Differing actions of beta-(2-thienyl)-gamma-aminobutyric acid in central and peripheral preparations. In the guinea-pig isolated ileum, beta-(2-thienyl)-gamma-aminobutyric acid (BTG; 100-500 microM) reversibly and competitively (pA2 = 4.3 +/- 0.1) antagonised the baclofen-induced (5-100 microM) depression of cholinergic twitch contractions, but not that to adenosine or morphine. By contrast, in rat neocortical slice preparations, BTG (100-500 microM) acted as an agonist, abolishing the frequency and amplitude of spontaneous discharges, sensitive to 2-hydroxysaclofen (100-500 microM). BTG exhibits differential actions at GABAB receptors in brain and periphery.


Effects of naloxone infusion upon spontaneous and amphetamine-induced activity. Naloxone-filled (1.0 mg/kg per h) or sham pumps were implanted subcutaneously (s.c.) in rats on day 0 and removed on day 7. Spontaneous locomotor activity was lower in naloxone-infused rats on day 3 only. Cumulative dose-response curves to D-amphetamine were constructed in separate groups on day 6 or day 8 (0.0-6.4 mg/kg s.c.). On day 6, naloxone infusion attenuated the gross movement response to D-amphetamine, but not the fine movement response. There were no differences in the response of naloxone- or sham-infused rats to D-amphetamine on day 8. Therefore, withdrawal from continuous naloxone infusion had no influence upon spontaneous or D-amphetamine-stimulated activity.


The molecular mechanism of central analgesia induced by morphine or carbachol and the L-arginine-nitric oxide-cGMP pathway. The role of the L-arginine-NO-cGMP pathway in morphine-induced central analgesia was investigated in two nociceptive tests: PGE2-induced hind paw hyperalgesia and tail-flick. The central analgesic effect of morphine was potentiated by MY5445, a specific cGMP phosphodiesterase inhibitor. I.c.v. injections of morphine or carbachol caused dose-dependent analgesia, which was prevented by methylene blue, an inhibitor of guanylate cyclase. The NO synthase inhibitor, N-iminoethyl-L-ornithine, prevented carbachol-induced analgesia, but did not affect morphine-induced analgesia. Our results suggest that activation of cGMP may underlies analgesia induced by morphine and carbachol. The activation of guanylate cyclase by carbachol seems to depend on the L-arginine-NO pathway, but that caused by morphine remains to be further characterized.


The effect of nedocromil sodium on the isolated rabbit vagus nerve. Nedocromil sodium depolarized the isolated rabbit vagus nerve. The depolarization was blocked by DIDS (4,4'-diisothiocyanostilbene-2,2'-disulphonic acid) and did not occur when the nerve was bathed in solutions low in chloride. After depolarization the nerve was refractory to nedocromil sodium for an hour. It is suggested that nedocromil sodium can affect a chloride channel.


Selective 5-HT1A and 5-HT2 receptor-mediated adrenocorticotropin release in the rat: effect of repeated antidepressant treatments. The 5-HT receptor agonists, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) and 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) produced dose-dependent increases in plasma adrenocorticotropin (ACTH) in the male rat by activation of 5-HT1A and 5-HT2 receptors respectively. The ACTH response to DOI was enhanced by repeated administration of electroconvulsive shock (five over 10 days) but abolished by the tricyclic antidepressant, amitriptyline (20 mg/kg for 14 days). In contrast 21 days lithium treatment failed to alter DOI-induced ACTH release. Neither repeated electroconvulsive shock, nor amitriptyline, nor lithium altered the ACTH response to 8-OH-DPAT. These data are consistent with results from ligand binding and behavioural studies which suggest that the sensitivity of brain 5-HT2 receptors is increased by repeated electroconvulsive shock but attenuated by tricyclic antidepressant treatment. In contrast, our data suggest that the antidepressant treatments studied do not alter the sensitivity of the 5-HT1A receptors involved in ACTH release.


Characterization of alpha 1-adrenoceptor subtypes labeled by [3H]prazosin in single cells prepared from rabbit thoracic aorta. alpha 1-Adrenoceptor agents with alpha 1-adrenoceptor subtypes sensitive and insensitive to inactivation by chloroethylclonidine were characterized in single cells prepared from rabbit thoracic aorta. WB 4101, 5-methylurapidil and spiperone interacted with high- and low-affinity sites labeled by [3H]prazosin. Chloroethylclonidine 10 microM pretreatment eliminated the low-affinity sites of displacement curves obtained with WB 4101 and 5-methylurapidil but had no effect on the high-affinity site for these agents. The treatment also reduced the site of the displacement curve obtained with spiperone but eliminated only the high-affinity site. Methoxamine and clonidine, alpha-adrenoceptor agonists, interacted with binding sites labeled by [3H]prazosin. The displacement curve for methoxamine was not affected by chloroethylclonidine 10 microM pretreatment, while that for clonidine was partially eliminated by the same type of pretreatment. These results suggest that, in single cells prepared from rabbit thoracic aorta: (1) WB 4101, 5-methylurapidil and spiperone interact with differing affinity at sites labeled by [3H]prazosin; (2) chloroethylclonidine-sensitive and -insensitive [3H]prazosin binding sites correspond to those with low- and high-affinity sites for WB 4101 and 5-methylurapidil, and a high- and low-affinity for spiperone, respectively; and (3) chloroethylclonidine treatment was shown to have no effect on the displacement curve of methoxamine but a partial effect on that of clonidine.


Effects of Na+ transport inhibitors on guinea-pig tracheal responses to spasmogens. The effects of ouabain, amiloride, K(+)-free solution and low Na+ (25 mM) solution on the responses to CaCl2 (in Ca(2+)-free, K(+)-depolarizing solution), KCl, acetylcholine, histamine and 5-hydroxytryptamine were studied in guinea-pig isolated trachea. Ouabain (10 microM) did not alter the contractile responses to CaCl2, KCl and acetylcholine but depressed those to histamine and 5-hydroxytryptamine produced in normal Ca2+ (2.5 mM) and Ca(2+)-free (EGTA 0.1 mM) media. Amiloride (0.1 mM), K(+)-free solution, and low Na+ solution depressed responses to acetylcholine, histamine and 5-hydroxytryptamine produced in normal Ca2+ and Ca(2+)-free media. Ouabain and amiloride had no effect on responses of skinned strips to Ca2+. The mechanism of the inhibitory effects of these interventions is uncertain but the findings suggest that the availability of Na+ influences the airway smooth muscle responses to spasmogens.


Ouabain-induced excitation of colonic smooth muscle due to block of K+ conductance by intracellular Na+ ions. The mechanism by which ouabain causes excitation of canine colonic circular smooth muscle was investigated. Ouabain-induced depolarization and increase in contractility were related to the concentration of extracellular sodium and prevented by complete substitution of sodium ions with N-methyl-D-glucamine or lithium ions. Absence of external sodium ions did not prevent the depolarization and increase in contractility induced by tetraethylammonium. Exposure of the muscle strips to sodium-free solutions produced a transient hyperpolarization and decrease in the input membrane resistance consistent with the hypothesis that intracellular sodium blocks potassium conductance. The relationship between the membrane potential and the extracellular potassium concentration indicated that the resting membrane potential is mainly determined by the membrane potassium conductance. Our data suggest the following mechanism of action for ouabain: (a) ouabain blocks Na+/K+ pump thereby increasing the intracellular sodium concentration; (b) increase in intracellular sodium inhibits membrane potassium conductance, which depolarizes the membrane and prolongs the slow wave plateau, resulting in an increase of the force of contraction. The direct contribution of the sodium pump to the resting membrane potential, if any, can only be minor (< 6 mV).


Effect of desipramine and amphetamine on noradrenergic neurotransmission: electrophysiological studies in the rat brain. The present electrophysiological experiments were undertaken to investigate the effect of desipramine and d-amphetamine on noradrenergic neurotransmission in the rat central nervous system. The effectiveness of electrical stimulation of the locus coeruleus and of microiontophoretic application of norepinephrine (NE) in suppressing the firing activity of CA3 pyramidal neurons was studied in the dorsal hippocampus. Desipramine (0.5 and 5 mg/kg i.v.) and d-amphetamine (0.25 and 5 mg/kg i.v.) decreased the effectiveness of locus coeruleus stimulation and prolonged the effect of microiontophoretically applied NE on the same pyramidal neurons. Subsequent i.v. administration of idazoxan, an alpha 2-adrenoceptor antagonist, reversed the effects of desipramine and d-amphetamine on the effectiveness of locus coeruleus stimulation and decreased that of microiontophoretically applied NE. In addition, idazoxan prevented the effect of subsequent administration of desipramine (5 mg/kg i.v.) on the effectiveness of locus coeruleus stimulation. High doses of d-amphetamine (5 and 10 mg/kg i.v.) decreased the firing activity of hippocampus pyramidal neurons by 70 and 98%, respectively, whereas low doses of desipramine (0.5 mg/kg i.v.) or of d-amphetamine (0.25 mg/kg i.v.) were without effect. After lesioning of NE projections with 6-hydroxydopamine, the effect of the 5 mg/kg dose of d-amphetamine on the firing activity of hippocampus pyramidal neurons was markedly reduced, whereas the cumulative 10 mg/kg dose of d-amphetamine completely suppressed, as in control rats, the firing activity of these neurons. This effect of d-amphetamine in 6-hydroxydopamine-pretreated rats was reversed by the administration of the 5-HT1A receptor antagonist BMY 7378. These data provide evidence that acute administration of desipramine and d-amphetamine decreases the effectiveness of locus coeruleus stimulation by increasing the activation of terminal alpha 2-adrenoceptor autoreceptors. In addition, acute administration of high doses of d-amphetamine decreases the firing rate of hippocampus pyramidal neurons by increasing NE and serotonin release.


Effects of endothelin on cytosolic Ca2+ level and mechanical activity in rat uterine smooth muscle. The effects of endothelin (ET) on cytosolic Ca2+ level ([Ca2+]i) and mechanical activity were examined in isolated rat uterine smooth muscle. ET-1, ET-2, ET-3 and sarafotoxin S6b (STX) induced rhythmic contractions superimposed on an increased muscle tone. The concentration needed to induce a half-maximum contraction (EC50) was 1.6-3.3 nM for ET-1, ET-2 and STX and higher than 200 nM for ET-3, suggesting that the ET(A) receptor is responsible for these contractions. The sensitivity to ET-1 of uterus at day 20 of gestation was higher than that of non-pregnant rat uterus. Contraction induced by ET-1 followed an increase in [Ca2+]i. The relation between [Ca2+]i and muscle tension, an an indicator of Ca2+ sensitivity of contractile elements, in the presence of ET-1 was identical to that in the presence of high K+ in non-pregnant and pregnant rat uteri. The ET-1-induced increases in [Ca2+]i and muscle tension were strongly inhibited by verapamil in non-pregnant rat uterus. In pregnant rat uterus, however, verapamil only partially inhibited the increases. The verapamil-insensitive portions of [Ca2+]i and contraction were inhibited by EGTA. In the absence of external Ca2+, ET changed neither [Ca2+]i nor muscle tension. These results suggest that ET-1 acts on ET(A) receptors, increase [Ca2+]i and induces contraction without changing Ca2+ sensitivity of contractile elements. The increase in [Ca2+]i seemed to be mediated by opening of L-type Ca2+ channels in non-pregnant rat uterus and also of non-L-type Ca2+ channels in pregnant rat uterus, but not by Ca2+ release from intracellular storage sites.


