Suppression of spontaneous hepatocellular carcinoma development in C3H/HeNCrj mice by the lipophilic ascorbic acid, 2-O-octadecylascorbic acid (CV-3611). The study was performed to examine the effects of the lipophilic ascorbic acid, 2-O-octadecylascorbic acid (CV-3611), with a strong scavenging capacity for active oxygen species, on the spontaneous development of liver tumors in male C3H/HeNCrj mice. Animals were given a diet containing 0.1% CV-3611 for a total experimental period of 16 months. Hepatocellular carcinomas developed in 2/39 (5%) of these experimental mice and in 11/43 (26%) of control mice fed a basal diet. The numbers of carcinoma per mouse were 0.05 +/- 0.22 and 0.33 +/- 0.61 respectively. Thus, CV-3611 clearly suppressed the development of hepatocellular carcinomas. However, the scavenger did not affect either the incidence or the number of hepatocellular adenomas, suggesting that active oxygen species might be involved in the conversion of adenomas to carcinomas in spontaneous liver carcinogenesis in C3H/HeNCrj mice.


Embryonic lesions of the substantia nigra prevent the patchy expression of opiate receptors, but not the segregation of patch and matrix compartment neurons, in the developing rat striatum. Unilateral lesions of the substantia nigra on embryonic day 19 prevent the development of the normal patchy distribution of opiate receptors in the ipsilateral rat striatum. Independent, early and permanent labelling of patch compartment neurons in the same brains on embryonic day 14 with [3H]thymidine revealed that the substantia nigra lesions did not prevent the aggregation of early born neurons into patches, but rather blocked the normal expression of one phenotype (dense opiate receptor binding) of these patches. Thus, early nigrostriatal connections may not be critical for the fundamental patch/matrix compartmentation of the striatum, but may be important in the maturation of phenotypic markers of these compartments.


Differential development of beta-endorphin and mu opioid binding sites in mouse brain. Mouse brains of various ages from embryonal day 14 (E14) to adult were analyzed for opioid receptor binding using the enkephalin analog Tyr-D-Ala-Gly-NMe-Phe-Gly-ol (DAMGE) and the opiate alkaloid dihydromorphine (DHM) as mu-selective radioligands. Binding parameters were estimated from homologous and heterologous competition binding curves. During the postnatal period, Kd values for [3H]DAMGE did not change but Bmax values (fmol/mg protein) increased 2.7 fold from postnatal day 3 (P3) to P7. Minor receptor density fluctuations were evident from P7 to adult. Similar results were obtained with [3H]DHM. In contrast, estimation of total mu binding sites (fmol/brain) revealed a continuous rise from P3 to the adult. The postnatal developmental profile of total mu binding sites was comparable to the weight gain of mouse brain and the increase in protein content. In contrast, during the same period beta-endorphin immunoreactivity (IR) levels undergo an increase that is inversely proportional to mu opioid receptor Bmax values. [3H]DAMGE binding to E14 membrane preparations was inhibited to a greater extent by Gpp(NH)p than that to P1 or adult. Additional characterization of mu receptors was accomplished by heterologous competition binding assays. IC50 values for beta-endorphin in competition with [3H]DHM and [3H]DAMGE were age dependent and differed for the two radioligands. These results suggest that mu receptor selectivity for mu-specific peptide and alkaloid ligands changes as a function of age.


The ouabain-dependent Na(+)-K+ pump and the brain renin-angiotensin system. The present study investigated the role of ouabain-dependent inhibition of the Na(+)-K+ pump and stimulation of the brain renin-angiotensin system by looking at 1) the short-term and long-term effects of ouabain on arterial blood pressure, and 2) the acute and chronic effects of angiotensin II (ANG II) intraventricularly (i.c.v.) on the release of an endogenous inhibitor of the Na(+)-K+ pump. Ouabain infused subcutaneously in a dose of 1.5 mg.kg-1. 24 h-1 for 7 days did not affect arterial blood pressure in rats, whereas increases in both blood pressure and weight were observed in rats infused with ouabain at the same dose for a 4-week period. Plasma supernate obtained from pentobarbital-anesthetized dogs acutely treated with ANG II (1 microgram i.c.v. every 30 min for 2 h) induced a 44% decrease in the ouabain-sensitive 86Rb uptake by the rat tail artery which was prevented by pretreatment with saralasin i.c.v. Plasma supernate obtained from dogs that were infused for 4 days with ANG II (20 ng/min i.c.v.) and received saline as the drinking fluid also reduced by 34% the ouabain-sensitive 86Rb uptake by the rat tail artery. The present study provides evidence that chronic inhibition of the Na(+)-K+ pump for 4 weeks leads to the development of hypertension and that the release of an endogenous inhibitor of the Na(+)-K+ pump is implicated in the hypertension resulting from chronic stimulation of the brain angiotensin-system and an increase in sodium chloride intake.


Effects of immobilization stress on renal sympathetic neurotransmission. The effects immobilization stress on renal sympathetic neurotransmission as well as on heart, spleen and adrenal catecholamine content were examined in the rat. A single 2.5 hr stress period produced significant increases in blood pressure, heart rate, plasma norepinephrine and plasma epinephrine concentrations. However, no changes in renal catecholamine content or in stimulus-induced (1 Hz, 120 pulses, supramax. V) overflow of catecholamines were observed when the isolated perfused rat kidney was studied immediately after the 2.5 hr stress period. In contrast, the single stress period produced a 3-4 fold increase in cardiac epinephrine content while no effects on spleen or adrenal catecholamine content were observed. When stress was applied for 7 daily 2.5 hr periods, the repetition of the stress failed to produce any changes in renal neurotransmitter content or stimulus-induced overflow from the isolated perfused rat kidney. The data suggest that the accumulation of epinephrine into peripheral sympathetic nerves as a result of stress-induced adrenal catecholamine release is not a phenomenon which can be generalized to all regions of the cardiovascular system.


