of the HOX2 cluster. The glioblastoma cell line, 251-MG expresses almost all of the genes of the HOX2 locus. Differentiation of these cells modulates the expression of the HOX2 genes in a manner that is dependent upon the cell type as well as the differentiation factor. Differentiation affects both the level of HOX2 gene expression and the distribution of transcript sizes. In conclusion, our analysis reveals a complex pattern of expression for the genes of the HOX2 locus in neuronal and glial cells and suggests that the cell-specific expression of these genes may be correlated with the phenotypic differences that are observed between different neuronal and glial cell populations within the nervous system.


Epithelial proliferation in Barrett's esophagus by proliferating cell nuclear antigen immunolocalization. Proliferating cell nuclear antigen (PCNA) is an auxiliary protein to DNA polymerase delta and is an absolute requirement for cellular proliferation. Specialized-type Barrett's columnar-lined esophagus (CLE) is associated with adenocarcinomatous change. In the present study, the cellular proliferation of three histological types of CLE was assessed by semiquantitative evaluation of PCNA immunolocalization in 93 biopsy specimens from 45 patients using the murine monoclonal PC10. Statistical comparison was performed by the Mann-Whitney U test. Luminal surface cell labeling was uncommon in all histological types other than specialized CLE where 25 of the 43 biopsy specimens had at least occasional luminal surface cell labeling. Mean crypt labeling score of 4.06 for specialized type exceeded that for junctional (mean, 3.12; P < 0.001) and fundic types (mean, 1.6; P < 0.001). Gland cell PCNA staining scores for specialized-type CLE (mean, 3.18) exceeded that of junctional (mean, 1.97; P < 0.001) and fundic (mean, 1.04; P < 0.001). Summated PCNA scores for specialized-type, mean of 8.29, exceeded junctional mean score of 5.45 (P < 0.001) and fundic mean score of 2.76 (P < 0.001). PCNA immunolocalization reveals a high proportion of cells in cycle in the specialized-type CLE and expansion of the proliferative compartment, which may explain the association of specialized-type CLE with malignancy.


Effects of somatostatin on renal function in cirrhosis. To investigate the renal effects of somatostatin in cirrhosis, renal function and plasma and urinary levels of endogenous neurohumoral vasoactive substances were measured in conditions of intravenous water overload (20 mL/kg body wt with 5% glucose) before and during the intravenous infusion of somatostatin (250-500 micrograms/h) in 6 cirrhotic patients without ascites and 17 nonazotemic cirrhotic patients with ascites. Somatostatin induced a significant reduction of renal plasma flow, glomerular filtration rate, and free water clearance in both groups of patients. In patients with ascites, somatostatin also reduced urinary sodium excretion. Changes in renal function were significantly more marked in patients with ascites than in those without ascites and occurred in the absence of changes in mean arterial pressure and plasma levels of renin, aldosterone, norepinephrine, antidiuretic hormone, and atrial natriuretic peptide. Somatostatin induced a significant reduction in the plasma concentration of glucagon and urinary excretion of prostaglandin E2 that was not related to changes in renal function. These findings indicate that somatostatin administration induces renal vasoconstriction and impairs glomerular filtration rate, free water clearance, and sodium excretion in cirrhosis by a mechanism unrelated to systemic hemodynamics and endogenous neurohumoral vasoactive systems.


Hypersensitivity with hepatotoxicity to mesalazine after hypersensitivity to sulfasalazine. A 21-year-old woman with Crohn's disease of the colon developed a skin rash after 3 weeks of treatment with sulfasalazine. Administration of sulfasalazine was discontinued. When mesalazine was instituted 1 week later, she developed a severe hypersensitivity reaction characterized by fever, diarrhea, skin rash with subsequent desquamation, marked atypical lymphocytosis, and severe hepatotoxicity. Recovery was complete. The clinical and biological features as well as liver pathology of this case bear a striking resemblance to earlier reports of hypersensitivity reaction with severe hepatotoxicity to sulfasalazine. The authors urge caution when mesalazine is given to a patient with known hypersensitivity to sulfasalazine.


[Overexpression of c-erbB-2 oncogene in primary ovarian cancers: incidence and prognostic significance in 243 patients]. In ovarian cancers, no valuable prognostic factors are available so far for predicting biological behaviour and clinical outcome. During the last years, overexpression of c-erbB-2-oncogene has been reported to be a prognostic factor in various human carcinomas. Its prognostic value in ovarian cancer is still a matter of controversy. The records and histological specimens of 243 patients with primary ovarian cancer were reviewed. Overexpression of c-erbB-2-oncogene encoded transmembrane protein p185 was determined immunohistochemically using polyclonal and monoclonal antibodies. Of 243 investigated tumours, 45 (19%) were positive. We examined the relationship between p185 and clinical stage, histological type, grading and prognosis. p185 overexpression differs within the histological subtypes of tumours. No correlation was found between p185 overexpression and stage or histological grade. The follow-up data demonstrate that patients with positive staining for p185 have a significantly worse prognosis (p = 0.001) than those with negative staining. These results suggest that overexpression of c-erbB-2 oncogene is associated with higher biological aggressiveness and unfavourable course of disease.


Dominant hemimelia and En-1 on mouse chromosome 1 are not allelic. Previous studies have shown that En-1, a homeobox-containing gene, maps close to or at the Dh locus in the mouse. Since homeobox-containing genes are key genes in the control of development the close proximity of En-1 to the developmentally significant gene Dh raised the possibility that the Dh mutation represented a mutant allele of En-1. A genetic analysis involving En-1, Dh, and other chromosome 1 markers (Emv-17, ln and Pep-3) shows that although Dh and En-1 are closely linked they are separable by recombination (4/563). The likely gene order and recombination frequencies of these loci are: ln (5.2 +/- 0.9) Emv-17 (1.1 +/- 0.4) Dh (0.7 +/- 0.4) En-1 (3.0 +/- 0.7) Pep-3. This shows that Dh is not a mutant allele of En-1.