Vascular smooth muscle cell detachment from elastin and migration through elastic laminae is promoted by chondroitin sulfate-induced "shedding" of the 67-kDa cell surface elastin binding protein. Impaired elastin fiber assembly is observed in the fetal ductus arteriosus (DA), associated with a reduced concentration of elastin binding protein (EBP), a 67-kDa galactolectin. It is also seen in cultured aortic (Ao) smooth muscle cells (SMC) following the release of the EBP by glycosaminoglycans rich in N-acetylgalactosamine, such as chondroitin sulfate (CS). In the DA, impaired elastin fiber assembly is observed in conjunction with intimal thickening associated with increased migration of SMC into the subendothelium, a feature we previously related to increased production of fibronectin. In this report, we determined whether SMC use the EBP to attach to an elastin substrate, whether shedding of the EBP promotes SMC migration through a three-dimensional network of pure elastic laminae prepared from sheep aorta, and whether the latter is associated with increased production of fibronectin. We observed reduced attachment to elastin-coated surfaces of DA SMC deficient in EBP compared to Ao SMC. Addition of CS but not heparan sulfate (a glycosaminoglycan which does not induce EBP shedding) decreased Ao SMC attachment to elastin, as did preincubation with VGVAPG elastin-derived peptides which saturate the EBP. The immunolocalization of cell surface EBP suggested that cells can quickly replace EBP released from their surfaces by CS treatment. The magnitude of CS-induced impaired attachment of SMC to elastin was dose dependent and could be further increased by the administration of cyclohexamide and sodium azide. Also, the reversibility of CS-induced detachment was prevented by monensin. This suggests that a process of new synthesis and intracellular transport of the EBP was necessary to replace the EBP molecules released from the cell surface by CS treatment. In the migration assay, both DA and Ao SMC attached to the top of an elastin membrane, but only DA SMC deficient in EBP migrated through the laminae. Addition of CS, which induced shedding of EBP, resulted in Ao SMC migration associated with increased synthesis of fibronectin. We postulate that CS-induced release of EBP from SMC surfaces causes cell detachment from elastin and an increase in fibronectin synthesis, processes which may be critical in promoting SMC migration associated with intimal thickening developmentally in the DA and perhaps also in vascular disease.


Impairment of the TSH signal transduction system in human thyroid carcinoma cells. In order to further evaluate the role of TSH in the proliferation and the differentiation of human thyroid carcinoma cells, we have analyzed the function of the TSH receptor in the established thyroid carcinoma cell lines NPA and WRO. The TSH signal transduction system in the carcinoma cells was also compared with that in normal thyroid cells. Although unresponsiveness to bovine and human TSH was demonstrated by measurement of cAMP production and [3H]thymidine incorporation after treatment of TSH, cAMP production was induced after stimulation of these cells by forskolin, cholera toxin, and isoproterenol. Specific binding to 125I-TSH was demonstrated in both NPA and WRO cells in addition to the existence of a TSH receptor mRNA and thyroglobulin mRNA species, although thyroid-specific gene expression in these cells was not regulated by TSH. These findings suggest that the unresponsiveness to TSH in these cells may be due to an abnormality of TSH receptor-G protein coupling rather than to a decreased level of TSH-receptor expression or a Gs protein abnormality.


Selective localization of receptors for urokinase amino-terminal fragment at substratum contact sites of an in vitro-established line of human epidermal cells. We have shown the presence of surface receptors for the amino-terminal fragment (ATF) of human urokinase-type plasminogen activator (u-PA) on an in vitro-established cell line of human epidermal origin by both radio-binding assays with human 125I-u-PA-ATF and transmission electron microscopy of a gold-u-PA complex. On the basis of cross-linking experiments with 125I-u-PA-ATF and subsequent autoradiography of the gels we have observed that such receptors are not spontaneously released into the culture medium. The treatment with phosphatidylinositol-specific phospholipase C induces the release of the receptor, which behaves as a glycosyl phosphatidyl inositol(GPI)-anchored protein. Phase-partitioning experiments on cell lysates have shown that the receptor partitions into the detergent phase. By detaching cell monolayers with the chelating agent EDTA we have prepared the cell-substratum contact sites of these cells, which represent only the 3.5% of the surface membrane of monolayered cells. Such plasma membrane remnants are highly selected since they contain about 43% of total u-PA-ATF binding sites. Such binding sites show the same biochemical and morphological characteristics of u-PA-ATF receptors observed in the monolayered cells, thus indicating that u-PA is selectively concentrated at the level of cell-substratum contacts. This is likely to enable directional proteolysis for cell migration and invasion.


Ribonucleotide reductase gene expression during cyclic AMP-induced cell cycle arrest in T lymphocytes. In both 3T3 mouse fibroblasts and S49 mouse T lymphocytes the genes encoding both subunits of ribonucleotide reductase are expressed beginning in late G1 phase. In studies reported here, we compared the expression of the genes that code for the M1 and M2 subunits of ribonucleotide reductase in S49 cells, which are arrested in G1 phase by agents that increase cyclic AMP, with those from CEM human T lymphoma cells that are unaffected by exposure to dibutyryl cyclic AMP. Dibutyryl cyclic AMP treatment results in a prompt steady diminution of M2 mRNA concentration to levels at or below that of elutriated G1 cell-cycle-specific populations in S49 cells, in contrast to CEM cell M2 mRNA, which is unchanged. M1 mRNA concentration decreases more slowly than M2 mRNA in S49 cells and marginally, if at all, in CEM cells. The time course of diminution of the M2 message concentration by dibutyryl cyclic AMP in S49 cells is similar to that obtained when cells are treated with actinomycin D and to the combination of the two agents. This suggests that cyclic AMP and actinomycin D may act similarly on ribonucleotide reductase gene expression. Furthermore, cycloheximide pretreatment diminishes the effect of dibutyryl cyclic AMP, indicating that the effect might be mediated by a labile protein. Transcription runoff assays suggest a diminution of transcription rate for the M2 gene in S49 cells treated with dibutyryl cyclic AMP and a transient decline in the M1 transcription rate. These data suggest that dibutyryl cyclic AMP diminishes the transcription of ribonucleotide reductase genes in sensitive cells and that this and the short half-life of the M2 message are major factors in the disappearance of the M2 messenger RNA from dibutyryl cyclic AMP-treated cells although other mechanisms may also play a role. These events clearly precede any alteration in cell cycle distribution and thus they may contribute to G1 arrest.


The EGF-inducible protein EIP-1 of migrating normal and malignant rat liver epithelial cells is identical to plasminogen activator inhibitor 1 and is a component of the ECM migration tracks. Induction of rat liver epithelial cell migration by epidermal growth factor (EGF) changes the expression pattern of secreted proteins. The expression of the early induced glycoprotein EGF-inducible protein No. 1 (EIP-1) correlates with the migratory behavior of both normal and Ha-ras-transformed, tumorigenic cells and is deposited into the ECM migration tracks. The sequence of two clones from a cDNA library of EGF-induced cells and the amino terminal sequence of the purified protein revealed that EIP-1 is identical to rat plasminogen activator inhibitor 1 (PAI-1). Based on the migration-linked expression pattern of EIP-1/PAI-1 it is proposed that the inhibitor is required for the migration of these cells, but not sufficient to stimulate it.


Endotoxin transduces Ca2+ signaling via platelet-activating factor receptor. Lipopolysaccharide (LPS) is a pathogenic substance causing severe multiple organ failures and high mortality. Although several LPS binding proteins have been identified, the molecular mechanism underlying the LPS signaling pathway still remains obscure. We have found that the LPS-induced Ca2+ increase in platelets and platelet aggregation is blocked by selective platelet-activating factor (PAF) receptor antagonists, thus suggesting a cross-talk between LPS and the PAF receptor. Next, we confirmed this hypothesis using the cloned PAF receptors [(1991) Nature 349, 342-346; (1991) J. Biol. Chem. 266, 20400-20405] expressed in Xenopus oocytes and Chinese hamster ovary (CHO) cells. In both systems, cells responded to LPS only when PAF receptors were expressed, and specific PAF binding was successfully displaced and reversibly dissociated by LPS. PAF receptor activation by LPS may represent a novel important pathway in the pathogenesis of circulatory collapse and systemic thrombosis caused by endotoxin.


Expression of GAP-43 mRNA in the adult mammalian spinal cord under normal conditions and after different types of lesions, with special reference to motoneurons. In situ hybridization histochemistry was used to detect cell bodies expressing mRNA encoding for the phosphoprotein GAP-43 in the lumbosacral spinal cord of the adult rat, cat and monkey under normal conditions and, in the cat and rat, also after different types of lesions. In the normal spinal cord, a large number of neurons throughout the spinal cord gray matter were found to express GAP-43 mRNA. All neurons, both large and small, in the motor nucleus (Rexed's lamina IX) appeared labeled, indicating that both alpha and gamma motoneurons express GAP-43 mRNA under normal conditions. After axotomy by an incision in the ventral funiculus or a transection of ventral roots or peripheral nerves, GAP-43 mRNA was clearly upregulated in axotomized motoneurons, including both alpha and gamma motoneurons. An increase in GAP-43 mRNA expression was already detectable 24 h postoperatively in lumbar motoneurons both after a transection of the sciatic nerve at knee level and after a transection of ventral roots. At this time, a stronger response was seen in the motoneurons which had been subjected to the distal sciatic nerve transection than was apparent for the more proximal ventral root lesion. An upregulation of GAP-43 mRNA could also be found in intact motoneurons located on the side contralateral to the lesion, but only after a peripheral nerve transection, indicating that the concomitant influence of dorsal root afferents may play a role in GAP-43 mRNA regulation. However, a dorsal root transection alone did not seem to have any detectable influence on the expression of GAP-43 mRNA in spinal motoneurons, while the neurons located in the superficial laminae of the dorsal horn responded with an upregulation of GAP-43 mRNA. The presence of high levels of GAP-43 in neurons has been correlated with periods of axonal growth during both development and regeneration. The role for GAP-43 in neurons under normal conditions is not clear, but it may be linked with events underlying remodelling of synaptic relationships or transmitter release. Our findings provide an anatomical substrate to support such a hypothesis in the normal spinal cord, and indicate a potential role for GAP-43 in axon regeneration of the motoneurons, since GAP-43 mRNA levels was strongly upregulated following both peripheral axotomy and axotomy within the spinal cord.(ABSTRACT TRUNCATED AT 400 WORDS)


Evidence that deprotonation of serine-55 is responsible for the pH-dependence of the parvalbumin Eu3+ 7F0-->5D0 spectrum. The Eu(III)7F0-->5D0 excitation spectra of the parvalbumins are highly pH-dependent. Below pH 6.0, they exhibit a sharp, partially resolved doublet centered near 5,795 A. However, as the pH is raised, the spectrum becomes increasingly dominated by a much broader signal near 5,784 A. This behavior has been traced to the Eu(III) ion bound at the CD site, but the identity of the moiety undergoing deprotonation remains uncertain. Site-specific mutagenesis studies on the parvalbumin-like protein known as oncomodulin now suggest that the species in question is a liganding serine hydroxyl group. Specifically, replacement of serine-55 by aspartate (the residue present at the corresponding position in the EF site) affords a protein that retains two functional lanthanide binding sites, but fails to undergo the pH-dependent spectral alteration. By contrast, replacement of aspartate-59 by glycine (the corresponding EF site residue) fails to abolish the pH-dependent behavior.