Erythrocyte sodium-lithium countertransport in Chinese: its relationship to family history of hypertension. Rates of sodium (Na+)-stimulated lithium (Li+) efflux (Na(+)-Li+ countertransport) and ouabain-sensitive Na+ efflux (Na+ pump) were determined in erythrocytes of Chinese normotensive and hypertensive subjects. Near-maximal rate of Na(+)-Li+ countertransport was found to be significantly higher in hypertensive than normotensive subjects. No significant difference was observed for the rate of Na+ pump between them. A second series of study involved normotensive subjects without and with hypertensive parent(s) (group A and B, respectively) and hypertensive subjects (group C). We found that the rate of Na(+)-Li+ countertransport in group A was significantly lower than that of group B and C, while no difference existed between group B and C. No significant difference was observed for the rate of Na+ pump among the three groups. Our results suggested that Na(+)-Li+ countertransport activity could be a genetic marker for essential hypertension in Chinese, similar to that as proposed in Caucasians.


Pernicious anaemia in Africans.

Pernicious anaemia in Africans. Ten cases of pernicious anaemia seen over a 15-year period (1973-1988) in a Lagos hospital are presented. Their ages ranged from 34 to 67 with a mean of 53.6 years. Females outnumbered males 6 to 4. Complications seen include gastric carcinoma, myelopathy, peripheral neuropathy, skin hyperpigmentation, hair depigmentation and diarrhoea. Reluctance to consider the diagnosis owing to firmly held notions of its rarity and a penchant for empirically treating chronic anaemias with all available haematinics and blood transfusion are probably contributory to its underdiagnosis. The fact that seven of the patients presented were seen in the last three years and three of them in the last one year raises the possibility of an increasing incidence of pernicious anaemia in Africans. The disease may be much less rare in Africans than once believed, and medical education should emphasize its existence and advocate greater care in the management of chronic anaemias.


Scarified photopatch testing in lomefloxacin photosensitivity. We report the results of scarified photopatch testing in 3 patients with photosensitivity to lomefloxacin, a new quinolone antibacterial agent introduced onto the Japanese market in April 1990. The patients developed pruritic eczematous lesions on sun-exposed areas 5 days to 3 weeks after they had started taking lomefloxacin. 2 cases reacted positively to lomefloxacin on scarified photopatch testing down to 0.1% pet., and 1 patient down to 10% pet. One of them also showed positive reactions on conventional photopatch testing. Scarified photopatch testing with 7 other pyridone carbonate derivatives in 2 of the 3 cases, however, did not show any positive reactions. Patch and scarified patch testing were negative in all patients. The clinical course, clinical and histological findings and results of skin tests in these patients suggest that photoallergic mechanisms are involved. This is the 1st report of positive reactions on scarified photopatch testing, which appears to be a useful method of detecting the causative drug in drug-induced photosensitivity.


Evidence that the insulin resistance of pregnancy may not involve a post-receptor defect in human adipocytes. Insulin binding and the capacity of insulin to stimulate the conversion of glucose to carbon dioxide and lipid, and to activate the protein tyrosine kinase associated with the insulin receptor have been investigated in adipocytes isolated from pregnant and non-pregnant women. Insulin binding and the conversion of glucose to lipid were the same for both groups. However, conversion of glucose to CO2 was higher in the non-pregnant group due to an elevated basal activity, and the increase produced by insulin was similar in both groups. The tyrosine kinase activity of the isolated receptor preparations was higher in the pregnant group due to an increase in the basal non-insulin dependent activity, and the increase produced by insulin was similar in both groups. These findings show the in vitro insulin responsiveness of isolated adipocytes is similar for both groups, and suggests that the in vivo insulin resistance of late pregnancy, as far as adipose tissue is concerned, is not due to any inherent defect in insulin action at the receptor or post-receptor level. In vivo insulin resistance may result from an increased level of circulating insulin antagonists.


Changes in polyphosphoinositides and phosphatidic acid of erythrocyte membranes in diabetes. We studied metabolic pool size of polyphosphoinositides and phosphatidate of erythrocyte membranes from normal and diabetic subjects using 32P for 20-h incubation, a sufficiently long period to reach isotopic equilibrium between monoesterphosphate bond and gamma-phosphate of ATP. Phosphatidylinositol 4-monophosphate (PtdIns4P), phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) and phosphatidate were the phospholipids labelled. Metabolic pools of individual phospholipids were estimated, based on their proportionate and absolute radioactivity. A significant decline in radioactivity of phosphatidate and PtdIns(4,5)P2 was seen in erythrocytes from the diabetic subjects, indicating suppression of the metabolically labile pool of these two phospholipids. There was no significant change in PtdIns4P radioactivity between the groups. The direct effect of insulin on phosphorylation of polyphosphoinositides and phosphatidate was also evaluated by a short incubation period of erythrocyte membranes with [gamma-32P]-ATP. Added insulin increased the incorporation of 32P into phosphatidate in a dose-dependent manner that reached a steady state at 2 nM. We conclude that the metabolically labile pool size of phosphatidate is decreased and that of polyphosphoinositides is altered in erythrocyte membranes from diabetic patients.


Utilization of three-dimensional culture for early morphometric and electrophysiological analyses of solitary cerebellar neurons. When cells from the cerebellum of late-gestation rats were grown at low density (50-100 cells/mm2) in a three-dimensional culture system, they rapidly expressed morphological and electrophysiological properties of neurons. Growth and differentiation of the population of solitary neurons as a whole was statistically assessed at 6, 12, 18, 24, 48 and 72 h using data obtained from image analysis. Mean length of axons and dendrites increased 17- and 220-fold, respectively, from 6 to 72 h. Average number of branch points rose 35-fold. Other indices of complexity increased 2- to 3-fold. Whole-cell voltage clamp revealed that as early as 24-30 h the cells displayed a tetrodotoxin-sensitive Na+ current (INa), a nifedipine-sensitive high-threshold Ca2+ current, a Ni(2+)-sensitive low-threshold Ca2+ current, and two voltage-dependent K+ outward currents consisting of a 4-aminopyridine-sensitive fast transient outward current and a CsCl-sensitive slow delayed component. These observations correlate closely with voltage-activated currents previously recorded in neonatal or young rat cerebellum, and demonstrate that the culture model is useful for analyzing the early rapid growth, differentiation and intrinsic ionic currents of these neurons as single cells.