Malignant change in trichilemmal cysts: a study of cell proliferation and DNA content. We have examined proliferative activity in a series of pilar and trichilemmal cysts using an antibody to proliferating cell nuclear antigen. In benign lesions proliferative activity was confined to the basal layers of the squamous epithelium. Lesions showing malignant change showed increased proliferative activity and were not confined to the basal layer. These findings were correlated with studies on DNA content using flow cytometry.


T cell maturation stage-linked heterogeneity of the glycosylphosphatidylinositol membrane anchor of Thy-1. We showed that some of Thy-1 molecules on murine thymocytes are resistant to phosphatidylinositol-specific phospholipase C (PI-PLC) derived from Bacillus thuringiensis. Both immature thymocytes with low CD3 expression and mature thymic T lymphocytes with high CD3 expression carried the PI-PLC-resistant Thy-1, and the PI-PLC-sensitivity of Thy-1 extensively varied among thymocyte subpopulations. In contrast, the same PI-PLC fully hydrolysed the anchor of Thy-1 on peripheral T lymphocytes. When the latter cells were activated with mitogen in vitro, however, some Thy-1 on them became resistant to PI-PLC. We then found that virtually all Thy-1 molecules on thymocytes became sensitive to PI-PLC when they were treated with hydroxylamine that should cleave ester-linked lipids. The result ruled out the possibility that the PI-PLC-resistant Thy-1 had a transmembranous peptide sequence, and suggested the presence of an additional fatty acyl group on the inositol ring of the Thy-1 anchor. In addition, the molecular size of the PI-PLC-resistant membrane-bound Thy-1 was only marginally larger than that of the PI-PLC-sensitive solubilized Thy-1 in detergent-partitioning SDS-PAGE analysis.


In vivo depletion of interferon-gamma leads to susceptibility of A/J mice to mouse hepatitis virus 3 infection. The possible role of interferon-gamma (IFN-gamma) in the resistance of A/J mice to MHV3 infection was investigated. Monoclonal antibodies specific for IFN-gamma, CD4 and CD8 molecules were administered in vivo to deplete selectively the IFN-gamma synthesized or the appropriate subset of T cells. The animals were then infected with MHV3 and the course of infection was followed by studying different parameters, such as, the mortality, the virus growth in the tissues and the IFN-gamma synthesis in sera and peritoneal exudates. After MHV3 infection, a full resistance of control A/J mice was observed, in contrast to the high mortality rate observed among the depleted animals, where higher virus titers were found in different tissues. The IFN-gamma synthesis in sera and peritoneal exudates of depleted mice, after MHV3 infection, drastically decreased when compared to that detected in control mice. The data presented are consistent with the hypothesis that IFN-gamma plays an essential role in the resistance of A/J mice to MHV3 infection.


Vasodilators inhibit acute alpha 1-adrenergic receptor-induced trophic responses in the vasculature. Cardiovascular hypertrophy plays an important role in the development and maintenance of hypertension. Hyperactivity of the sympathetic nervous system may be one of the initiating factors responsible for the stimulation of growth processes involved in these structural alterations. We have used a well-established early biochemical marker of cellular growth processes, induction of ornithine decarboxylase (ODC), to determine whether alpha 1-adrenergic receptor-induced vascular trophic responses are dependent on arterial pressure elevation. Hydralazine or felodipine were coadministered to control the alpha 1-adrenergic receptor agonist-induced rise in mean arterial pressure (MAP). Methoxamine (2, 5, or 10 mg/kg s.c.) increased the average MAP (up to 20 mm Hg) and vascular ODC activity (up to ninefold) above control rats over 4 hours. Concomitant administration of hydralazine (0.5, 1.25, or 5 mg/kg s.c.) or felodipine (100 or 250 micrograms/kg s.c.) with methoxamine (10 mg/kg) attenuated the alpha 1-adrenergic receptor-induced activation of ODC in the aorta and mesenteric resistance vasculature, as well as the MAP increases. Vasodilators alone did not lower basal vascular ODC activity. The major findings include: 1) alpha 1-adrenergic receptor activation dose-dependently induces vascular ODC activity concomitantly with MAP elevation, 2) vasodilators inhibited both the alpha 1-adrenergic receptor-induced MAP increases and the activation of mesenteric vascular and aortic ODC, and 3) the stimulus-response correlation between MAP elevation and mesenteric (r = 0.78) and aortic (r = 0.92) ODC activation was characterized by a logistic function.(ABSTRACT TRUNCATED AT 250 WORDS)


Regulation of intestinal guanylate cyclase by the heat-stable enterotoxin of Escherichia coli (STa) and protein kinase C. The heat-stable enterotoxin of Escherichia coli (STa) stimulates membrane-bound guanylate cyclase in intestinal epithelium and induces fluid and ion secretion. Using the T84 human colon carcinoma cell line as a model, we observed that phorbol esters markedly enhanced STa-stimulated cyclic GMP accumulation in T84 cells (C. S. Weikel, C. L. Spann, C. P. Chambers, J. K. Crane, J. Linden, and E. L. Hewlett, Infect. Immun. 58:1402-1407, 1990). In this study we document that the phorbol ester treatment increases 125I-STa-binding sites as well as membrane-bound guanylate cyclase activity in T84 cells and provide evidence that both effects are mediated by phosphorylation. Guanylate cyclase activity was increased approximately 50% in membranes prepared from intact T84 cells treated with phorbol-12,13-dibutyrate (beta-PDB) and after treatment of homogenates with beta-PDB in a manner dependent on ATP, MgCl2, and cytosol. Similarly, treatment of membranes with purified bovine brain protein kinase C in the presence of appropriate cofactors and beta-PDB resulted in an increase in STa-stimulated guanylate cyclase activity of about 70%. Likewise, the number of 125I-STa-binding sites was increased by about 25 to 40% in membranes prepared from intact cells or homogenates treated with beta-PDB; no effect on binding affinity (Kd = 0.15 nM) was noted. These experiments suggest that protein kinase C may phosphorylate the STa receptor-guanylate cyclase or a closely related protein and increase guanylate cyclase activity. The stimulatory effects of protein kinase C on STa-sensitive guanylate cyclase are opposite in direction to the profound inhibitory effects of the kinase on atrial natriuretic peptide-stimulated guanylate cyclase, demonstrating differential regulation by protein kinases within the guanylate cyclase-receptor family.