Thrombin-induced proliferation and expression of platelet-derived growth factor-A chain gene in human vascular smooth muscle cells. Treatment of human vascular smooth muscle cells (SMC) with human alpha-thrombin greatly increased DNA synthesis and cell proliferation. Both the integrity of the catalytic site and that of the anion binding exosite were required for expression of this activity. Experiments employing Northerns indicated induction of c-fos expression as well as a time-dependent induction of platelet-derived growth factor-A (PDGF-A) gene by thrombin. The thrombin mitogenic activity was potentiated by PDGF-BB, insulin and the vasoconstrictor peptide endothelin-1 suggesting synergism by convergence of intracellular growth-promoting signals. SMC treatment with pertussis toxin and forskolin indicated that the mitogenic activity of thrombin may be induced via signal transduction mechanism(s) involving changes in cAMP levels and activation of a Gi-like protein. These results suggest that thrombin may play a functional role in the regulation of human vascular SMC proliferation.


Assignment of the redox potentials to the four haems in Desulfovibrio vulgaris cytochrome c3 by 2D-NMR. Using 2D-NMR the four haems of Desulfovibrio vulgaris (Hildenborough) cytochrome c3 within the X-ray structure were fully cross assigned according to their redox potential. The strategy used was based on a complete network of chemical exchange connectivities between the NMR signals obtained for all oxidation levels to the corresponding ones in the fully reduced spectrum [1992, Eur. J. Biochem., in press]. This unequivocal cross-assignment disagrees with earlier results obtained for the similar protein from Desulfovibrio vulgaris (Miyazaki F.).


The oxygen reactive species of cytochrome-c-oxidase: an alternative view. In a recent review article Babcok and Wikström (Nature, 1992, 356, 301-309) proposed that the species of cytochrome-c-oxidase which binds molecular oxygen during turnover is the so-called mixed valence enzyme, in which the binuclear center cytochrome a3-CuB is reduced, while the cytochrome a/CuA sites are oxidized. This proposal is based on earlier work (Morgan and Wikström, Biochemistry 1991, 30, 948-958) in which it was found that the steady-state reduction levels of cytochrome c and cytochrome a in respiring rat liver mitochondria (sustained by ascorbate and TMPD) are quite different, the latter being much more oxidized than the former; evaluation of the steady-state reduction levels demanded a large correction due to the optical contribution of oxidized TMPD+ which overlaps with the cytochromes. We report below that application of transient spectroscopy and SVD analysis to respiring rat heart myocytes, under conditions in which the contribution of TMPD+ is very small or absent, allows to show that the steady-state reduction levels of cytochrome c and cytochrome a are comparable at all times accessible to measurement in the rapid-scanning stopped-flow spectrophotometer. Our conclusion, in agreement with previous results, is that mixed valence cytochrome-c-oxidase as defined above is not the prevailing oxygen binding species of cytochrome-c-oxidase, unless electron donation to cytochrome c becomes rate limiting.


Cross-linking of apolipoproteins is involved in a loss of the ligand activity of high density lipoprotein upon Cu(2+)-mediated oxidation. A recent study demonstrated that Cu(2+)-mediated oxidation of high density lipoprotein (HDL) resulted in a loss of the capacity to reduce cholesterol from macrophage foam cells [(1991) Proc. Natl. Acad. Sci. USA 88, 6457-6461]. In the present study we characterized the physicochemical properties of oxidized HDL and correlated them with the ligand activity toward the HDL receptor. Among them, the cross-linking of apolipoproteins and an increase in lipid peroxides were characteristic and closely similar to those of tetranitromethane-treated HDL, an abortive ligand for the HDL receptor. Cellular experiments with murine peritoneal macrophages revealed that both the cellular binding activity of HDL and its capacity to enhance cholesterol efflux from macrophage foam cells were markedly reduced upon oxidation. These results suggest that cross-linking of HDL apolipoproteins is involved in the loss of the ligand activity of oxidized HDL.


Effects of phosphodiesterase inhibitors caffeine and pentoxifylline on spontaneous and stimulus-induced acrosome reactions in human sperm. To determine whether the phosphodiesterase inhibitors caffeine and pentoxifylline influence the acrosome reaction in the conditions in which they are currently used as sperm movement enhancers. The frequency of acrosome reaction occurring spontaneously in capacitating media or induced by physiological (follicular fluid [FF]) and artificial (ionophore A23187) stimuli was compared in the presence and absence of the phosphodiesterase inhibitors. Private hospital and research laboratory. Patients undergoing routine semen examination before in vitro fertilization (no pathology detected) and healthy sperm donors. None. Percentage of acrosome-reacted sperm determined with the use of fluorescein-labeled Pisum sativum agglutinin as acrosomal stain. Caffeine alone augmented the frequency of acrosome reaction, but this effect was not observed with pentoxifylline alone. However, pentoxifylline increased sperm responsiveness to the acrosome reaction-inducing stimuli, FF and ionophore A23187. The promotion of spontaneous acrosome reaction may counteract the benefits from application of caffeine as motility stimulant. On the other hand, the sensitization to physiological acrosome reaction stimuli is expected to contribute to the improvement of sperm fertilizing ability by pentoxifylline and make this drug a potential candidate for the treatment of acrosome reaction anomalies.


Comparison of three methods to detect white blood cells in semen: leukocyte esterase dipstick test, granulocyte elastase enzymeimmunoassay, and peroxidase cytochemistry. Comparison of three methods for the detection of WBC in semen revealed a low concordance of positive test results. Among 557 semen samples, most positives were observed with the leukocyte esterase dipstick test (n = 95; 17.1%) followed by the peroxidase test (n = 51; 9.2%). There was little overlap between positives in the esterase-dipstick and the peroxidase method (29/117; 24.8%). With only 4 of 557 samples (0.7%), the PMN-elastase ELISA showed a surprisingly low incidence of positives. Because of lack of a gold standard, none of the three methods could be identified as superior. Because of its simplicity, specificity, and cost-effectiveness, the peroxidase method appeared most suited for clinical application.


[A case of chronic thyroiditis with transient painful thyroiditis occurring after the administration of lipiodol]. We reported a female case of painful thyroiditis occurring after hysterosalpingography and investigated whether the destructive thyroiditis was iodine-induced. The patient, aged 28, had TBG deficiency and the previous episode of thyrotoxicosis caused by Graves' disease. Lipiodol (containing 4.8g of iodide in 10ml solution) was administered via vagina for hysterosalpingography. One month after the radiography, serum inorganic iodide and Tg were elevated abnormally, but she was asymptomatic. After the subsequent 3 months she developed a painful and 3rd grade-sized goiter with concomitant marked elevation of thyroid hormones and inorganic iodide and also high titer of MCHA (320(2)X). A dramatic response was obtained with steroid. Thereafter she was treated with acupuncture on the thyroid gland, resulting in a sudden reappearance of tender goiter. This traumatic thyroiditis disappeared successfully in 2 weeks with steroid treatment. The painful thyroiditis subsided in 5 months throughout the course and she remained euthyroid for the ensuing 2 years. Aspiration biopsy was performed twice and revealed lymphocytic thyroiditis. Values of serum Tg varied in good correlation with those of serum inorganic iodide or rT3 throughout the course, respectively (P < 0.01, P < 0.05). Significant correlations between FT4 and FT3, and also T4 and T3 were observed, respectively (P < 0.01, P < 0.05). Serum inorganic iodide was elevated to 316 micrograms/dl at the symptomatic stage of the thyroiditis and decreased to 170 micrograms/dl at the resolving phase 2 months after the inflammation. Iodide disappearance curve showed a diphasic slope. The BHL was calculated as 60.3 days during the symptomatic stage and 6.9 months in euthyroid state.(ABSTRACT TRUNCATED AT 250 WORDS)


[Hyperresponsiveness of TSH and prolactin and impaired responsiveness of GH in Japanese patients with isolated ACTH deficiency]. Two hundred and forty-one cases of isolated ACTH deficiency have been reported in Japan since 1969. Pituitary hormone responsiveness to stimulation tests before and after hydrocortisone supplementation was investigated in these cases. Plasma ACTH level showed no or little change in response to lysine vasopressin, metyrapone, CRF or insulin-induced hypoglycemia in 97.3-100% of the cases. Serum GH level changed little or not at all in response to GRF, insulin-induced hypoglycemia, glucagon, 1-dopa and arginine in 26.9, 29.3, 40.0, 50.0 and 56.1%, respectively. Serum TSH and prolactin (PRL) levels showed hyperresponse to TRH in 34.7 and 35.6%, respectively. After hydrocortisone therapy, GH secretion was more responsive than before therapy in 78.9% of the cases. After supplementation, TSH level was less responsive to TRH stimulation than before therapy in 59.3% of the cases. After hydrocortisone supplementation, TSH response to TRH decreased in 75% of ACTH-deficient patients without primary hypothyroidism but did not decrease in more than half of those with primary hypothyroidism. TSH response to TRH decreased after supplementation in 76.5% of the patients with TSH hyperresponsiveness before therapy, and increased after therapy in 66.7% of those with normal TSH responses before therapy. After supplementation, PRL response to TRH was less than that before therapy in 43.5% of ACTH--deficient patients, and greater than that before therapy in 30.4%. PRL response to TRH decreased after therapy in 66.7% of the patients with PRL hyperresponsiveness before therapy, and increased in 63.6% of those with normal PRL response before therapy. Primary hypothyroidism and Hashimoto's thyroiditis were complicated in 21.6 and 11.6%, respectively, of the 241 patients with isolated ACTH deficiency. In patients who had TSH hyperresponsiveness and/or high basal TSH levels and PRL hyperresponsiveness and/or high basal PRL levels, primary hypothyroidism was complicated in 58.4 and 42.3%, respectively. Hashimoto's thyroiditis was complicated in 29.8 and 20.5%, respectively, of these patients. Pituitary cell antibody (PCA) was detected in 36.6% of ACTH-deficient patients who were examined. Pituitary cell surface antibody (PCSA) to AtT-20 cells and GH3 cells was detected in 50.0 and 28.0% of the examined cases, respectively. The prevalence of PCA and PCSA did not differ between TSH-hyperresponsive patients and those with normal TSH basal levels and response, whereas PCA and PCSA were significantly more prevalent in PRL-hyperresponsive patients than in those with normal PRL levels and response. An empty sella was found in 30.2% of the examined case.(ABSTRACT TRUNCATED AT 400 WORDS)


Comparison of enoxaparin, a low-molecular-weight heparin, and unfractionated heparin, with or without dihydroergotamine, in abdominal hysterectomy. The effects of administration of low-molecular-weight heparin (enoxaparin 20 mg) once a day, of unfractionated heparin (5000 IU twice a day, and of unfractionated heparin (2500 IU) plus dihydroergotamine (0.5 mg) twice a day were assessed in 100 patients undergoing abdominal hysterectomy. The test medications were given subcutaneously 2 hours before operation and for 3 days thereafter. There were no thromboembolic complications. Intraoperative blood loss, wound haematomas and blood loss via drains during four days after operation were similar in the three groups. None of the 37 patients receiving enoxaparin experienced major postoperative bleeding. Six out of 31 patients receiving unfractionated heparin without dihydroergotamine and two out of 32 patients receiving dihydroergotamine in addition experienced major bleeding necessitating re-operation and/or blood transfusion, (P < 0.05). Enoxaparin caused less major bleeding than unfractionated heparin with or without dihydroergotamine in patients undergoing hysterectomy.