The 46/50 kDa phosphoprotein VASP purified from human platelets is a novel protein associated with actin filaments and focal contacts. Vasoactive agents which elevate either cGMP or cAMP inhibit platelet activation by pathways sharing at least one component, the 46/50 kDa vasodilator-stimulated phosphoprotein (VASP). VASP is stoichiometrically phosphorylated by both cGMP-dependent and cAMP-dependent protein kinases in intact human platelets, and its phosphorylation correlates very well with platelet inhibition caused by cGMP- and cAMP-elevating agents. Here we report that in human platelets spread on glass, VASP is associated predominantly with the distal parts of radial microfilament bundles and with microfilaments outlining the periphery, whereas less VASP is associated with a central microfilamentous ring. VASP is also detectable in a variety of different cell types including fibroblasts and epithelial cells. In fibroblasts, VASP is concentrated at focal contact areas, along microfilament bundles (stress fibres) in a punctate pattern, in the periphery of protruding lamellae, and is phosphorylated by cGMP- and cAMP-dependent protein kinases in response to appropriate stimuli. Evidence for the direct binding of VASP to F-actin is also presented. The data demonstrate that VASP is a novel phosphoprotein associated with actin filaments and focal contact areas, i.e. transmembrane junctions between microfilaments and the extracellular matrix.


Muscular fatigability in mitochondrial myopathies. An electrophysiological study. A frequent occurrence of ophthalmoplegia and muscle fatigability in mitochondrial myopathy (MAM) often makes its differential diagnosis from myasthenia rather difficult. Neuromuscular transmission was investigated in 9 patients with MAM, presenting marked fatigability. The aim of the study was to see whether there were any other causes of muscle fatigability in addition to the metabolic factors. Classical electrostimulation as well as the SFEMG, which is very sensitive in detecting neuromuscular transmission disorders, were used. The findings were far from uniform: we found normal neuromuscular transmission in 5 cases, in 3 patients we observed slight abnormalities of neuromuscular transmission, in 1 case neuromuscular transmission disturbances seemed to be of neurogenic origin. Our results allow an assumption that the causes of muscle fatigability in MAM are of a much more complex nature than it has been anticipated. They might depend not only on the metabolic disorders within the muscle fibre itself but also on the impaired function of the peripheral nerve or of the neuromuscular junction. All the mechanisms combined may also play a role, though in individual patients the contribution of particular factors responsible may vary.


MAP kinase activator from insulin-stimulated skeletal muscle is a protein threonine/tyrosine kinase. A 'MAP kinase activator' was purified several thousand-fold from insulin-stimulated rabbit skeletal muscle, which resembled the 'activator' from nerve growth factor-stimulated PC12 cells in that it could be inactivated by incubation with protein phosphatase 2A, but not by protein tyrosine phosphatases and its apparent molecular mass was 45-50 kDa. In the presence of MgATP, 'MAP kinase activator' converted the normal 'wild-type' 42 kDa MAP kinase from an inactive dephosphorylated form to the fully active diphosphorylated species. Phosphorylation occurred on the same threonine and tyrosine residues which are phosphorylated in vivo in response to growth factors or phorbol esters. A mutant MAP kinase produced by changing a lysine at the active centre to arginine was phosphorylated in an identical manner by the 'MAP kinase activator', but no activity was generated. The results demonstrate that 'MAP kinase activator' is a protein kinase (MAP kinase kinase) and not a protein that stimulates the autophosphorylation of MAP kinase. MAP kinase kinase is the first established example of a protein kinase that can phosphorylate an exogenous protein on threonine as well as tyrosine residues.


Cell cycle regulated phosphorylation of RPA-32 occurs within the replication initiation complex. The transition from G1 to S phase of the cell cycle may be regulated by modification of proteins which are essential for initiating DNA replication. One of the first events during initiation is to unwind the origin DNA and this requires a single-stranded DNA binding protein. RPA, a highly conserved multi-subunit single-stranded DNA binding protein, was first identified as a cellular protein necessary for the initiation of SV40 DNA replication. The 32 kDa subunit of RPA has been shown to be phosphorylated at the start of S phase. Using SV40 replication as a model, we have reproduced in vitro the S phase-dependent phosphorylation of RPA-32 and show that it occurs specifically within the replication initiation complex. Phosphorylated RPA-32 is predominantly associated with DNA. Phosphorylation is not a pre-requisite for association with DNA, but occurs after RPA binds to single-stranded DNA formed at the origin during the initiation phase. The protein kinase(s) which phosphorylates RPA-32 is present at all stages of the cell cycle but RPA-32 does not bind to the SV40 origin or become phosphorylated in extracts from G1 cells. Therefore, the cell cycle-dependent phosphorylation of RPA-32 may be regulated by its binding to single-stranded origin DNA during replication initiation.


cdc2 family kinases phosphorylate a human cell DNA replication factor, RPA, and activate DNA replication. RPA is a single-stranded DNA binding protein complex purified from human cells and is essential for the initiation and elongation stages of SV40 DNA replication in vitro. In both human and yeast cells, the 34 kDa polypeptide subunit of RPA is phosphorylated in the S and G2 phases of the cell cycle and not in G1. One of the major RPA kinases present in extracts of human cells was purified and shown to be the cyclin B-cdc2 complex. This purified kinase, and a closely related cyclin A associated cdc2-like kinase, phosphorylated RPA p34 on a subset of the chymotryptic peptides that were phosphorylated in vivo at the G1-S transition. Two serines near the N-terminus of RPA p34 were identified as possible sites of phosphorylation by cdc2 kinase. These same serines were necessary for RPA phosphorylation in vivo. The purified cdc2 kinase stimulated SV40 DNA replication in vitro when added to G1 cell extracts. The kinase also stimulated unwinding at the origin of replication, one of the earliest steps in DNA replication requiring RPA, but only in the presence of an additional factor present in G1 cell extracts. Thus, one or more members of the cyclin-cdc2 kinase family may be required for the initiation and maintenance of S phase, in part due to their ability to phosphorylate and activate a cellular DNA replication factor, RPA.


Interference between pathway-specific transcription factors: glucocorticoids antagonize phorbol ester-induced AP-1 activity without altering AP-1 site occupation in vivo. Phorbol esters stimulate and glucocorticoid hormones down-regulate a variety of promoters such as that of the collagenase gene through the transcription factor AP-1 (Fos/Jun). We now show by genomic footprinting of the collagenase promoter that phorbol ester treatment of cells results in the binding of AP-1 to its cognate DNA binding site in vivo. The DNA-protein contacts obtained in living cells are also found in vitro using cloned DNA and purified AP-1. Although in vitro synthesized glucocorticoid receptor can disturb the DNA binding of Jun homodimers, it does not interfere with the binding of Fos-Jun heterodimers or of purified AP-1 in vitro. Consistently, fully inhibitory doses of glucocorticoid hormone cause no change in apparent occupation of the AP-1 binding site in vivo. The hormone receptor acts without itself binding to DNA.