T-cell-independent stimulation of immunoglobulin secretion in resting human B lymphocytes by the mannose-specific adhesin of Escherichia coli type 1 fimbriae. Purified Escherichia coli type 1 fimbriae have been shown previously to stimulate T-cell-independent proliferation of human B lymphocytes. The response is mediated by the mannose-specific, lectin-like adhesin protein FimH. Here we show that type 1 fimbriae also stimulate immunoglobulin (Ig) secretion by B cells. The response was maximal at three days of culture and consisted predominantly of the IgM isotype. It was independent of serum components, T lymphocytes, monocytes, and natural killer cells. Highly purified resting B cells were induced to proliferate and secrete Ig in response to the fimbriae. The role of FimH in the response was shown by the failure of FimH- type 1 fimbriae to stimulate and by inhibition of the response with alpha-methyl mannoside. In light of the fact that carbohydrate-binding adhesins have been found on a wide variety of microorganisms, these studies suggest the possibility that responses of other cell types to other microbial adhesins will be discovered.


Comparison of different follicle-stimulating hormone and luteinizing hormone ratios for ovulation induction during in vitro fertilization. Five normally menstruating women were treated in an attempt to induce development of multiple follicles. They were randomly divided into two groups. The first group, consisting of three women, was treated with a combination of follicle stimulating hormone (FSH) and human menopausal gonadotropin (hMG) (combination FSH/hMG). The second group, two women, was treated with hMG only. Following a nonconceptual cycle, the treatments were exchanged. The increment patterns of serum estradiol during the follicular phase and progesterone levels in both groups were identical. Ultrasonographic scanning revealed similar number and size of the growing follicles, which subsequently terminated in the same oocyte recovery rate in both groups (7.6 +/- 3.4 oocytes/procedure in the combination FSH/hMG and 8.0 +/- 2.5 in the hMG-only group). Fertilization and cleavage rates were also similar. These data indicate that high FSH/LH levels in different ratios do not alter ovarian response and oocyte fertilization potential.


A conservative, low-cost superovulation regimen. The protocol reported decreases the cost and inconvenience of inducing the growth of multiple ovulatory follicles in selected infertility patients. Safety was maintained by treating patients with relatively small amounts of exogenous gonadotropins and using patient response to slowly increase gonadotropin stimulation in subsequent cycles. The pregnancy rate was 26% per cycle. For couples with male factor infertility, it was 20% per cycle. This regimen may be especially suitable for the practitioner with only a few patients requiring this therapy who may not have the capacity to obtain daily estradiol levels.


Life styles of men in barren couples and their relationship to sperm quality. This study is based on a questionnaire which focused on the possible association between life-style factors and male fertility in a group of 252 men attending our laboratory in connection with a fertility investigation. Their answers were correlated to sperm quality. No association could be documented between sperm quality and smoking habits, coffee drinking, a moderate alcohol intake, exposure to heat (sauna, hot baths, type of underwear, sedentary activities), or physical activities in their leisure time. In contrast, the reported average ejaculation frequency was significantly positively correlated to the motility of the sperm (% progressive), and inversely related to the proportion of sperm with abnormal morphology and semen volume. This indicates that the life style of the subject has little if any impact on semen quality, at least within the limits recorded in the present study.


Should androgen levels be measured in hirsute women with normal menstrual cycles? In a study of 129 consecutively referred hirsute women, 40% had regular menstrual cycles. About one half of such individuals had elevated levels of one or more androgens (DHEAS, testosterone or free testosterone index), whereas a higher proportion (69%) of hirsute women with oligo-amenorrhea were abnormal. Mean androgen levels in regularly cycling hirsute women were higher than in controls, but lower than or equal to those in oligo-amenorrheic hirsutism. Thus, menstrual status does not predict androgen status in hirsute women.


Effect of boar seminal plasma immunosuppressive factor on NK cell activity and skin graft survival. The B 10 strain of mice was used to test the effect of the boar seminal vesicle immunosuppressive factor on the female mouse response to the male-specific transplantation antigen. Influence of this factor on human natural killer (NK) cell activity was also studied. No inhibitory effect of the immunosuppressive factor on graft survival was apparent during a time of more than 200 days, nor did the factor suppress NK cell activity.


Ultrastructural comparison of human spermatozoa along a Percoll density gradient. We studied the possible deleterious effects of Percoll gradient centrifugation processing on spermatozoal integrity in 18 semen samples from patients participating in our IVF program, using optical and electron microscopy. Substantial recovery by sperm count was obtained in the 100% Percoll layer (32%), with greater numbers of motile, alive spermatozoa (P < .01). Whether samples studied were from fertile or infertile men, no significant differences, using nonparametric tests with transmission electron microscopy, appeared in spermatozoal membranes, acrosomes, or nuclei either before or after Percoll treatment. No deleterious effects of Percoll use on spermatozoa were detected during the study.