Magnetic resonance imaging of Krukenberg tumor from gastric cancer. The magnetic resonance imaging (MRI) findings in a 42-year-old woman with a giant Krukenberg tumor from gastric cancer are reported. The macroscopic features of the Krukenberg tumor resulting from gastric cancer were clearly and precisely shown on MRI. Furthermore, MRI was more useful than ultrasonography or CT for determining the origin and further tissue characterization of the tumor. These findings suggest that MRI can be especially useful in the evaluation of giant ovarian tumors as in the present case.


The natriuretic peptide system in the brain: implications in the central control of cardiovascular and neuroendocrine functions. The natriuretic peptide system consists of three endogenous ligands, i.e., atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP), and at least three subtypes of receptors. All of the peptides and receptors exist in the central nervous system (CNS). ANPs in the brain are N-terminally truncated forms: ANP (4-28) and ANP (5-28). The primary structure of BNP varies considerably among species, whereas that of CNP is highly conserved. ANP, BNP, and CNP are distributed in discrete brain regions, although the distribution varies in different species. Few immunohistochemical studies have so far been performed on BNP and CNP. There are three subtypes of receptors: ANP-A and ANP-B, which are bioactive, and the C receptor, which does not seem to be directly related to bioactivity. In the rat, the major subtype of ANP receptor in the CNS is the ANP-B receptor, based on the results of Northern blotting. Since the ligand for ANP-B receptor is CNP, the CNP-ANP-B receptor system may be most important, at least in rat brain. It is still unknown whether or not a specific receptor for BNP exists in central or peripheral tissues. Further studies should clarify the exact localization of ANP, BNP, and CNP and the three receptor subtypes in the CNS. Although natriuretic peptides and their receptors are distributed widely in the CNS, the AV3V regions, basal medial hypothalamus, brainstem, and circumventricular organs are the most prominent sites. This suggests an important physiological role of the natriuretic peptide system in the central control of cardiovascular homeostasis. The natriuretic peptide system seems to be involved in the regulation of water and salt intake, blood pressure, and secretion of vasopressin in the direction of reducing body fluid and lowering blood pressure. Such actions of natriuretic peptides are antagonistic to the central actions of angiotensin II (AII). In fact, the distribution of ANP and AII and their receptors in the CNS overlaps considerably. It is highly likely, therefore, that the central natriuretic peptide system and the renin-angiotensin system play important roles in the central control of cardiovascular and body fluid homeostasis in opposite directions. The natriuretic peptide system may also be involved in neuroendocrine control and some other CNS functions, although the physiological significance of these actions is less clear at the present time. It is now clear that there is considerable plasticity in the regulation of natriuretic peptides and their receptors.(ABSTRACT TRUNCATED AT 400 WORDS)


The involvement of serotonin in regulation of pituitary-adrenocortical function. Adrenocorticotropin (ACTH) secretion from the anterior pituitary gland influences glucocorticoid secretion from the adrenal cortex and in turn is controlled mainly by corticotropin-releasing factor (CRF) release from the hypothalamus. CRF-containing neurons projecting from the paraventricular nucleus to the median eminence, which are involved in controlling pituitary-adrenocortical function, receive synaptic input from serotonin neurons projecting from the midbrain raphe nuclei. Serotonin stimulates the release of bio- or immunoassayable CRF from isolated rat hypothalamus in vitro. Corticosterone, ACTH, and CRF release in vivo is increased in rats by drugs that enhance serotonin function, including serotonin precursors, serotonin uptake inhibitors, serotonin releasers, and direct-acting serotonin agonists. Among the multiple serotonin receptors that exist in brain, at least two--5HT1A and 5HT2 or 5HT1C receptors--seem to mediate activation of pituitary-adrenocortical function. The physiological role of this stimulatory serotonergic influence on pituitary-adrenocortical function is still not well understood, but serotonin may play a role in circadian rhythmicity of adrenocortical secretion and in the activation of pituitary-adrenocortical function by certain types of stress. Measurement of ACTH or cortisol levels in humans after administration of a direct- or indirect-acting serotonin agonist provides one means of probing the functional state of brain serotonergic systems in disease or after drug treatment.


Structure-function correlates in the corticotropes of the anterior pituitary. Investigators have described changes in pituitary corticotropes that correlate with changes in the physiological state of the animal. The stellate subtype degranulated and enlarged initially after adrenalectomy. This was followed by repopulation of the granules during the first 3 weeks after surgery with larger granules. There was also an increase in the percentage of corticotropes. More recent studies have shown that chronic stimulation with corticotropin-releasing hormone (CRH) produces some of the same changes; however, the magnitude differs because of corticosterone feedback. Corticotropes are heterogeneous in size, shape, storage patterns, and secretory responses. Specific changes are evident within a short time after stimulation as well. Their average cellular area increases within 1-2 h of stimulation by CRH in vitro or cold stress in vivo. Whereas many corticotropes acutely stimulated by cold or a novel environment are better granulated, others are depleted of granules. Cold stress for 30 min also stimulates an increase in the percentage of immunoreactive corticotropes and cells that bind CRH or arginine vasopressin (AVP). Secretagogues like CRH or epidermal growth factor (EGF) act in vitro to increase percentages of cells that store adrenocorticotropin (ACTH) or express mRNA for pro-opiomelanocortin. AVP or angiotensin II (A-II), or their activated second messengers, also increase percentages of cells that bind CRH and store ACTH. Inhibition of ACTH secretion by ion channel blockers or corticosterone has potent inhibitory effects on percentages of CRH-bound cells. AVP binding is not affected. Some of the inhibitory states reduce the average area of corticotropes. However, about 30% of the cells remain unaffected by these inhibitors. The rapid changes in cell percentages with the different treatments have led workers to postulate the existence of reserve cells that may be sensitive to certain levels of types of stimuli. Several candidate reserve cells are proposed. One group of cells that store ACTH with gonadotropins may function in the proestrous female to stimulate adrenal progesterone. Another multihormonal cell may function during cold stress to release both ACTH and thyroid-stimulating hormone (TSH) under the influence of AVP. There may be subpopulations of corticotropes that act in synchrony with other cell populations. They may be awaiting the proper type or combination of secretagogues to support the pituitary-adrenal and other axes.


Chloride conductance of apical membrane in cultured porcine thyroid cells activated by cyclic AMP. The thyroid epithelium transports fluid bidirectionally using active transport of Na+ ions from apical to basal poles and active transport of Cl- in the reverse direction. In these studies we sought evidence for cyclic AMP activated Cl- channels on the apical membranes of thyroid cells in monolayer culture. A Cl(-)-dependent basal-positive short-circuit current (ISC) was demonstrated in bicameral chambers after blocking Na+ transport with phenamil, and responded to prostaglandin (PG) E2 with a spike of 5-10 min duration followed by a plateau. The onset of the spike coincided with an increase in the conductance of the epithelium. Application of an external Cl- concentration gradient, by replacing the medium in the apical compartment with Cl(-)-free medium, resulted in an increase in ISC after, but not before, addition of PGE2. Forskolin and thyroid-stimulating hormone (TSH), but not A23187, also stimulated Cl- transport. In conjunction with previous observations that Cl- transport was mediated by a bumetanide-sensitive NaKCl2 symporter on the basal membrane, these observations indicated the presence of a cyclic AMP activated Cl- conductance in the apical membrane of thyroid cells.


Pancreastatin increases cytosolic Ca2+ in insulin secreting RINm5F cells. We have investigated the effect of pancreastatin on cytosolic Ca2+ concentration in the insulin secreting cell line RINm5F. Changes in [Ca2+]i induced by pancreastatin were detected by Fluo-3 fluorescence using both flow cytometry and batch analysis measurements, and turned out to be from 90 to 315 nM equivalent to 80% of that caused by ATP, which increased [Ca2+]i from 90 nM to 400 nM. This effect of pancreastatin did not depend on extracellular calcium and was not mediated by alpha-adrenergic receptors since it was not prevented by the alpha-blocker yohimbine. It is concluded that pancreastatin has a role in the homeostasis of free cytosolic calcium in the insulin secreting cell line Rinm5F.


Testosterone and 5 alpha-dihydrotestosterone interact differently with the androgen receptor to enhance transcription of the MMTV-CAT reporter gene. Testosterone and its 5 alpha-reduced derivative 5 alpha-dihydrotestosterone exert different actions in the male during embryogenesis and in postnatal life. Nevertheless the two hormones bind to the same intracellular androgen receptor, and genetic and endocrinological studies in the Tfm mouse suggest that the actions of both hormones are mediated by this single receptor. Previous studies indicate that dihydrotestosterone binds more tightly to the androgen receptor but that the Bmax of binding of the two hormones is the same. To determine whether these differences in binding parameters could explain the mechanism by which the two hormones exert different physiological actions via the same receptor, we introduced a plasmid encoding the androgen receptor cDNA and a reporter plasmid encoding MMTV-CAT into Chinese hamster ovary cells. These cells do not express endogenous androgen receptor and do not convert testosterone to dihydrotestosterone. Therefore, it was possible to examine the relation between the concentration of each of the steroids and reporter gene expression. Both hormones enhanced CAT activity, but dihydrotestosterone was approximately 10 times as potent (half maximal of 0.018 nM) as testosterone (half maximal of 0.2 nM); the maximal activity achieved was the same for the two androgens. These findings are nearly identical to the apparent Kd values for the interaction of the two hormones with the androgen receptor. Although testosterone and dihydrotestosterone may influence the expression of other genes differently, these findings are compatible with a model system in which the differential effects can be explained as a consequence of different binding affinities to the receptor.(ABSTRACT TRUNCATED AT 250 WORDS)