Transcriptional enhancer factor (TEF)-1 and its cell-specific co-activator activate human papillomavirus-16 E6 and E7 oncogene transcription in keratinocytes and cervical carcinoma cells. The human papillomavirus (HPV)-16 oncogenes, E6 and E7, are transcribed preferentially in keratinocytes and cervical carcinoma cells due to a 5' enhancer. An abundant peptide binding to a 37 nt enhancer element was purified from human keratinocytes by sequence-specific DNA chromatography. This protein was identified as transcriptional enhancer factor (TEF)-1 by complex mobility, binding to wild-type and mutant SV40 and HPV-16 enhansons and antigenic reactivity with two anti-TEF-1 antibodies. TEF-1 is cell-specific, but its transactivation also depends on a limiting, cell-specific TEF-1 'co-activator'. We show that both TEF-1 and the TEF-1 co-activator are active in human keratinocytes and essential for HPV-16 transcription. TEF-1 binding in vivo was necessary for HPV-16 P97 promoter activity. Excess TEF-1 and chimeric GAL4-TEF-1 specifically inhibited the P97 promoter by 'squelching', indicating that HPV-16 transcription also requires a limiting TEF-1 co-activator. TEF-1 and the TEF-1 co-activator functions mirrored HPV-16 transcription by their presence in keratinocytes and cervical carcinoma cells and their absence from lymphoid B-cells, but also functioned in liver cells where the HPV-16 promoter is inactive. TEF-1 and its associated co-activator are thus part of a complex mechanism which determines the restricted cell range of the HPV-16 E6 and E7 oncogene promoter.


Retinoic acid-mediated repression of human papillomavirus 18 transcription and different ligand regulation of the retinoic acid receptor beta gene in non-tumorigenic and tumorigenic HeLa hybrid cells. Human papillomavirus type 18 (HPV18) belongs to the group of genital papillomaviruses involved in the development of cervical carcinomas. Since retinoic acid (RA) is a key regulator of epithelial cell differentiation and a growth inhibitor in vitro of HPV18-positive HeLa cervical carcinoma cells, we have used HeLa and HeLa hybrid cells in order to analyse the effects of RA on expression of the HPV18 E6 and E7 oncogenes and of the cellular RA receptor genes RAR-beta and -gamma. We show here that RA down-regulates HPV18 mRNA levels apparently due to transcriptional repression. Transient cotransfection assays indicated that RARs negatively regulate the HPV18 upstream regulatory region and that the central enhancer can confer RA-dependent repression on a heterologous promoter. RA treatment resulted in induction of RAR-beta mRNA levels in non-tumorigenic HeLa hybrid cells, but not in tumorigenic hybrid segregants nor in HeLa cells. No alterations of the RAR-beta gene or of the HeLa RAR-beta promoter could be revealed by Southern and DNA sequence analysis, respectively. As determined by transient transfection assays, however, the RAR-beta control region was activated by RA more strongly in non-tumorigenic hybrid cells than in HeLa cells, thus indicating differences in trans-acting regulatory factors. Our data suggest that the RARs are potential negative regulators of HPV18 E6 and E7 gene expression, and that dysregulation of the RAR-beta gene either causatively contributes to or is an indicator of tumorigenicity in HeLa and HeLa hybrid cells.


Platelet alpha 2 adrenoceptor characteristics during the morning increase in platelet aggregability. The incidence of myocardial infarction and sudden cardiac death increases in the morning, as do platelet aggregability and sympathetic activity. We considered the possibility that increased platelet aggregability in the morning was due to an increase in platelet alpha 2 adrenergic receptor number or agonist binding affinity, or to a temporal disparity between the increase in sympathetic activity that accompanies arising and the anticipated decrease in platelet high affinity alpha 2 adrenoceptors. To evaluate these possibilities, we studied eight healthy male volunteers (20-35 years) before and 1-1.5 and 3-4 h after they arose at 08.00 h and performed routine morning activities. Platelet aggregability was determined in platelet-rich plasma; alpha 2 adrenoceptor density and agonist binding affinity were determined in intact platelets and in membrane preparations using the alpha 2 selective ligand [3H]-yohimbine. The threshold concentration of epinephrine required to produce biphasic aggregation decreased (i.e. platelet aggregability increased) from 3.6 +/- 1.2 to 0.9 +/- 0.3 microM (P less than 0.05) after arising. However, alpha 2 adrenoceptor density (380 +/- 71 to 365 +/- 51 sites per platelet) and agonist binding affinity assessed simultaneously did not change after arising, suggesting that the increase in platelet aggregability is due to factors extrinsic to the platelets or to an intra-platelet mechanism distal to the receptor level.


The fate of the three subunits of major histocompatibility complex class I molecules. At the surface of the murine T lymphoma cell line RMA-S, the expression of "empty" class I molecules can be dramatically enhanced by culture at 26 degrees C. These class I molecules are unstable following transfer to 37 degrees C unless they are loaded with exogenously added peptides. Class I heterodimers that have failed to bind peptide ("empty" class I molecules) dissociate and the class I heavy chains are degraded. Internalization, if it precedes breakdown, would be the rate-limiting step. Radioiodinated peptides (VSV NP 8-mer or Sendai NP 9-mer) dissociate from the class I molecules in the absence of exogenous peptide or beta 2 microglobulin and appear in the medium. Release of the iodinated peptides does not result in a reduction in the quantity of stable assembled class I molecules. This paradox may be explained by a more rapid off-rate for radioiodinated peptides, when compared with their unlabeled counterparts, which constitute about 99% of the total in our radiolabeled preparations. In the medium the peptide is rapidly modified by serum- and cell-derived proteases. The short half-life of empty class I molecules and of free ligand would effectively preclude sensitization of innocent bystanders for lysis by cytotoxic T lymphocytes.