Late luteal phase progestogen-dependent endometrial protein levels in women with in-phase biopsies--can low levels predict a subfertile group? A group of infertile women who had luteal phase defects (LPD), but in whom follicular maturation was deemed normal, were treated with progesterone until the endometrial biopsy was corrected. At the time the corrected biopsy was obtained, serum was taken and the progestogen-dependent endometrial protein (PEP) concentration was determined. Serum PEP concentration in patients who successfully conceived was 102.5 +/- 62.6% units/mL, while PEP concentrations in patients who failed to conceive were 57.9 +/- 34.4% (P = .003). In patients whose PEP value was more than two standard deviations below the corresponding mean control PEP, pregnancy was achieved in 6/17 (35.3%). The conception rate was significantly greater (25/35, 71.4%) in patients with values higher than this. Thus, the PEP concentration in serum may identify a group of patients with persistent LPD despite apparent normalization of the morphology of late secretory phase endometrium, which might explain some cases of cryptic, unexplained infertility.


Ovarian consequences of the transient interruption of combined oral contraceptives. The combined oral contraceptive pill is an efficient means of contraception. It acts at different levels of the genital tract. Despite its efficiency, it is universally suggested that patients take the pill at regular daily intervals. Little attention has been given to the question of what happens if you miss the pill one day or more. A study was undertaken to evaluate the consequences of pill misses at different times of the cycle. Forty-seven young, healthy, normally menstruating patients voluntarily enrolled. All were given Cilest (ethinyl estradiol 35 micrograms and norgestimate 250 mg, Cilag France) for 21 days without any misses. Then, after a 7-day interval, they were prescribed one (group 1), two (group 2), three (group 3) or four days of pill misses, to occur respectively on day 1 (group a), 6 (group b), 12 (group c) or 18 (group d) of a new 21 day cycle; supplementary contraceptive means were recommended. Four patients had no miss prescribed and served as controls. Ovarian function was evaluated with daily estrogen measurements (E1 + E2 enzymatic dosage, BioMérieux, France) and ultrasound examinations. When required, because of significant increase in estrogen or because of follicular growth detected on ultrasound, LH and progesterone were measured. None of the patients experienced a normal ovulation. Four patients (1 control, 1 from group 2a, and 2 from group 3a) had a significant increase in estrogen levels and had a follicular image on ultrasounds. One of them (group 3a) had a follicular rupture, but none had a LH surge or increase in progesterone.(ABSTRACT TRUNCATED AT 250 WORDS)


Comparative effects of different methods of anastomosis on rat uterine horn. This study compares the results of microsurgical uterine horn anastomosis performed using fibrin glue (FK-1), 8/0 Prolene sutures, or fibrin glue in combination with 8/0 Prolene sutures. Thirty-two rats were divided into three groups. Three results were studied: frequency of adhesion formation in the area of anastomosis, uterine horn patency, and pregnancy rates. Compared with microsurgical sutures, fibrin glue used for the anastomosis of uterine horns in rats results in identical pregnancy and patency rates. Moreover, the formation of adhesions was significantly lower and the duration of surgery was shortened.


Predictive value of sperm hyperactivation measurements based on the dilution effect method in clinical in vitro fertilization. Sperm hyperactivation motility characterized by wide oscillatory movements of the sperm head, nonlinear directions, and rapid motility with occasional star-shaped pattern of movement was measured during routine semen analyses prior to an in vitro fertilization procedure. The method consisted of diluting liquefied semen 1:20 with Ham's F-10 supplemented with processed human cord sera followed by incubation at 37 degrees C for 30 minutes. At the end of the incubation period, aliquots of semen samples were evaluated by phase contrast microscopy for sperm hyperactivation. The results indicated that (1) sperm samples exhibiting 15% or more hyperactive motility were associated with a significantly higher percent fertilization of oocytes during the IVF procedure; (2) sperm hyperactive motility was correlated to sperm fertilizability during IVF treatment cycles.


Spermidine/spermine N1-acetyltransferase, a new biochemical marker for epithelial proliferation in rat bladder. We examined the activity of spermidine/spermine N1-acetyltransferase (SAT), a rate-limiting enzyme of the biodegradation of polyamines, in N-butyl-N-(4-hydroxybutyl)nitrosamine-induced transitional cell carcinoma (TCC) and melamine-induced papillomatosis of rat bladder, and compared the activity to that of ornithine decarboxylase (ODC). Both activities were higher in both lesions than in control rats. The difference between SAT and ODC activities in cancerous tissue and papillomatosis was not significant. Cells stained for proliferating cell nuclear antigen (PCNA) were abundant in papillomatosis. TCC had areas with much PCNA. The results indicated that an elevation of SAT activity occurs in both reversible and irreversible proliferation of bladder epithelium and could be important in bladder carcinogenesis.


Determination of diphenylmethane antihistaminic drugs and their analogues in body fluids by gas chromatography with surface ionization detection. Eleven diphenylmethane antihistaminic drugs and their analogues were tested for their detection by capillary gas chromatography (GC) with surface ionization detection (SID). The GC-SID response was highest for doxylamine, diphenhydramine and orphenadrine and lowest for terodiline, clemastine and pipethanate. The detection limits for drugs with the highest response were 2-5 pg (ca. 6-20 fmol) on-column (100-250 pg/ml of body fluid). The detection limits with GC-SID were 10-100 times higher than those with GC with nitrogen-phosphorus detection. A detailed procedure for the isolation of the antihistaminics from human whole blood and urine by the use of Sep-Pak C18 cartridges, prior to GC-SID, is also presented. The recoveries of the drugs (50 or 500 pmol), which had been added to 1 ml of body fluids, were > 60%. The baselines remained steady as the column temperature was increased and the background was clean, especially for whole blood extracts.