Transcriptional regulation of androgen receptor gene expression in Sertoli cells and other cell types. Regulation of androgen receptor (AR) mRNA expression was studied in Sertoli cells and peritubular myoid cells isolated from immature rat testis, and in the lymph node carcinoma cell line derived from a human prostate (LNCaP). Addition of dibutyryl-cyclic AMP (dbcAMP) to Sertoli cell cultures resulted in a rapid transient decrease in AR mRNA expression (5 h), which was followed by a gradual increase in AR mRNA expression (24-72 h). This effect of dbcAMP mimicked follicle-stimulating hormone (FSH) action. In peritubular myoid cells, there was only a moderate but prolonged decrease during incubation in the presence of dbcAMP, and in LNCaP cells no effect of dbcAMP on AR mRNA expression was observed. When Sertoli cells or peritubular myoid cells were cultured in the presence of androgens, AR mRNA expression in these cell types did not change. This is in contrast to LNCaP cells, that showed a marked reduction of AR mRNA expression during androgen treatment. In the present experiments, transcriptional regulation of AR gene expression in Sertoli cells and LNCaP cells was also examined. Freshly isolated Sertoli cell clusters were transfected with a series of luciferase reporter gene constructs, driven by the AR promoter. It was found that addition of dbcAMP to the transfected Sertoli cells resulted in a small but consistent increase in reporter gene expression (which was interpreted as resulting from AR promoter activity); a construct that only contained the AR 5' untranslated region of the cDNA sequence did not show such a regulation. The same constructs, transfected into LNCaP cells, did not show any transcriptional down-regulation when the synthetic androgen R1881 was added to the cell cultures. A nuclear transcription elongation experiment (run-on), however, demonstrated that androgen-induced AR mRNA down-regulation in LNCaP cells resulted from an inhibition of AR gene transcription. The present results indicate that in Sertoli cells and LNCaP cells, hormonal effects on AR gene transcription play a role in regulation of AR expression. However, AR gene transcription in these cells is differentially regulated.


Elements required for activation of the major promoter of the human insulin-like growth factor II gene. The human insulin-like growth factor II (IGF-II) gene contains four promoters, P1-P4. In fetal liver promoter P3 is the major promoter, which consists of a proximal region that supports general transcription, and a cell-specific region located more upstream. In addition to the TATA box, the proximal region contains four binding sites for nuclear proteins, designated PE3-1 to PE3-4. To determine the influence of the proteins binding to these elements, the transcriptional activity of the proximal region of P3 was investigated. Promoter P3 was analyzed in Hep3B cells, which express high levels of IGF-II mRNA derived from this promoter, and in HeLa cells, that have an inactive IGF-II gene. By analysis of 5'-deletion constructs in an in vitro transcription system and in transient expression assays, and by competition with specific oligonucleotides it was shown that the factors binding to the elements PE3-4, PE3-2 and PE3-1 play an important role in the regulation of promoter P3.


Sites of glandular kallikrein gene expression in fetal mice. As part of an ongoing study of the cell-specific expression of glandular kallikrein genes in mice, we have investigated cellular sites of expression of the renal/pancreatic kallikrein gene, mGK-6, during fetal life. Expression of alpha I and beta-subunit genes of Na+K+ATPase and bradykinin binding were used as an indication of the functional maturity of the fetal epithelial tubules in which mGK-6 expression was identified. mGK-6 mRNA was first observed at embryonic day 16 (E16) in the submandibular main duct, then at E18 in the sub-lingual main duct, at E19 in renal tubules and at E19 in ducts of the nasal glands. All of these ducts contained detectable epithelial Na+K+ATPase mRNAs from an earlier gestational age than mGK-6 mRNA, suggesting their capacity for electrolyte transport. Bradykinin binding was evident in renal tubules at E18. This study established that renal/pancreatic kallikrein is synthesized in fetal epithelial tubules which are mature functionally.


Assessment of liver function using a novel galactose single point method. A novel, simple, clinically useful quantitative liver function test, called the galactose single point (GSP) method, was developed by measurement of galactose blood concentration 1 h after galactose was administered (0.5 g/kg). It was quickly infused intravenously in 55 normal healthy volunteers, 73 patients with chronic hepatitis (CH), 36 with cirrhosis and 41 with hepatocellular carcinoma (HCC). Patients with CH diagnosis were assessed by liver biopsy. Cirrhosis was diagnosed by histological examination or a chronic hepatitis history with esophageal varices or ascites, whereas HCC was diagnosed either histologically, or cytologically proved, or as implied in the 'one imagine study' being positive with AFP > 300 ng/dl. Highly significant galactose blood levels were observed between normal healthy volunteers and patients 50, 60 and 70 min after galactose was administered. Galactose elimination capacity (GEC), modified GEC (MGEC) and consecutive GSP tests were performed in 6 healthy volunteers for 2 days. 0.64-16.87% variation was observed for each subject. The significant differences (p < 0.001) in average GSP values were 247 +/- 18.1, 422 +/- 27.3, 629 +/- 42.8 and 579 +/- 43.6 micrograms/ml for normal healthy volunteers, CH, cirrhosis and HCC patients, respectively. Highly significant correlations (p < 0.001) were obtained among GSP, GEC and MGEC for all patients. Positive correlations were observed between GSP, GEC, MGEC and AST (serum aspartate aminotransferase), ALT (serum alanine aminotransferase), serum bilirubin, albumin, prothrombin time and r-globulin. According to results obtained from 202 normal healthy volunteers and patients, the GSP method may be a simple, clinically useful quantitative measurement of liver function for the determination of a patient's residual liver function, the prognosis of liver function for patients with cirrhosis, postoperational follow-up and, finally, the timing of a liver transplant.


Discoidin proteins of Dictyostelium are necessary for normal cytoskeletal organization and cellular morphology during aggregation. The onset of aggregation of bacterially-grown Dictyostelium discoideum amoebae is accompanied by the accumulation of the discoidin proteins. An immunofluorescent analysis demonstrates that discoidin is distributed throughout the cytoplasm, but is excluded from vesicles and nucleoli. There is no indication of either extracellular or membrane localization. Translocating amoebae of mutants lacking discoidin form more dispersed pseudopodial regions at the cell periphery, possess an abnormally centered microtubule organizing center, are blunt rather than elongate, and lack the tapered posterior uropod characteristic of translocating wild-type cells. However, in spite of the loss of the normal elongate morphology, discoidinless mutants translocate with instantaneous velocities and directional persistence comparable to wild-type cells, and they respond normally to the rapid addition of cAMP. These results demonstrate that the discoidin proteins are cytoplasmic components essential for the maintenance of the elongate cell morphology, cytoskeletal organization and the ability to align with other cells during aggregation. However, the elongate morphology is not a requisite for rapid and persistent single cell translocation.


Retinoic acid regulates gene expression of retinoic acid receptors alpha, beta and gamma in F9 mouse teratocarcinoma cells. The regulatory effects of retinoic acid (RA) on retinoic acid receptor (RAR) alpha, beta and gamma mRNA were examined in the F9 mouse teratocarcinoma cells. Northern blot hybridization showed that RA regulated all three types of RAR gene expression. The different transcripts for each RAR alpha and beta were differentially regulated by RA. The maximum induction of the 2.8 kb RAR alpha and 3.3 kb RAR beta transcript levels by RA took longer than the induction of the 3.8 kb RAR alpha and 3.5 kb RAR beta transcript levels. The data suggest that these transcripts originated from different promoters. Short term treatment (< or = 24 hours) of RA induced both 3.1 and 3.3 kb RAR gamma transcripts. Long term treatment (> 24 hours) of RA resulted in the inhibition of 3.1 kb mRNA, whereas the 3.3 kb mRNA remained elevated. In addition, a new RAR gamma transcript of 2.9 kb was induced. In contrast to RAR alpha and beta, the effect on RAR gamma gene expression was irreversible. Cycloheximide did not prevent the effect of RA on RAR gene expression, whereas actinomycin D totally abolished the RA effect on the expression of all three receptor genes. The data suggest that the biological effects of RA may be constrained or augmented by differential regulation of its own receptor gene expression.


The effects of alpha, beta-unsaturated aldehydes on hepatic thiols and thiol-containing enzymes. The effects of series of alpha, beta-unsaturated aldehydes on hepatic glutathione, cytochrome P450, and NADPH-cytochrome c reductase activity were compared with time. Male F-344 rats were dosed with muconaldehyde (36 mumol/kg), acrolein (89 mumol/kg), crotonaldehyde (450 mumol/kg), or the saturated aldehyde propionaldehyde (89 mumol/kg) and terminated 0.5, 4, or 24 hr later. Acrolein or muconaldehyde reduced glutathione to 51 and 75% of controls, respectively, at 4 hr; glutathione returned to control values at 24 hr. Only at 24 hr, acrolein, muconaldehyde, or crotonaldehyde decreased cytochrome P450 to 61, 71, and 67% of control values, respectively; ethylmorphine N-demethylation was decreased to a greater extent, i.e., to 35, 60, and 23% of controls. The reductase activity was unchanged at any time following the treatment with reactive aldehydes which were not hepatotoxic (as shown by glucose 6-phosphatase activity, histological changes, or serum enzymes). Propionaldehyde changed none of these activities. Acrolein (44.5 mumol/kg) given 4 hr prior to phenobarbital (50 mg/kg) for two consecutive days decreased the phenobarbital induction of cytochrome P450 to 45% of phenobarbital alone. This treatment also decreased the 2 alpha, 2 beta, 6 beta, 16 alpha, and 16 beta hydroxylation of testosterone as well as androstenedione formation showing effects on individual cytochrome P450 isozymes. NADPH-cytochrome c reductase induction was not decreased by this treatment, thus indicating that in vivo these changes are due to a mechanism other than generalized inhibition of protein synthesis.


The pulmonary response and clearance of Ludox colloidal silica after a 4-week inhalation exposure in rats. Rats were exposed to Ludox colloidal silica (CS) at concentrations of 0, 10, 50, and 150 mg/m3 for 6 hr/day, 5 days/week for 4 weeks. Rats were killed after 4 weeks of exposure and 10 days or 3 months postexposure (PE). The exposure concentration of 10 mg/m3 Ludox CS is considered to be the no-effect concentration. There were no exposure-related clinical signs in any group. After 4 weeks exposure, lung weights were increased significantly in rats exposed to 50 and 150 mg/m3 Ludox CS, but lung weights were similar to those of controls at 3 months PE. After 4 weeks exposure to 50 mg/m3 Ludox CS, a slight alveolar macrophage response, polymorphonuclear leukocytic infiltration, and Type II pneumocyte hyperplasia in alveolar duct regions were present. After 3 months PE, these pulmonary lesions had almost disappeared with removal of most dust-laden alveolar macrophages (AMs). The pulmonary response to 150 mg/m3 Ludox CS was similar in character but increased in magnitude from that seen at 50 mg/m3. At 3 months PE, most particle-laden AMs had disappeared and the remaining AMs were aggregated and sharply demarcated. A few aggregates of particle-laden AMs appeared to transform into silicotic nodules comprising macrophages, epithelioid cells, and lymphocytic infiltration in some animals. Some silicotic nodules showed reticular fiber networks with minute collagen fiber deposition. Tracheobronchial lymph nodes were enlarged with aggregates of particle-laden AMs and hyperplastic histiocytic cells. Lung-deposited Ludox cleared rapidly from the lungs with half-times of approximately 40 and 50 days for the 50 and 150 mg/m3 groups, respectively.