A viral peptide can mimic an endogenous peptide for allorecognition of a major histocompatibility complex class I product. Alloreactive class I-restricted T cells may recognize the class I structure alone, in association with a specific peptide, or with any stabilizing peptide. We have tested the role of endogenous peptides in the recognition of H-2Kb molecules by two alloreactive cytolytic T lymphocyte (CTL) clones using the mutant tumor line RMA-S, which expresses its surface H-2b molecules devoid of peptides and is not lysed by these two CTL clones. Empty H-2b molecules on RMA-S cells can be stabilized by binding exogenously added peptides. H-2Kb-specific recognition of the RMA-S cells by one of the CTL clones was restored by endogenous peptide extracts which only minimally stabilized H-2Kb on the surface of RMA-S cells, indicating the requirement for a specific peptide on a limited number of H-2Kb molecules. In addition, one out of three peptides which greatly enhance the expression of H-2Kb, the nucleoprotein peptide 52-59 from vesicular stomatitis virus (VSV), was also able to restore the lysis of RMA-S cells by the clone. The recognition of a common motif by an alloreactive clone (H-2k anti-H-2Kb) and virus-specific Kb-restricted clones suggests that both H-2k and H-2b thymic environments allow selection of T cells capable of recognizing H-2Kb+VSV and that tolerance to self, as would be the case in the (H-2k x H-2b)F1 mice, would partially delete the repertoire of antiviral T cells.


High affinity histamine H3 receptors regulate ACTH release by AtT-20 cells. The distribution of high affinity histamine H3 receptors in various tissues from guinea pig has been determined using [3H]N alpha-methylhistamine binding. In the course of those studies, it was observed that the pituitary gland contains H3 receptors. Using this radioligand, we have now identified and characterized H3 receptors on' the AtT-20 cell line from a murine anterior pituitary tumor. This line has approximately 5000 high affinity (KD = 0.7 nM) H3 binding sites per cell. Competition binding with standard H1, H2 and H3 agents has confirmed that these sites are, indeed, H3 receptors. The H3 receptor specific agonist, (R)-alpha-methylhistamine increased the release of adrenocorticotropic hormone (ACTH) from AtT-20 cells in a dose- and time-dependent manner, while histamine and the H2 agonist dimaprit were significantly less potent. Furthermore, this response was blocked by thioperamide, an H3 receptor specific antagonist, but not by the H1 and H3 antagonists, chlorpeniramine and cimetidine. These results identify, for the first time, a cell line expressing H3 receptors and indicate that the high affinity histamine H3 receptor regulates ACTH release from that cell.


Effects of selective opioid receptor agonists and antagonists during myocardial ischaemia. The antiarrhythmic activities of 16-methylcyprenorphine (M8008), nor-binaltorphimine (NBT) and naltrexone, which are relatively specific opioid receptor antagonists for delta, kappa and mu receptors, respectively, were examined during the 30 min following coronary artery occlusion in anaesthetised rats. The haemodynamic and electrocardiographic effects of the opioid receptor agonists [D-Ala2,D-Leu5]enkephalin (DADLE) (relatively selective for delta receptors), ICI-204448 (kappa) and glyol (mu) were also investigated over the 30-90 min post ligation period. When administered intravenously 5 min before ligation, M8008 (0.5 mg kg-1 and 2.5 mg kg-1) reduced the number of ventricular ectopic beats but had no effect on the incidence or duration of ventricular fibrillation. NBT and naltrexone were not antiarrhythmic at a dose of 0.5 mg kg-1 but at 2.5 mg kg-1 (a concentration at which both drugs block kappa receptors) the number of ventricular ectopic beats, the incidence of ventricular fibrillation and mortality were all reduced. All of the opioid receptor agonists caused a transient decrease in heart rate and in arterial blood pressure but none exhibited an arrhythmogenic effect. These studies suggest that the delta and kappa opioid receptor antagonists used may be antiarrhythmic as a result of blockade of the action of endogenously released peptides acting on these receptors or that they have a non-specific 'direct' antiarrhythmic action.


The electrophysiological effects of opioid receptor-selective antagonists on sheep Purkinje fibres. The cardiac electrophysiological effects of 16-methylcyprenorphine (M8008), nor-binaltorphimine (NBT) and naltrexone, which are relatively specific opioid antagonists for delta, kappa and mu receptors, respectively, were studied in paced (1.5 Hz) sheep Purkinje fibres in vitro. M8008 (1 ng ml-1-10 micrograms ml-1) caused a concentration-dependent reduction in the maximum rate of depolarisation of phase 0 (MRD) and in the action potential duration measured at 50% repolarisation, APD50. Neither NBT (10 ng ml-1-10 micrograms ml-1) nor naltrexone (1 ng ml-1-10 micrograms ml-1) produced any significant effect on the cardiac action potential. In the presence of a physiological salt solution modified to mimic some of the changes that occur during myocardial ischaemia (i.e. hypoxia, acidosis, hyperkalaemia), M8008 caused a more marked reduction in MRD and prolonged rather than shortened APD50. These results suggest that the reported antiarrhythmic activity of M8008, but not NBT or naltrexone, may be, at least in part, explained by a direct cardiac electrophysiological action.


Endothelium-independent potentiating effects of neuropeptide Y in the rat tail artery. The role of the endothelium in the potentiating action of neuropeptide Y (NPY) to contraction induced by KCl, alpha, beta-methylene ATP (mATP), and noradrenaline (NA) was tested on rat tail arteries. Endothelium-intact and denuded ring segments and freshly isolated single smooth muscle cells were used in the study. Contraction responses to KCl and mATP were potentiated by NPY (50 nM) in both intact and denuded arteries. Contraction to NA was potentiated by NPY at 500 nM but not at 50 nM. The potentiation effect of NPY was antagonized by nifedipine. Similarly, the shortening of single smooth muscle cells in response to KCl and mATP was potentiated by NPY (50 nM). The noradrenaline response was potentiated by NPY at 500 nM but not at 50 nM. Our results suggest that the potentiating effect of NPY is more specific to contraction mediated by nifedipine-sensitive calcium channels and is not dependent on the presence of an intact endothelium.