Influence of transcutaneous electrical nerve stimulation on memory in patients with dementia of the Alzheimer type. This study examined the effect of transcutaneous electrical nerve stimulation (TENS) on memory in patients with dementia of the Alzheimer type. It was hypothesized that, in the early stage of the illness, electrical stimulation could activate the affected cortical regions by stimulating the neurotransmitter systems projecting to these areas. The results reveal that electrical stimulation improves the verbal long-term memory in these patients. Moreover, verbal fluency improves more in patients who received electrical stimulation than in patients who received control treatment. However, electrical stimulation does not influence the visual long-term memory of the patients, nor does it affect their verbal and nonverbal short-term memory. Underlying theoretical mechanisms are discussed.


Use of randomly amplified polymorphic DNA markers to distinguish isolates of Aspergillus fumigatus. Forty-four oligonucleotide decamers were tested for their abilities to generate randomly amplified polymorphic DNA (RAPD) markers from genomic DNAs of three different isolates of Aspergillus fumigatus. Seven primers generated RAPDs that allowed the three isolates to be differentiated; one of the primers also yielded a unique RAPD pattern in each of an additional six fungal isolates, demonstrating the utility of this technique for distinguishing between A. fumigatus isolates.


Epidemiology of nosocomial acquisition of Candida lusitaniae. Candida species are important nosocomial pathogens; however, little is known about the epidemiology of Candida lusitaniae, an organism frequently resistant to amphotericin B. We evaluated 98 patients admitted to the bone marrow transplant and medical intensive care units of a tertiary-care hospital. Each patient with C. lusitaniae was matched with control patients. Restriction fragment analysis of DNA was used to determine strain relatedness. Seven patients (7.1%) with C. lusitaniae were identified; five acquired C. lusitaniae after admission to the study unit. All isolates were susceptible to amphotericin B. There were no differences between patients and controls with regard to duration of stay in the study unit, antibiotic administration, antifungal therapy, immunosuppressive therapy, catheter use, or underlying disease. Temporal and geographic clustering of five patients with identical strains occurred. No common source was identified. Restriction fragment analysis revealed a total of eight strains, and five patients shared one strain type. These results demonstrate exogenous acquisition of C. lusitaniae. The mechanism of acquisition is probably indirect contact transmission between patients.


Mycobacteria in Crohn's disease: DNA probes identify the wood pigeon strain of Mycobacterium avium and Mycobacterium paratuberculosis from human tissue. Mycobacterium paratuberculosis is known to cause Johne's disease, a granulomatous ileitis in ruminants, and may be involved in some cases of Crohn's disease. Like M. paratuberculosis, the wood pigeon strain of Mycobacterium avium may also show mycobactin dependence on primary isolation that is attenuated on further subculturing. A wood pigeon strain, M. avium restriction fragment length polymorphism (RFLP) type A/I, is also capable of causing granulomatous ileitis in experimental animal models but is not known to cause disease in humans. M. avium RFLP type A is associated with disease in immunocompromised hosts. Three DNA probes, pMB22 and the two subclones pMB22/S4 and pMB/S12, were found to be capable of distinguishing among M. paratuberculosis, M. avium type A, and M. avium type A/I (wood pigeon strain) on the basis of RFLPs. These DNA probes were used to identify two mycobacterial isolates (M. paratuberculosis and M. avium type A/I, wood pigeon strain) derived from the intestinal tissues of two patients with Crohn's disease. In addition, the wood pigeon strain of M. avium was identified from a patient with ulcerative colitis, and M. avium RFLP type A was identified from a patient with colonic carcinoma. This is the first time that M. avium A/I (wood pigeon strain) is known to have been isolated from human tissue. There are too few isolates to speculate about the etiological significance of mycobacteria and inflammatory bowel disease, but it is reasonable to conjecture that M. paratuberculosis may be responsible for some cases of Crohn's disease and that the wood pigeon strain of M. avium may also be an inflammatory bowel disease pathogen in humans.


Detection of mRNA by in situ hybridisation and in northern blot analysis using oligodeoxynucleotide probes labelled with alkaline phosphatase. To assess whether a reduction in intensity of signal observed using an alkaline phosphatase labelled oligodeoxynucleotide probe could be explained on the basis of procedural steps rather than reduced sensitivity. Signal intensity was assessed on in situ hybridisation for pro-opiomelanocortin (POMC) mRNA in rat pituitary and for somatostatin mRNA in human pancreas and in northern blot analysis for POMC mRNA in the presence and absence of formamide. The direct effects of formamide on the alkaline phosphatase detection step were assessed using histochemical enzyme detection in rat kidney. All signals were reduced in systems containing formamide. In the absence of formamide clear, strong signals for specific mRNAs can be obtained by in situ hybridisation and northern blot analysis using oligodeoxynucleotide probes directly labelled with alkaline phosphatase. Formamide seems to inhibit the activity of alkaline phosphatase.