Effect of dithiocarbamates and dithiocarbamate-induced cadmium mobilization on essential trace metal metabolism in the female rat. The effects of two dithiocarbamates (both of which induce an increase in the excretion of cadmium) on the biliary and urinary excretion of the essential trace elements zinc, copper, iron, magnesium, and calcium have been examined in the female Sprague-Dawley rat to estimate what alterations in the excretion of essential metals accompanies the use of these compounds. The dithiocarbamates studied were sodium diethyldithiocarbamate (DDTC) and sodium N-(4-methylbenzyl)-4-O-(beta-D-galactopyranosyl)-D-glucamine-N-carbod ithioate (MeBLDTC). DDTC induced a modest decrease in the biliary and urinary excretion of copper. The biliary excretion of both zinc and iron was significantly enhanced when MeBLDTC was given ip to normal rats, while those of copper, magnesium, and calcium were not significantly affected by this compound. DDTC treatment of normal female rats which had not been administered cadmium resulted in a slight decrease in the iron level of the liver. Treatment of rats with cadmium chloride resulted in a significant increase in the zinc and iron levels of the kidney, liver, and pancreas and an increase in the copper levels of the kidney and the liver. After a treatment with MeBLDTC, which reduced hepatic cadmium levels, only some of the levels of these essential metals were modified toward the levels found in untreated controls. Cadmium-loaded animals from which hepatic cadmium had been mobilized by MeBLDTC did not differ in renal or hepatic histopathology from the control (untreated) animals or from the group which had received cadmium only.


Biological role of an arginine residue present in a histidine-rich peptide which inhibits hemagglutination of Porphyromonas gingivalis. Inhibitory effects of synthetic fragments in histatin 8, having the sequence Lys-Phe-His-Glu-Lys-His-His-Ser-His-Arg-Gly-Tyr, on hemagglutination by Porphyromonas gingivalis 381 were examined. The hemagglutinating activity was reduced much more by the peptide Lys-His-His-Ser-His-Arg-Gly-Tyr than by the peptides Lys-His-His-Ser-His and/or Lys-Phe-His-Glu-Lys. These results suggest that the arginine residue may have an important role in the inhibition of hemagglutination by P. gingivalis.


Oscillatory metabolism of Saccharomyces cerevisiae in continuous culture. Short-period (40-50 min) synchronized metabolic oscillation was found in a continuous culture of yeast Saccharomyces cerevisiae under aerobic conditions at low-dilution rates. During oscillation, many parameters changed cyclically, such as dissolved oxygen concentration, respiration rate, ethanol and acetate concentrations in the culture, glycogen, ATP, NADH, pyruvate and acetate concentrations in the cells. These changes were considered to be associated with glycogen metabolism. When glycogen was degraded, the respiro-fermentative phase was observed, in which ethanol was produced and the respiration rate decreased. In this phase, the levels of intracellular pyruvate and acetate became minimum, ATP became high and intracellular pH at its lowest level. When glycogen metabolism changed from degradation to accumulation, the respiratory phase started, during which ethanol was re-assimilated from the culture and the respiration rate increased. Intracellular pyruvate and acetate became maximum, ATP decreased and the intracellular pH appeared high. These findings may indicate new aspects of the control mechanism of glycogen metabolism and how respiration and ethanol fermentation are regulated together under aerobic conditions.


Gene dosage analysis in Azotobacter vinelandii. For more than a decade, Azotobacter vinelandii has been considered a polyploid bacterium on the basis of physical studies of chromosome size and DNA content per cell. However, as described in the present work, many genetic operations can be performed in A. vinelandii without the constraints expected in a polyploid bacterium: (i) reversion of transposon-induced mutations is usually associated with loss of the transposable element; (ii) revertants retaining the transposon always carry secondary transpositions; (iii) heterozygotic transconjugants and transformants are unstable and segregate homozygotic colonies even in the absence of selection. Physical monitoring of segregation, achieved by colony hybridization, indicates that phenotypic expression of an allele is always correlated with its physical presence, thus ruling out the existence of either threshold dosage requirements or transcriptionally inactive DNA. Chromosomal lac fusions constructed by double crossover with a linearized plasmid show a segregation pattern consistent with the inheritance of one or several chromosomes per daughter cell. Analysis of the delay required for the expression of recessive chromosomal mutations such as rif, nal and str provides further evidence that A. vinelandii is not a polyploid bacterium.


DNA repair and the evolution of transformation in Haemophilus influenzae. Under certain environmental conditions, naturally transforming bacteria are induced to pick up DNA released into the environment by other cells of the same or closely related species and, by homologous recombination, integrate that DNA into their chromosome. The selective pressures responsible for the evolution and maintenance of this form of genetic outcrossing, or sex, in bacteria are not known. A prominent hypothesis is that transformation, and sex in general, evolved as a means of obtaining DNA templates to repair damaged regions of the chromosome. Previous results obtained with Bacillus subtilis were consistent with the repair hypothesis. In an effort to explore the generality of those results, I have tested the repair hypothesis with Haemophilus influenzae, a naturally transforming, gram-negative species of bacteria. The results of UV damage-survivorship experiments with H. influenzae were also consistent with that hypothesis. However, additional experiments demonstrate that the higher survival of transformed cultures cannot be accounted for by use of the transforming DNA as templates for repair. I consider alternative hypotheses for the means by which transformation can increase cell survival following UV exposure and discuss the implications of these results with respect to the DNA repair hypothesis and the evolution of transformation.


Characterization of insertion mutations in the Saccharomyces cerevisiae MSH1 and MSH2 genes: evidence for separate mitochondrial and nuclear functions. The MSH1 and MSH2 genes of Saccharomyces cerevisiae are predicted to encode proteins that are homologous to the Escherichia coli MutS and Streptococcus pneumoniae HexA proteins and their homologs. Disruption of the MSH1 gene caused a petite phenotype which was established rapidly. A functional MSH1 gene present on a single-copy centromere plasmid was incapable of rescuing the established msh1 petite phenotype. Analysis of msh1 strains demonstrated that mutagenesis and large-scale rearrangement of mitochondrial DNA had occurred. 4',6-Diamidino-2-phenylindole (DAPI) staining of msh1 yeast revealed an aberrant distribution of mtDNA. Haploid msh2 mutants displayed an increase of 85-fold in the rate of spontaneous mutation to canavanine resistance. Sporulation of homozygous msh2/msh2 diploids gave rise to a high level of lethality which was compounded during increased vegetative growth prior to sporulation. msh2 mutations also affected gene conversion of two HIS4 alleles. The his4x mutation, lying near the 5' end of the gene, was converted with equal frequency in both wild-type and msh2 strains. However, many of the events in the msh2 background were post-meiotic segregation (PMS) events (46.4%) while none (< 0.25%) of the aberrant segregations in wild type were PMS events. The his4b allele, lying 1.6 kb downstream of his4x, was converted at a 10-fold higher frequency in the msh2 background than in the corresponding wild-type strain. Like the his4x allele, his4b showed a high level of PMS (30%) in the msh2 background compared to the corresponding wild-type strain where no (< 0.26%) PMS events were observed. These results indicate that MSH1 plays a role in repair or stability of mtDNA and MSH2 plays a role in repair of 4-bp insertion/deletion mispairs in the nucleus.


A mutated retinoic acid receptor-alpha exhibiting dominant-negative activity alters the lineage development of a multipotent hematopoietic cell line. The retinoic acid receptor (RAR alpha) is expressed in virtually all hematopoietic lineages, but the role of this transcription factor in regulating the growth and differentiation of hematopoietic progenitors is unknown. We have constructed a mutant RAR alpha that both exhibits dominant-negative activity against the normal RAR alpha in transient expression assays in mouse fibroblasts and inhibits retinoic acid-induced neutrophilic differentiation of the HL-60 human promyelocytic leukemia cell line. When this dominant-negative RAR alpha construct is introduced into the multipotent interleukin-3-dependent FDCP mix A4 murine hematopoietic cell line, there is a rapid switch from spontaneous neutrophil/monocyte differentiation to basophil/mast cell development. Thus, in this multipotent hemopoietic cell line the normal RAR alpha transcription factor and/or related molecules appear to promote the differentiation of neutrophils and monocytes but suppress the development of basophils/mast cells.


Prepattern in the developing Drosophila eye revealed by an activated torso--sevenless chimeric receptor. Induction of the R7 photoreceptor cell fate in the developing eye of Drosophila depends on the activation of the sevenless receptor tyrosine kinase in the R7 precursor cell. The sevenless protein is expressed transiently in 8 of the 20 precursors of an ommatidium. Activation of the sevenless kinase in these eight cells indicates that six of them are competent to become R7 cells. To test the competence of all 20 ommatidial precursors in a temporally unrestricted manner we have used a constitutively activated sevenless kinase created by fusing the extracellular domain of a mutant torso protein, another Drosophila receptor tyrosine kinase, to the sevenless kinase. Our results show that competence to develop as neuronal cells in response to sevenless activity is spatially and temporally limited to the cells expressing sevenless. Therefore, the expression of sevenless marks a preexisting pattern of developmental potential in the disc epithelium.


SIT4 protein phosphatase is required for the normal accumulation of SWI4, CLN1, CLN2, and HCS26 RNAs during late G1. In Saccharomyces cerevisiae, the RNA levels of the G1 cyclins CLN1, CLN2, and HCS26 increase dramatically during the late G1 phase of the cell cycle. The SIT4 gene, which encodes a serine/threonine protein phosphatase, is required for the normal accumulation of CLN1, CLN2, and HCS26 RNAs during late G1. This requirement for SIT4 in normal G1 cyclin RNA accumulation is at least partly via SWI4. Strains containing mutations in SIT4 are sensitive to the loss of either CLN2 or CLN3 function. At the nonpermissive temperature, temperature-sensitive sit4 strains are blocked for both bud emergence and DNA synthesis. Heterologous expression of CLN2 in the absence of SIT4 function results in DNA synthesis, but most of the cells are still blocked for bud emergence. Therefore, SIT4 is required for at least two late G1 or G1/S functions: the normal accumulation of G1 cyclin RNAs (which is required for DNA synthesis) and some additional function that is required for bud emergence or cell cycle progression through late G1 or G1/S.


Super-unstable mutations associated with P-M hybrid dysgenesis in Drosophila melanogaster. Super-unstable mutations occasionally appear either in natural populations of Drosophila melanogaster or in P-M hybrid dysgenesis. We found that they may be reproducibly obtained with a high frequency from crosses between males from the pi 2 strain and females from the waG* strain or its derivatives. Super-unstable mutations in the ocelliless, singed, white, yellow and other loci have been obtained. Each super-unstable mutation gives rise to a large family of new super-unstable mutations with a wide range of phenotypic expression. Mutations with the same phenotype often differ in the specificity of their potential for further mutation. As a rule, a super-unstable mutation is associated with a specific reversible mutation and paired alleles are formed in this way. Other mutations are usually irreversible, but new mutations of these may also form paired alleles. Active transposase encoded by transposable P elements is necessary to maintain super-instability. Finally, some preliminary molecular data are discussed which suggest that this type of super-instability is a result of interaction between P elements and a novel mobile element, designated as X.