Maintenance of acute morphine tolerance in mice by selective blockage of kappa opioid receptors with norbinaltorphimine. In this study we investigated the effect of the highly selective kappa opioid antagonist, norbinaltorphimine (norBNI) on the development of tolerance to a single dose of morphine. Mice were pretreated with 100 mg/kg of morphine sulfate (morphine), s.c. and 2 h later, norBNI (20 mg/kg s.c.) was administered and various times after this pretreatment, antinociceptive ED50 value of morphine was determined in the tail-flick assay. Twenty-four and 72 h after morphine injection, ED50 values of morphine were significantly increased by about 2.5-fold from those of their control mice that received saline instead of the tolerance-inducing dose of morphine. In a second set of experiments, animals were pretreated similarly with morphine and norBNI and 72 h after morphine injection, various opioid agonists were applied by the i.c.v. or i.t. route to see whether or not any cross-tolerance had developed to these agonists. The ED50 of i.c.v.-administered morphine was significantly greater than that of the non-pretreated controls. A small degree of cross-tolerance was observed with U-50,488H but not with DPDPE [D-Pen2,D-Pen5]enkephalin (DPDPE) at the supraspinal site. At the spinal site, tolerance to morphine was not observed. These results suggest that antagonism at kappa opioid sites after morphine administration, modulates positively the development of opioid tolerance.


Nitric oxide does not mediate the neurotrophic effects of excitatory amino acids in cultured cerebellar granule neurons. Excitatory amino acids, such as N-methyl-D-aspartate (NMDA) and kainate, promote neuritogenesis and viability in primary cultures of cerebellar granule cells. In view of the recent demonstration that excitatory amino acids activate the synthesis of nitric oxide, the present study examined a potential role of nitric oxide in mediating the neurotrophic effects of excitatory amino acids. NMDA enhanced the viability of 8-day-old cerebellar granule cell cultures in a concentration-dependent fashion, whereas kainate showed a concentration-dependent biphasic effect. A specific inhibitor of nitric oxide synthase, NG-nitroarginine (0.5 mM), did not antagonize the neurotrophic effects of NMDA (0.5 mM) or kainate (0.05 mM). The concentration of NG-nitroarginine was sufficient to inhibit NMDA or kainate stimulated nitric oxide synthesis and was stable in the culture media throughout the 8-day culture period. Using a specific chemiluminescence detection method, endogenous nitric oxide was directly measured in the headspace gas phase of homogenized cultured cerebellar granule cells, and was not detected when homogenates were incubated with NG-nitroarginine (0.5 mM). Furthermore, addition of a nitric oxide precursor, S-nitroso-N-acetylpenicillamine (1-200 microM), was not neurotrophic, but rather, was neurotoxic in granule cells. These findings indicate that nitric oxide is not a neurotrophic factor in primary cultures of cerebellar granule cells.


Splanchnic vascular endothelial dysfunction in rat endotoxemia: role of superoxide radicals. Intravenous lipopolysaccharide, 30 mg/kg, results in rapid systemic hypotension in anesthetized rats. Interaction of lipopolysaccharide with the vascular endothelium and blood borne cells results in the elaboration of cytokines and oxygen-derived free radicals, all of which can be injurious to normal endothelial function. To evaluate endothelial function, superior mesenteric artery rings were isolated from endotoxemic rats just prior to death. Endotoxemia significantly blunted superior mesenteric artery ring vasorelaxations to acetylcholine and to A23187 but not to NaNO2. Contraction of superior mesenteric artery rings from endotoxemic rats induced by U46619 was not altered. Treatment with human superoxide dismutase or U74006F, an aminosteroid, significantly preserved vasorelaxation to acetylcholine and A23187. However, the hydroxyl radical scavenger N-(2-mercaptopropionyl)-glycine did not protect the endothelium. Thus, intravenous lipopolysaccharide can induce endothelial dysfunction in superior mesenteric artery rings. Furthermore, because superoxide dismutase but not N-(2-mercaptopropionyl)-glycine preserves endothelial function, it is likely that superoxide radicals mediate the endothelial dysfunction observed in endotoxemic rats.


Pertussis toxin induces bronchopulmonary hyperresponsiveness in guinea-pigs while antagonizing the effects of formyl-L-methionyl-L-leucyl-L-phenylalanine. Pertussis toxin injected i.v. at 0.8-20 micrograms/kg markedly enhanced bronchoconstriction induced by the i.v. administration of histamine or serotonin (5-HT) (0.5-16 micrograms/kg) to propranolol-treated guinea-pigs, under conditions where propranolol or pertussis toxin alone were poorly effective. In contrast, bronchoconstriction and the accompanying leukopenia induced by the i.v. administration of the secretagogue formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) were suppressed by pertussis toxin. Bronchoconstriction induced by histamine or 5-HT was not enhanced when perfused lungs from pertussis toxin-treated guinea-pigs were studied in vitro, under conditions where bronchoconstriction and thromboxane A2 release evoked by fMLP were suppressed. Pertussis toxin negatively modifies signal transduction in cells involved in the lung responses to fMLP both in vivo and in vitro, but positively and only in vivo it modifies signal transduction in cells involved in the lung responses to the direct constricting agents histamine and 5-HT. As hyperresponsiveness to histamine and 5-HT were exclusively found in vivo, the target for pertussis toxin is probably not the adrenergic nor the cholinergic systems, since neither hexamethonium, isoprenaline, atropine nor vagotomy were effective. In addition, since dexamethasone and nedocromil sodium were inactive and enrichment of bronchoalveolar lavage with inflammatory cells was not noted, despite lung invasion by neutrophils and lymphocytes, acute inflammation does not account for pertussis toxin-induced hyperresponsiveness.


Epithelial modulation of the relaxant activity of atriopeptides in rat and guinea-pig tracheal smooth muscle. Three peptide components of atrial natriuretic factor (ANF) caused relaxation of carbachol-contracted guinea-pig isolated tracheal smooth muscle. These were the 1-28, 5-28 and 5-27 peptide sequences (ANF(1-28), ANF-(5-28) and ANF-(5-27)). The peptides were 10-30 times more potent in epithelium-denuded than in epithelium-intact preparations. In the absence of airway epithelium, ANF-(1-28) was the most potent relaxant (mean pD2 = 7.40 +/- 0.08), with ANF-(5-27) and ANF-(5-28) 2-3 fold less potent. The neutral endopeptidase inhibitor phosphoramidon (1 microM) increased the potency of ANF-(5-27) in both epithelium-intact and epithelium-denuded guinea-pig tracheal rings. In contrast, removal of the epithelium from rat trachea, or pretreatment with phosphoramidon (1 microM) decreased relaxant responsiveness to ANF-(5-27). Thus, in rat trachea, epithelial endopeptidase may convert ANF-(5-27) to a more active relaxant peptide. Human bronchial preparations with or without epithelium, obtained from non-diseased lung samples and from a single sample of asthmatic lung, were virtually unresponsive to ANF-(5-27). Consistent with the spasmolytic effects of ANF in guinea-pig trachea, autoradiographic analysis revealed the presence of a sparse population of specific binding sites for [125I]ANF-(1-28) over both tracheal smooth muscle and epithelium. The present study shows that the relaxant effects of atriopeptins in rat and guinea-pig airway smooth muscle were modulated by the epithelium and the activity of neutral endopeptidase. However, marked species differences in airway smooth muscle responsiveness to ANF and in the modulatory role of the airway epithelium were evident.