Directional atherectomy for treatment of restenosis within coronary stents: clinical, angiographic and histologic results. The safety and long-term results of directional coronary atherectomy in stented coronary arteries were determined. In addition, tissue studies were performed to characterize the development of restenosis. Directional coronary atherectomy was performed in restenosed stents in nine patients (10 procedures) 82 to 1,179 days after stenting. The tissue was assessed for histologic features of restenosis, smooth muscle cell phenotype, markers of cell proliferation and cell density. A control (no stenting) group consisted of 13 patients treated with directional coronary atherectomy for restenosis 14 to 597 days after coronary angioplasty, directional coronary atherectomy or laser intervention. Directional coronary atherectomy procedures within the stent were technically successful with results similar to those of the initial stenting procedure (2.31 +/- 0.38 vs. 2.44 +/- 0.35 mm). Of five patients with angiographic follow-up, three had restenosis requiring reintervention (surgery in two and repeat atherectomy followed by laser angioplasty in one). Intimal hyperplasia was identified in 80% of specimens after stenting and in 77% after coronary angioplasty or atherectomy. In three patients with stenting, 70% to 76% of the intimal cells showed morphologic features of a contractile phenotype by electron microscopy 47 to 185 days after coronary intervention. Evidence of ongoing proliferation (proliferating cell nuclear antigen antibody studies) was absent in all specimens studied. Although wide individual variability was present in the maximal cell density of the intimal hyperplasia, there was a trend toward a reduction in cell density over time. Although atherectomy is feasible for the treatment of restenosis in stented coronary arteries and initial results are excellent, recurrence of restenosis is common. Intimal hyperplasia is a nonspecific response to injury regardless of the device used and accounts for about 80% of cases of restenosis. Smooth muscle cell proliferation and phenotypic modulation toward a contractile phenotype are early events and largely completed by the time of clinical presentation of restenosis. Restenotic lesions may be predominantly cellular, matrix or a combination at a particular time after a coronary procedure.


Silymarin protects against paracetamol-induced lipid peroxidation and liver damage. The effect of silymarin on liver damage induced by acetaminophen (APAP) intoxication was studied. Wistar male rats pretreated (72 h) with 3-methylcholanthrene (3-MC) (20 mg kg-1 body wt. i.p.) were divided into three groups: animals in group 1 were treated with acetaminophen (APAP) (500 mg kg-1 body wt. p.o.), group 2 consisted of animals that received APAP plus silymarin (200 mg kg-1 body wt. p.o.) 24 h before APAP, and rats in group 3 (control) received the equivalent amount of the vehicles. Animals were sacrificed at different times after APAP administration. Reduced glutathione (GSH), lipid peroxidation and glycogen were measured in liver and alkaline phosphatase (AP), gamma-glutamyl transpeptidase (GGTP) and glutamic pyruvic transaminase (GPT) activities were measured in serum. After APAP intoxication, GSH and glycogen decreased very fast (1 h) and remained low for 6 h. Lipid peroxidation increased three times over the control 4 and 6 h after APAP treatment. Enzyme activities increased 18 h after intoxication. In the group receiving APAP plus silymarin, levels of lipid peroxidation and serum enzyme activities remained within the control values at any time studied. The fall in GSH was not prevented by silymarin, but glycogen was restored at 18 h. It was concluded that silymarin can protect against APAP intoxication through its antioxidant properties, possibly acting as a free-radical scavenger.


Investigation of peroxisomal lipid beta-oxidation enzymes in guinea pig liver peroxisomes by immunoblotting and immunocytochemistry. We investigated the immunoreactivity of the peroxisomal lipid beta-oxidation enzymes acyl-CoA oxidase, trifunctional protein, and thiolase in guinea pig liver and compared it with that of homologous proteins in rat, using immunoblotting of highly purified peroxisomal fractions and monospecific antibodies to rat proteins. In addition, the immunocytochemical localization of beta-oxidation enzymes in guinea pig liver was compared with that of catalase. All antibodies showed crossreactivity between the two species, indicating that these peroxisomal proteins have been well conserved, although all exhibited some differences with respect to molecular size and, in the case of acyl-CoA oxidase, in frequency of the immunoreactive bands. In the latter case, a distinct second band in the 70 KD range was observed in guinea pig, in addition to the regular band due to subunit A present in rat liver. This novel band could be due either to trihydroxycoprostanoyl-CoA oxidase or to the non-inducible branched chain fatty acid oxidase described recently. All three beta-oxidation enzymes were immunolocalized by light and electron microscopy to the matrix of peroxisomes, in contrast to catalase, which is also found in the cytoplasm and the nucleus of hepatocytes in guinea pig liver.


A new fluorescence method for alkaline phosphatase histochemistry. We have developed a new fluorescence method for the histochemical localization of alkaline phosphatase activity. Calcium phosphate deposited at the sites of alkaline phosphatase activity in a Gomori-type reaction are identified by calcium binding fluorochromes. The calcium binding fluorochromes calcein, calcein blue, and xylenol orange were investigated, with each fluorochrome being included in the alkaline phosphatase incubating medium and used in a single-step procedure. Alkaline phosphatase activity was studied in freeze-substituted, resin-embedded human liver and jejunal biopsies, and each fluorochrome produced intense fluorescence of different colors at sites of alkaline phosphatase activity. Calcein, calcein blue, and xylenol orange produced green, blue, and red fluorescence, respectively. Sites of enzyme activity were accurately localized without evidence of diffusion, and there was an absence of non-enzyme-catalyzed binding of any of the fluorochromes to tissue. This fluorescence method, which is particularly suited to investigating the localization and distribution of the activity of different enzymes in the same section, was used to investigate the distribution and co-localization of alkaline phosphatase and aminopeptidase M in human liver and jejunum.


The use of unfixed cryostat sections for electron microscopic study of D-amino acid oxidase activity in rat liver. Unfixed cryostat sections of rat liver were incubated to demonstrate D-amino acid oxidase activity at the ultrastructural level. Incubation was performed by mounting the sections on a semipermeable membrane which was stretched over a gelled incubation medium containing D-proline as substrate and cerium ions as capture reagent for hydrogen peroxide. After an incubation period of 30 min, ultrastructural morphology was retained to such an extent that the final reaction product could be localized in peroxisomes, whereas the crystalline core remained unstained. Control incubations were performed in the absence of substrate; the lack of final reaction product in peroxisomes indicated the specificity of the reaction. We conclude that the semipermeable membrane technique opens new perspectives for localization of enzyme activities at the ultrastructural level without prior tissue fixation, thus enabling localization of the activity of soluble and/or labile enzymes.