Genes involved in the development of bristles and hairs in Drosophila melanogaster. Mutations in three loci influencing the development of bristles and hairs were detected in experiments with strains containing either a mobilized Stalker or a mobilized P-element. The mutations in two genes, suppressor of scute and putative microchaete, modify phenotypic expression of mutations in the scute locus. In particular, su(sc) mutations suppress the sc-phenotype in the scutellum and enhance the Hw-phenotype in the thorax. Mutations in the third gene, pseudoscute, lead to reduction of all bristles and hairs. The latter locus seems to control the development of bristles independently of the achaete-scute complex control.


Oxidation-reduction reactions in Ehrlich cells treated with copper-neocuproine. The interaction of 2,9-dimethyl-1,10-phenanthroline (neocuproine or NC) and its copper complex with Ehrlich ascites tumor cells was studied. NC is frequently used as a negative control in studies of in vitro DNA degradation by copper phenanthroline and has also found use as a potential inhibitor of damage from oxidative stress in biological systems. NC inhibited Ehrlich cell growth in monolayer culture over 48 h treatment by 50% at 0.05 nmol/10(5) cells. Addition of 5- to 100-fold ratios of CuCl2 to NC (at 0.035 nmol NC/10(5) cells) produced progressively more growth inhibition. Addition of 1:0.5 ratios of NC to CuCl2 over the range of NC concentrations 0.08-0.2 nmol/10(5) cells/mL resulted in DNA single-strand breakage during 1-h treatments as measured by DNA alkaline elution. Concomitant addition of catalase or dimethyl sulfoxide (DMSO) inhibited DNA strand scission, while superoxide dismutase enhanced breakage. Catalase and DMSO also inhibited induction of membrane permeability by the copper complex of NC. These cellular effects apparently result from the intracellular generation of hydroxyl radical from H2O2. NC facilitated the uptake of copper into cells, though it was initially bound as a copper-histidine-like complex. The internalized copper was reduced to Cu(I), bound mostly as (NC)2Cu(I). To explain the (NC)2Cu-dependent generation of hydroxyl radical, it is hypothesized that glutathione successfully competes for Cu(I), converting it to a redox-active form that can catalyze the reduction of molecular oxygen to .OH. Model studies support this view. Radical scavengers did not reverse growth inhibition produced by NC or NC + CuCl2.


Antioxidant and free radical scavenging activities of the iron chelators pyoverdin and hydroxypyrid-4-ones in iron-loaded hepatocyte cultures: comparison of their mechanism of protection with that of desferrioxamine. The protective effect on iron-supplemented hepatocyte cultures of three iron chelators, pyoverdin Pa and hydroxypyrid-4-one derivatives CP20 and CP22, was compared to that of the widely known desferrioxamine B (Desferal:DFO), on the basis of two criteria: (a) their effectiveness in inhibiting free malondialdehyde (MDA) production as an index of iron-induced lipid peroxidation; and (b) their ability to reduce intracellular enzyme leakage. In view of these two markers of iron toxicity, the protective effect of these chelators was classified as follows: DFO > CP20 > or = CP22 > Pa. The mechanism of cellular protection was elucidated by investigating both the iron-chelating activity and the free radical scavenging property of these agents. As concerns the iron chelation, DFO and Pa exerted the same rank order as for cytoprotection (DFO > Pa). The free radical scavenging property toward hydroxyl radical .OH and peroxyl radical ROO. was investigated in a cell-free experimental model. The two siderophores, DFO and Pa, appeared to have a lower antiradical activity toward .OH than hydroxypyrid-4-one CP22. This .OH scavenging activity was classified as follows: CP22 >> Pa > DFO. Moreover, the chelators exhibited for the quenching of ROO. the same order of effectiveness as that observed for cellular protection: DFO > CP20 > or = CP22 > Pa. These data indicate that, in addition to the iron-chelating activity which represents the most important property for determining the protection capacity of these iron chelators, their free radical scavenging ability also must be taken into account. This direct demonstration of a strong association between the free radical scavenging activity and the protective effect of iron chelators further increases the prospects for the development and clinical applications of new oral chelating drugs.


Antioxidant properties of nitecapone (OR-462). Nitecapone [3-(3,4-dihydroxy-5-nitrophenyl)methylene-2,4-pentanedione] [OR-462] is a catechol-O-methyltransferase inhibitor with gastroprotective properties. Recently, its antioxidant properties have been discovered: It scavenges peroxyl radicals (ROO.) and thus spares glutathione. Further examination of the properties of nitecapone demonstrated a remarkable ability of this compound to act as an antioxidant: (1) to scavenge ROO. in solution with a stoichiometry factor of 2; (2) to scavenge ROO. in membranes; (3) to inhibit lipid peroxidation; (4) to act as a competitive inhibitor for xanthine oxidase with Ki of 8.8 microM; (5) to scavenge O2- with a second order kinetic rate constant of 1.0 x 10(4) M-1 s-1; and (6) to scavenge HO.. Nitecapone also interacts with oxidation product of ascorbate to participate in recycling of vitamin E. Thus, nitecapone potentially is an effective therapeutic antioxidant, and the use of this compound in a combination with other antioxidants may be beneficial.


Metabolism of oxygen radicals in peroxisomes and cellular implications. Peroxisomes are subcellular respiratory organelles which contain catalase and H2O2-producing flavin oxidases as basic enzymatic constituents. These organelles have an essentially oxidative type of metabolism and have the potential to carry out different important metabolic pathways. In recent years the presence of different types of superoxide dismutase (SOD) have been demonstrated in peroxisomes from several plant species, and more recently the occurrence of SOD has been extended to peroxisomes from human and transformed yeast cells. A copper,zinc-containing SOD from plant peroxisomes has been purified and partially characterized. The production of hydroxyl and superoxide radicals has been studied in peroxisomes. There are two sites of O2- production in peroxisomes: (1) in the matrix, the generating system being xanthine oxidase; and (2) in peroxisomal membranes, dependent on reduced nicotinamide adenine dinucleotide (NADH), and the electron transport components of the peroxisomal membrane are possibly responsible. The generation of oxygen radicals in peroxisomes could have important effects on cellular metabolism. Diverse cellular implications of oxyradical metabolism in peroxisomes are discussed in relation to phenomena such as cell injury, peroxisomal genetic diseases, peroxisome proliferation and oxidative stress, metal and salt stress, catabolism of nucleic acids, senescence, and plant pathogenic processes.


Intracranial microdialysis of salicylic acid to detect hydroxyl radical generation through dopamine autooxidation in the caudate nucleus: effects of MPP+. Ringer's solution containing salicylic acid (5 nmol/microliters/min) was infused directly through an intracranial microdialysis probe to detect the generation of hydroxyl radicals (.OH) reflected by the formation of dihydroxybenzoic acids (DHBA) in the caudate nucleus of anesthetized rats. Brain dialysate was assayed for dopamine, 2,3-, and 2,5-DHBA by a high-pressure liquid chromatography-electrochemical (HPLC-EC) procedure. 1-Methyl-4-phenylpyridinium ions (MPP+, 0 to 150 nmol) increased dose-dependently the release of dopamine and the formation of DHBA. A positive linear correlation between the release of dopamine and the formation of 2,3- or 2,5-DHBA was observed (R2 = .98). The present results demonstrate the validity of the use of not only 2,3-DHBA but also 2,5-DHBA as an in vivo index of oxidative damage generated by reactive .OH radicals. In conclusion, the present study demonstrates a novel use of intracranial microdialysis of salicylic acid to assess the oxidative damage elicited by .OH in living brain.


Formation of the excited ferryl species following Fenton reaction. A concomitant chemiluminescence burst can be detected when hydrogen peroxide is injected into the solution containing either free or chelated ferrous iron. The duration of the burst is slightly longer than the time for the oxidation of Fe2+. The luminescence intensity is a quadratic function of the initial concentration of H2O2 and is dependent on Fe2+ concentration. The scavengers of hydroxyl radical effectively inhibit, while the previously added ferric ions enhance, the luminescence. It is proposed that the excited species responsible for the luminescence is created by the reaction of hydroxyl radical with ferric iron. The theoretical analysis based on such a mechanism can fairly well interpret the experimental observation. The present study suggests that an excited ferryl species may be formed in Fenton reaction.


Redox cycling of potential antitumor aziridinyl quinones. The formation of reactive oxygen intermediates (ROI) during redox cycling of newly synthesized potential antitumor 2,5-bis (1-aziridinyl)-1,4-benzoquinone (BABQ) derivatives has been studied by assaying the production of ROI (superoxide, hydroxyl radical, and hydrogen peroxide) by xanthine oxidase in the presence of BABQ derivatives. At low concentrations (< 10 microM) some BABQ derivatives turned out to inhibit the production of superoxide and hydroxyl radicals by xanthine oxidase, while the effect on the xanthine-oxidase-induced production of hydrogen peroxide was much less pronounced. Induction of DNA strand breaks by reactive oxygen species generated by xanthine oxidase was also inhibited by BABQ derivatives. The DNA damage was comparable to the amount of hydroxyl radicals produced. The inhibiting effect on hydroxyl radical production can be explained as a consequence of the lowered level of superoxide, which disrupts the Haber-Weiss reaction sequence. The inhibitory effect of BABQ derivatives on superoxide formation correlated with their one-electron reduction potentials: BABQ derivatives with a high reduction potential scavenge superoxide anion radicals produced by xanthine oxidase, leading to reduced BABQ species and production of hydrogen peroxide from reoxidation of reduced BABQ. This study, using a unique series of BABQ derivatives with an extended range of reduction potentials, demonstrates that the formation of superoxide and hydroxyl radicals by bioreductively activated antitumor quinones can in principle be uncoupled from alkylating activity.