An endothelial cell-line contains functional vasoactive intestinal polypeptide receptors: they control inwardly rectifying K+ channels. Bovine endothelial cells cultured from pulmonary artery (ATCC cell line No. 209) were found to contain a high density of 125I-VIP (vasoactive intestinal polypeptide) binding sites. These were found to be saturable and to be fit by a single binding site model (Kd 1.8 nM; Bmax 534 fmol/mg protein). Studies of association and dissociation of 125I-VIP to this site revealed that binding was fully reversible and yielded a Kd value similar to that from equilibrium binding. However competition studies showed that VIP competed for binding at two sites (Ki1 1.2 x 10(-11) M, Ki2 4.7 x 10(-9) M; N1 = 21%, N2 = 77%; Ki a dissociation constant for inhibitor; N percentage of occupied receptors). [Phe1]VIP also competed at two sites, but VIP-(10-28), PHM, [4-Cl-D-Phe6,Leu17]VIP and [D-Ala4]VIP displaced all specific VIP binding in a simple competitive manner. These VIP binding sites were shown to be functional. In patch clamp studies VIP 10(-8)-10(-7) M inhibited opening of inwardly rectifying K+ channels on hyperpolarization. These channels were affected appropriately by alteration in the K(+)-gradient and by Ba2+ or Cs+. The VIP antagonist [4-Cl-D-Phe6, Leu17]VIP prevented or reversed the effects of VIP. These results show that functional VIP receptors are present in high density in a endothelial cell line and provide a possible model for analysis of the molecular biology of these receptors.


Neurotensin: dual effect on the motor activity of rat duodenum. The effects of neurotensin on mechanical activity of rat duodenum were investigated using an isometric-isovolumic preparation. Neurotensin (1 pM to 10 nM) induced a concentration-dependent, tetrodotoxin (TTX)-insensitive fall in both endoluminal pressure and isometric tension. At higher concentrations of neurotensin (1 nM to 1 microM) the relaxation was followed by a concentration-dependent TTX-insensitive contraction, detected only by an increase in endoluminal pressure. Different concentrations of neurotensin were required to desensitize the relaxant and the contractile actions of the neuropeptide. The relaxation was antagonized by apamin, while the contractile response was blocked by nifedipine. Neurotensin, when tested separately on longitudinal and circular muscular strips, caused relaxation of the longitudinal strips. Circular strips showed contractions in response to neurotensin, following an inhibitory phase, if the strips were spontaneously or pharmacologically activated. The results suggest the presence of two sets of neurotensin receptors with a differential localization between the two muscular layers in rat duodenum.


Endothelium-dependent relaxant effect of neurokinins on rabbit aorta is mediated by the NK1 receptor. Several neurokinins, namely substance P, neurokinin A, neurokinin B, [beta-Ala8]neurokinin A-(4-10) and senktide, were tested on noradrenaline-precontracted rabbit aortic rings to characterize the receptor mediating their endothelium-dependent relaxant effect in this preparation. CP-96,345, the new nonpeptide antagonist selective for the NK1 receptor, was also studied. Substance P, neurokinin A and neurokinin B, in that order of potency, were effective in relaxing precontracted rings, indicating the involvement of the NK1 receptor; [beta-Ala8]neurokinin A-(4-10) and senktide, which are selective agonists for NK2 and NK3 receptors, respectively, had no significant relaxant effect. The relaxant effects of substance P, neurokinin A and neurokinin B were competitively antagonized by nanomolar concentrations of CP-96,345. These findings support the view that the NK1 receptor mediates the endothelium-dependent relaxant effect of the neurokinins in rabbit aorta.


Effects of Cl- channel blockers on beta-adrenoceptor-mediated decreases in resting potential and intracellular Cl- activity in guinea-pig heart. In order to find a more specific blocker of the cardiac Cl- channel, we examined the effects of anthracene-9-carboxylic acid (9AC) and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) on the beta-adrenoceptor-mediated decreases in resting potential and intracellular chloride ion activity (aiCl) in guinea-pig papillary muscles by using Cl- ion selective microelectrodes. 9AC (1 mM) significantly inhibited the isoproterenol (1 microM)-induced decreases in resting potential and aiCl in quiescent preparations. However, 1 mM DIDS did not significantly affect the changes in aiCl and resting potential during beta-adrenergic stimulation. Thus, in cardiac cells, 9AC is a more potent blocker of the Cl- channels activated by beta-adrenergic stimulation than DIDS.


The mu-opioid activity of kappa-opioid receptor agonist compounds in the guinea pig ileum. On the basis of their in vivo activity and binding affinity, nalorphine and (-)SKF 10,047 were classified as mixed agonist/antagonist compounds. However, in isolated tissue preparations without a selective antagonist to block their agonist effect, the characterization of these compounds and the determination of their antagonist activity were very difficult. Nor-binaltorphimine, a selective kappa-opioid receptor antagonist, was used in the longitudinal muscle preparations of the guinea pig ileum to block the kappa-agonist activity of nalorphine and (-)SKF 10,047. In the absence of their kappa-agonist activity, we were able to determine the mu-antagonist activity using the mu-selective agonist DAMGO ([D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin). The pA2 values for nalorphine and (-)SKF 10,047 were 7.50 and 7.69, respectively.