Reduction of cardiovascular side effects associated with ocular administration of metipranolol by inclusion in polymeric nanocapsules. A new formulation of metipranolol base for ophthalmic administration was developed consisting of a colloidal suspension of polyepsiloncaprolactone nanocapsules with an oily core (Migliol 840) in which the drug is dissolved. Physicochemical properties of the nanocapsules show that the polymer coating around the oily droplets causes an important reduction of the droplet size, with no significant modification of the surface charge noted. When this formulation was administered to rabbits, a reduction of intraocular pressure similar to that seen with commercial eye drops was observed. Nevertheless, the evaluation of the cardiovascular side effects clearly showed lower conjunctival absorption of the encapsulated drug compared with the commercial drops. The direct (bradycardia) and the indirect evaluation (based on the study of the influence on the hypotensive and positive chronotropic effects of isoprenaline) showed that blockage of beta-adrenoreceptors was reduced greatly by the topical administration of the new formulation.


A comparison of models used for calculation of RFLP pattern frequencies. In recent years the application of DNA typing information to criminal investigations has gained widespread acceptance. The primary method currently in use relies on length variation of DNA restriction fragments between individuals. These variations are identified using variable number tandem repeat (VNTR) DNA probes. As this technology becomes more widely used, it is crucial that scientifically valid methods of interpreting the significance of a DNA typing result be adopted. The method chosen should not only give a reliable approximation of the statistical likelihood of a particular RFLP pattern occurring, but should also be easy to present and for the court to understand. In this manuscript five methods of calculating a frequency of occurrence of a RFLP pattern will be presented (fixed bin genotype, floating bin phenotype, floating bin genotype, National Research Council (NRC) method using fixed bins and the NRC method using floating bins). The calculations discussed here demonstrated that the fixed bin genotype method produces a frequency very similar to that obtained from floating bin phenotypes. In addition, regardless of the method chosen or the database size, the frequency of any particular banding pattern in the population over several loci was found to be very rare.


Studies on Formosan soft corals. II. Cytotoxic cembranolides from the soft coral Lobophytum michaelae. Bioactivity-guided fractionation of a CHCl3 extract of the soft coral Lobophytum michaelae afforded a new cytotoxic cembranolide, lobomichaolide (1), and a known cytotoxic cembranolide, crassolide (2). The structure of 1 was determined by spectral and X-ray crystallographic analysis.


Proliferating cell nuclear antigen (PCNA) expression in Hodgkin's disease. Previous studies of the proliferating cell fraction in Hodgkin's disease (HD) have been directed towards the classical Hodgkin and Reed-Sternberg cells (HRS) to the exclusion of the background population and have not included cases of nodular lymphocyte predominant Hodgkin's disease (NLPHD). Using an antibody to proliferating cell nuclear antigen (PCNA), we have determined the growth fraction of HRS cells and L&H cells in paraffin sections of 15 cases of classical HD [12 nodular sclerosis (NS), 3 mixed cellularity (MC)] and eight cases of NLPHD. By double staining with anti-PCNA and antibodies to B cells (CD20) and T cells (CD45RO), we also determined the growth fraction and immunophenotype of the background population in each case. In classical HD, 50.4 per cent of HRS cells were PCNA-positive and judged to be proliferating, which is comparable to previous studies, while in NLPHD 76.9 per cent of L&H cells were PCNA-positive. In both classical HD and NLPHD, the majority of PCNA-positive cells in the background were T cells, which showed a growth fraction of 57.8 and 68.5 per cent, respectively; in comparison, only 4 per cent of B cells were PCNA-positive in each type of HD. L&H cells are widely accepted to be B cells and there is growing evidence that HRS cells are also B cell-derived. Our results underline a relationship between classical HD and NLPHD and suggest that the characteristic histological features of both diseases may be caused by the production and release of cytokines from altered B cells.


Mitotic indices, anti-PCNA immunostaining, and AgNORs in thick cutaneous melanomas displaying paradoxical behaviour. Those melanomas which fail to behave as expected from their Breslow thickness provide interesting material for study. In an attempt to explain differences in behaviour, we have evaluated three distinct proliferative markers in 23 thick melanomas which failed to metastasize and in 20 well-matched control tumours with documented metastasis. The test group demonstrated significantly greater numbers of mitoses when expressed as an index (mitoses per 1000 cells), whilst no difference was found when the results were expressed in terms of mitoses per unit area. Tumours showing epidermal ulceration possessed higher mitotic indices than those of non-ulcerated lesions. High fractions of PCNA immunolabelling combined with low mitotic indices were observed frequently in the non-metastasizing group. This result and its possible relation to survival advantage are discussed in detail. Both AgNOR numbers and patterns failed to act as prognostic variables--indeed, AgNORs failed to correlate with the other proliferative indices, suggesting that their value as a marker of tumour growth is severely limited.


Diagnostic usefulness of dipeptidyl aminopeptidase IV monoclonal antibody in paraffin-embedded thyroid follicular tumours. Monoclonal antibodies to dipeptidyl aminopeptidase IV (DAP IV, EC 3.4.14.5) were raised and selectively applied to paraffin-embedded sections of thyroid carcinoma. Five monoclonal antibodies were found to stain paraffin sections of thyroid carcinomas. Using one of these antibodies (44-4), we studied retrospectively aberrant expression of DAP IV in thyroid carcinoma to determine whether immunohistochemical staining with DAP IV antibody is useful in pathological diagnosis. In almost all cases of thyroid follicular and papillary carcinoma, tumour cells were positive (99.0 per cent) with DAP IV, whereas the cases of follicular adenoma showed a low incidence (27.1 per cent) of positive staining. Follicular adenoma with incomplete capsular invasion had a higher positive incidence (50 per cent) than follicular adenoma without incomplete capsular invasion (9.6 per cent). In positive staining cases previously diagnosed as benign tumours, 11 benign cases reacting positively with DAP IV were rediagnosed as carcinoma after re-examination of more thyroid paraffin block sections or serial sections. These findings suggest that DAP IV monoclonal antibody is very useful in distinguishing thyroid follicular carcinoma from follicular adenoma.