Biological effects of cigarette smoke, wood smoke, and the smoke from plastics: the use of electron spin resonance. This review compares and contrasts the chemistry of cigarette smoke, wood smoke, and the smoke from plastics and building materials that is inhaled by persons trapped in fires. Cigarette smoke produces cancer, emphysema, and other diseases after a delay of years. Acute exposure to smoke in a fire can produce a loss of lung function and death after a delay of days or weeks. Tobacco smoke and the smoke inhaled in a burning building have some similarities from a chemical viewpoint. For example, both contain high concentrations of CO and other combustion products. In addition, both contain high concentrations of free radicals, and our laboratory has studied these free radicals, largely by electron spin resonance (ESR) methods, for about 15 years. This article reviews what is known about the radicals present in these different types of smokes and soots and tars and summarizes the evidence that suggests these radicals could be involved in cigarette-induced pathology and smoke-inhalation deaths. The combustion of all organic materials produces radicals, but (with the exception of the smoke from perfluoropolymers) the radicals that are detected by ESR methods (and thus the radicals that would reach the lungs) are not those that arise in the combustion process. Rather they arise from chemical reactions that occur in the smoke itself. Thus, a knowledge of the chemistry of the smoke is necessary to understand the nature of the radicals formed. Even materials as similar as cigarettes and wood (cellulose) produce smoke that contains radicals with very different lifetimes and chemical characteristics, and mechanistic rationales for this are discussed. Cigarette tar contains a semiquinone radical that is infinitely stable and can be directly observed by ESR. Aqueous extracts of cigarette tar, which contain this radical, reduce oxygen to superoxide and thus produce both hydrogen peroxide and the hydroxyl radical. These solutions both oxidize alpha-1-proteinase inhibitor (a1PI) and nick DNA. Because of the potential role of radicals in smoke-inhalation injury, we suggest that antioxidant therapy (such as use of an inhaler for persons brought out of a burning building) might prove efficacious.


Nitroxide-stimulated H2O2 decomposition by peroxidases and pseudoperoxidases. Nitroxide free radicals interact with Hb/metHb, Mb/metMb and with peroxidases/phenols to induce a catalase-like conversion of H2O2 to O2 (catalatic activity), without being substantially consumed in the process. The mechanism of this reaction is postulated to involve a one-electron oxidation of the nitroxide to the immonium oxene, which then reacts further to release oxygen and the nitroxide. An involvement of the immonium oxene in the reaction mechanism is consistent with ferryl heme reduction by nitroxides and a detection of the reduced nitroxide when the reaction mixture is supplemented with the two-electron reductant sodium borohydride. The nitroxide-induced catalatic activity is completely inhibited when the reaction mixture is supplemented with glutathione. Nitroxides suppress free radical formation by hydroperoxide-activated heme proteins, as inferred from their inhibition of the spin-trapping of glutathionyl radicals. H2O2 decomposition and a suppression of reactive free radical formation by heme proteins appears to be an antioxidant activity of nitroxides, which is distinct from their previously reported superoxide dismutating activity and which may be a factor in their protective action in models of cardiac reperfusion injury.


Platelet activating factor stimulates and primes the liver, Kupffer cells and neutrophils to release superoxide anion. Platelet activating factor (PAF) is considered a key mediator in eliciting the immunologic and metabolic consequences of endotoxic shock and sepsis. Release of oxygen-derived radicals is one of the important and relevant actions of PAF. This study examines the direct and priming effects of PAF on superoxide anion release by perfused liver, isolated Kupffer cells and blood neutrophils. One hour after PAF infusion at a dose of 2.2 micrograms/kg body weight a significant amount of superoxide release (0.71 +/- 0.1 nmol/min/g liver) was measured in the perfused liver compared with the control livers (0.2 +/- 0.01). In the in vitro presence of either phorbol ester or opsonized zymosan, superoxide release following PAF treatment in vivo was significantly increased to 1.36 +/- 0.2 and 4.29 +/- 0.36, respectively. The administration of PAF receptor antagonist (SDZ 63-441) almost completely inhibited the release of this radical. Kupffer cells (KC1, KC2, KC3) and blood neutrophils isolated from PAF-treated rats were also primed for increased production when these cells were challenged in vitro by the activator of protein kinase C, opsonin-coated zymosan as well as the chemotactic factors, complement 5a and F-met-leu-phe. PAF added in vitro to the perfused livers, isolated Kupffer cells or neutrophils from normal animals stimulated the release of superoxide with or without the above agonists. The direct stimulatory effect of PAF on superoxide release was inhibited by the PAF receptor antagonist in vitro. The role of PAF in the LPS-induced superoxide release by the perfused liver was also examined by the administration of PAF antagonist in endotoxic rats. The antagonist inhibited the LPS-mediated superoxide release at 1 hr, but not at 3 hr post-treatment. These results indicate that PAF stimulates and primes the hepatic elements to release superoxide. PAF may be an important factor during the early phase of endotoxemia, while other bioactive substances may take over at a later phase. Therefore, PAF is a key mediator that can directly enhance the release of toxic oxygen-derived radicals which may contribute to organ failure during endotoxemia or sepsis.


Interferon therapy for non-A, non-B hepatitis: a pilot study and review of the literature. We treated four cases of acute unresolving non-A, non-B hepatitis, and eleven cases of chronic non-A, non-B hepatitis with recombinant interferon alpha-2a for up to one year. The dose of interferon was initially 3 million units daily, and was gradually decreased to 1 million units three times weekly. The overall response rate was 80 percent (twelve out of fifteen cases) at the end of treatment. Relapse occurred after the cessation of treatment in seven of the eight cases of chronic disease responding to interferon therapy. In contrast, three of the four cases of acute unresolving hepatitis became (sero)negative for antibody to hepatitis C virus, and in three completely normal serum aminotransferase levels persisted for more than one year after therapy. It is urged that early recognition of non-A, non-B hepatitis should be striven for, because interferon therapy may lead to an improved prognosis of the disease, particularly in cases of possible transitional phase from acute to chronic disease.


Androgen receptor in cirrhotic liver, adenomatous hyperplastic nodule and hepatocellular carcinoma in the human. Androgen receptors (AR) were assayed for cirrhotic liver, adenomatous hyperplastic nodule (AHN), and hepatocellular carcinoma (HCC) which were removed by partial hepatic resection from five patients. There were three men and two women. Age ranged from 59 to 68 years. Underlying cirrhosis was macronodular in three and micronodular in two. AHN was present within HCC in two patients, but these two lesions were found in different lobes of the same liver in another two patients. Only AHN was seen in the last patient. ARs in the cytosol of cirrhotic liver ranged from nil to 12.8 fmol/mg of protein with the dissociation constant (Kd) of 3.2-20.3 x 10(-10) M. AHNs possessed ARs ranging from 4.0 to 27.6 fmol/mg of protein (Kd values, 7.4-21.0 x 10(-10) M. All HCC nodules had ARs ranging from 11.8 to 72.8 fml/mg of protein with Kd values of 5.3-27.1 x 10(-10) M. Thus, cytosolic AR concentrations were highest in HCC, lowest in cirrhotic liver, and intermediate in AHN. The present study seems to indicate that AHN may be a precancerous lesion and that androgen and its receptor play an important role in human hepatocarcinogenesis.


Mucin-producing cystic adenocarcinoma of the pancreas, a case report, 7-year follow-up period. Mucinous ductal ectasia is recognized as a premalignant disease. Recently, we have encountered a patient with a mucin-producing neoplasm of the pancreas, who had been under periodic observation for chronic pancreatitis for 7 years prior to diagnosis of carcinoma and surgery. A 54-year-old male who had been investigated with US, CT and ERCP for chronic pancreatitis developed a mucin-producing ductal carcinoma of the pancreas. In this case a series of US and ERCP images obtained during the 7-year period had demonstrated insidious growth of the tumor. Pancreaticoduodenectomy was carried out, and the histopathological diagnosis was mucin-producing adenocarcinoma of the pancreas. The patient has been well and has suffered no recurrence for 2 years after surgery.


Internalization of monoclonal antibodies selected for immunotoxin activity against small-cell lung cancer. Two hybridomas producing MOABs with anti-SCLC activity were selected for immunotoxin activity by an indirect screen and were twice cloned. Binding activity of the MOABs to SCLC cells was demonstrated by immunoperoxidase activity, which could be blocked by streptavidin. The MOABs mediated the internalization of a biotinylated Fab' anti-mouse Ig marker at 37 degrees C. Internalization of the biotinylated marker by the SCLC target cells resulted in protection of the marker from streptavidin-blocking. These results show that MOABs selected for immunotoxin activity against SCLC can mediate internalization of an antibody fragment with a mass about 50% greater than that of the toxin. MOABs selected for immunotoxin activity may be useful for delivering agents other than toxins to the inside of SCLC cells.


Ultrastructural studies on interaction of infectious bursal disease virus (IBDV) and aflatoxin B1 on chick embryo fibroblast cell culture. Light and electron microscopic evaluation of chick embryo fibroblast (CEF) cell culture inoculated with graded doses (0.25, 2.5 and 25 micrograms/ml medium) of aflatoxin B1 with and without infectious bursal disease virus (IBDV) was undertaken. The light microscopy revealed degeneration, detachment and necrosis of fibroblasts and multiple plaques formation in IBDV infected group without and with (0.25, 2.5 micrograms) aflatoxin B1. The cultures infected with virus, with or without 25 micrograms aflatoxin B1 showed complete detachment from glass surface. Electron microscopy of these cultures showed marked pyknotic or bizarre shaped nuclei, pronounced degenerative changes in the rough endoplasmic reticulum (RER), mitochondria and the presence of multiple vacuoles in the cytoplasm. The viruses were spherical, arrayed, complete, generally closer to nuclei and RER and indistinctly membrane bound. The viruses were either localised or scattered in the cytoplasm. Cultures containing 25 micrograms aflatoxin B1 without or infected with virus showed marked necrosis of cells. In latter group only a few viruses were seen either in infected cells or free in culture. Control cultures failed to show cytopathic changes as observed in the other three groups.


Hypoglycemic effects of Coccinia indica: inhibition of key gluconeogenic enzyme, glucose-6-phosphatase. Coccinia indica (Family: Cucurbitaceae, locally known as telakucha) leaves were extracted with 95% ethanol. Following evaporation of the solvents, the residue was suspended in distilled water. When this suspension was fed orally to male normal-fed and 48-hr starved rats, the blood glucose was lowered 21% (P less than 0.01) in normal-fed and 24% (P less than 0.001) in 48-hr starved animals respectively. Starvation had induced a 3-fold increase in the activity of glucose-6-phosphatase and this activity was depressed 19% (P less than 0.05) by extract feeding while basal activity of the enzyme in normal-fed rats remained unaffected. Consistent with the depression of glucose-6-phosphatase, urea cycle enzyme arginase was also depressed 21% (P less than 0.001) and 12% (P less than 0.01) in the liver of 48 hr-starved and normal-fed animals respectively. Unlike glucose-6-phosphatase, starvation induced levels of gluconeogenic enzymes alanine aminotransferase and aspartate aminotransferase were not affected by Coccinia extract. These results suggest that the hypoglycemic effect of C. indica is partly due to the repression of the key gluconeogenic enzyme glucose-6-phosphatase.


Adenosine triphosphate content of Mycobacterium leprae by percoll buoyant density centrifugation. Thirty nine untreated patients of bacilliferous leprosy with a mean bacteriological index of 4.8 and morphological index of 1.3% formed the study group. Adenosine triphosphate assay was carried out by (i) enzyme treatment method in 18 patients and (ii) percoll buoyant density gradient method in 21 patients. ATP content obtained by percoll buoyant density gradient method was significantly higher than that obtained by enzyme treatment method. Percoll buoyant density centrifugation for purification and isolation of bacilli from human leproma is simplier, quicker and can serve as an alternate method of enzyme treatment.