Preventive medicine for HIV-infected patients: an analysis of isoniazid prophylaxis for tuberculin reactors and for anergic patients. To analyze the policies of isoniazid prophylaxis for human immunodeficiency virus (HIV)-infected tuberculin reactors and for HIV-infected anergic patients with unknown tuberculin status. Transition-state model of clinical immune deterioration of HIV-infection over ten years, review of published data, and a survey of AIDS experts. Outcome measures are the numbers of tuberculosis cases and deaths prevented and isoniazid toxicity cases and deaths occurring with prophylaxis. Hypothetical cohorts of HIV-infected 40-year-olds. Because the tuberculosis activation rate is so high in HIV-infected patients, the benefits of prophylaxis far outweigh the risks of isoniazid toxicity for tuberculin reactors with HIV infection at any stage of immune function: 1,469-2,868 tuberculosis cases and 170-274 deaths are prevented per 10,000 cohort over ten years, depending upon the cohort's initial immune state. The benefits of prophylaxis outweigh the risks of isoniazid toxicity for anergic HIV-infected patients if they come from a community with a 2% to 3% or greater prevalence of Mycobacterium tuberculosis infection. Isoniazid prophylaxis is a reasonable prevention measure for HIV-infected tuberculin reactors and for many HIV-infected anergic patients.


In situ hybridization demonstrates the stability of mRNA in post-mortem rat tissues. In situ hybridization was used to detect messenger RNA (mRNA) in a variety of rat tissues which were fixed in formalin either immediately after death or after a 24 h period of storage at 5 degrees C. A synthetic polydeoxythymidine [poly d(T)] oligonucleotide probe was used to demonstrate polyadenylated [poly (A)] mRNA in the small intestine, pancreas, liver, cerebellum, and pituitary. Of these tissues, only the liver showed a small reproducible reduction in hybridization signal following delayed fixation. Synthetic oligonucleotide probes complementary to albumin and pro-opiomelanocortin (POMC) mRNAs were hybridized to liver and pituitary, respectively. There was no significant reduction in hybridization signal in post-mortem tissues. The results suggest that some mRNAs may be remarkably stable under certain post-mortem conditions and this should encourage the wider application of in situ hybridization techniques to post-mortem material.


Proliferative activity determined by DNA flow cytometry and proliferating cell nuclear antigen (PCNA) immunohistochemistry as a prognostic factor in prostatic carcinoma. Proliferative activity was measured in 165 paraffin-embedded prostatic carcinomas using DNA flow cytometric analysis of the S-phase (SPF) and G2/M-phase fractions and CAS 200 image analysis of the proliferating cell nuclear antigen (PCNA) expression defined immunohistochemically by PC10 and 19A2 monoclonal antibodies. No significant associations were found between the flow cytometric and the two immunohistochemical measures of cell proliferation. Of the four indices, only SPF, S + G2/M, and immunostaining with 19A2 antibody were associated with the poor histological grade of the tumour. High SPF and S + G2/M were significantly associated with poor 10-year overall survival (P < 0.001) and prostatic carcinoma-specific survival (P < 0.01). Multivariate analyses of prostatic carcinoma-specific survival in patients with non-metastatic disease (M0-stage) indicated that only S + G2/M, T-stage, and histological grade (only if re-evaluated by a single pathologist) had independent prognostic significance. High-level PCNA staining (> 16 per cent of cells stained) with 19A2 antibody was associated with poor prognosis only in univariate analysis, and PC10 immunostaining had no prognostic value. In conclusion, a high proliferative activity as defined by flow cytometric S+G2/M is an independent predictor of poor survival in patients with non-metastatic prostatic carcinoma. PCNA immunostaining from formalin-fixed, paraffin-embedded prostatic carcinomas has little, if any, prognostic value.


Immunoreactivity of proliferating cell nuclear antigen compared with bromodeoxyuridine incorporation in normal and neoplastic rat tissue. Monoclonal antibodies (MoAbs) against proliferating cell nuclear antigen (PCNA) represent a potentially useful tool for cell kinetic analysis of tumours. Because in paraffin-embedded tissue the relationship between PCNA immunoreactivity and tumour cell proliferation is not well characterized, we have compared PCNA positivity as detected by the PC10 MoAb with the bromodeoxyuridine labelling index (BrdUrd-LI) in two different transplantable hormone-dependent rat mammary tumours. Together, these two tumour models (MCR-83 and EMR-86) cover a wide range of S-phase fractions. Evaluating 31 methacarn-fixed tumours, a strong but non-linear relationship (r = 0.98) was obtained. PCNA-positive fractions were invariably higher than corresponding BrdUrd-LIs and also higher than the estimated growth faction: growth fractions as determined by continuous BrdUrd labelling of the tumour and stromal cell population in EMR-86 carcinomas were 12 and 26 per cent lower than PCNA-positive fractions, implying that a certain fraction of non-cycling cells can also express PCNA. A dramatic disturbance in the relationship of PCNA positivity and the BrdUrd-LI was observed in the EMR-86 model after growth arrest induced by hormonal ablation: PCNA immunoreactivity remained detectable for at least 3 days, whereas the BrdUrd-LI decreased almost immediately. In comparison, PCNA immunoreactivity persisted for a much shorter period in small intestinal cells that had stopped DNA replication when moving from the crypt towards the villus. It is concluded that although differences in PCNA expression exist between various tissues, PCNA as detected by the PC10 MoAb may be used in tumours as an operational marker for the growth fraction.(ABSTRACT TRUNCATED AT 250 WORDS)