Gamma-hydroxybutyrate hyperpolarizes hippocampal neurones by activating GABAB receptors. gamma-Hydroxybutyrate, a naturally occurring substance present in the mammalian central nervous system caused a dose-dependent (0.25-10 mM) hyperpolarization and small membrane conductance increase when applied to hippocampal CA1 pyramidal neurones in vitro. This action was reversibly inhibited by the GABAB antagonist, CGP 35348 (20-100 microM) and divalent cations, Zn2+ and Ba2+, but not by the GABAA antagonist bicuculline (50 microM). These results suggest that GABAB receptors may mediate the actions of gamma-hydroxybutyrate.


Trans-ACPD selectively inhibits excitability of hippocampal CA1 neurones. The selective agonist of metabotropic glutamate receptors, t-ACPD (trans-1-aminocyclopentyl-1,3-dicarboxylic acid) (100-250 microM), reversibly inhibited extracellularly recorded EPSP (excitatory postsynaptic potentials) in the CA1 layer of rat hippocampus. This effect was accompanied by depression of electrical excitability of CA1 neurons as revealed by their antidromic stimulation. The excitability of CA3 neurons remained uneffected. Peculiarly, excitatory postsynaptic currents recorded in voltage clamped, internally perfused CA1 cells remained unaltered. Selective depolarization of CA1 neurons may account for the phenomena described.


The epidemiology of varicella-zoster virus infections: a mathematical model. Herpes-zoster is caused by the reactivation of varicella-zoster virus (VZV). In this paper different hypotheses of how this re-emergence of virus comes about are reviewed and discussed. From these hypotheses, and epidemiological data describing the initial transmission of the virus, a mathematical model of primary disease (varicella) and reactivated disease (zoster) in developed countries is derived. The steady-state age distributions of zoster cases predicted by this model are compared with the observed distribution, derived from a review and analysis of published epidemiological data. The model allows differentiation between published hypotheses in which age of host may or may not influence the probability of viral reactivation. The results indicate that the probability of reactivation must increase with age to allow the observed pattern of zoster cases. The basic mathematical model presented provides a conceptual framework, which may be extended to assess possible control programmes.


The epidemiology of varicella-zoster virus infections: the influence of varicella on the prevalence of herpes zoster. This paper uses mathematical models and data analysis to examine the epidemiological implications of possible immunologically mediated links between patterns of varicella and herpes-zoster incidence in human communities. A review of previously published reports does not clarify whether or not there is a relationship between the incidence of varicella and the incidence of zoster. However, new analysis of data collected by the Royal College of General Practitioners provides indirect evidence for the hypothesis that a high intensity of varicella transmission suppresses viral reactivation. The significance of this finding for proposed varicella vaccination campaigns is explored by a review of published data on the use of the vaccine. No significant difference is shown to exist between the risk of zoster caused by the vaccine and the wild virus. A mathematical model is then developed to take into consideration the influence of the prevalence of varicella on viral reactivation and the impact of vaccination with attenuated virus, which may be able to recrudesce. Under some conditions, mass application of such vaccines may have the impact of increasing zoster incidence. The results presented here indicate that, before starting any vaccination programme against varicella, its consequences need to be assessed in much more depth.


Enteroviruses in recreational waters of Northern Ireland. Virus surveillance of Northern Ireland recreational waters, between April 1986 and May 1989 demonstrated widespread enteroviral contamination of coastal and inland waters. In 1986, enteroviruses were detected in 4 of 46 (8.7%) water samples, collected from 6 coastal bathing waters. In 1987, 49 of 107 (45.8%) samples, from 16 coastal bathing waters, yielded enteroviruses; 33 of the enterovirus positive samples passed one or both of the coliform standards outlined by the European Economic Community (EEC) bathing water directive (76/160/EEC). Enteroviruses were also detected in 33 of 39 (84.6%) samples tested from 3 inland recreational waters.


Sodium-hydrogen exchange in erythrocytes of patients with acute deep venous thromboses. The rate of delta microH(+)-induced Na/H-exchange in erythrocytes of patients with occlusive and with floating types of acute deep venous thromboses, and in control volunteers, was estimated. In patients with occlusive thrombi Na/H-exchange was revealed to be fourfold higher in comparison with patients with floating thrombi and with controls, while no difference was observed between the two latter groups.


Resistance of in vivo-selected spontaneously transformed cells and Rous sarcoma virus-transformed cells to macrophage-mediated cytotoxicity. The cytotoxic activity (CTA) of activated peritoneal macrophages (MP) on variant lines of Syrian hamster embryo (HE) cells of differing malignant characteristics was studied. The target cells were a line of low-malignant cells resulting from spontaneous transformation of HE cells in vitro (STHE strain), and malignant variants selected from them in vivo (STHE-LM-4, STHE-LM-8, and STHE-75/18 strains). In addition, we used cells of the HET-SR-1 strain; these are HE cells transformed in vitro by a tumorigenic Rous sarcoma virus (Schmidt-Ruppin strain, RSV-SR), or the TU-SR strain induced by RSV-SR in vivo. Thioglycollate-elicited peritoneal MP from Syrian hamsters were activated in vitro with bacterial levan, LPS or MDP and used as effector cells. MP-mediated cytolysis was determined by means of a 42-h radioactivity release assay with 3H-thymidine-labeled target cells. We found that only the parental STHE cells were susceptible towards fully-activated MP-mediated CTA. All three of the in vivo-selected malignant variants of the STHE cell sublines, as well as the tumorigenic RSV-SR transformants, were resistant to cytolysis by activated MP. Non-activated thioglycollate-elicited MP did not lyse any of the tumor cells studied.


Interaction of mannose-6-phosphate with the hysteretic transition in glucose-6-phosphate hydrolysis in intact liver microsomes. We showed previously that glucose-6-phosphatase activity was characterised in intact liver microsomes by a hysteretic transition between a rapid and a slower catalytic form of the enzyme. We have now further investigated the substrate specificity of these two kinetic forms. It was found that the pre-incubation of intact microsomes with mannose-6-phosphate or glucose-6-phosphate (50 microM for 30 s) suppressed the burst in glucose-6-phosphatase activity, that the hysteretic transition was reversible and that mannose-6-phosphate inhibited glucose-6-phosphate hydrolysis during the first seconds of incubation, but not anymore after the burst. Our results indicate (i) that mannose-6-phosphate is recognised by the enzyme and can promote the hysteretic transition and (ii) that the transient phase is part of the catalytic mechanism itself.


Lethal oxidative damage to human immunodeficiency virus by human recombinant myeloperoxidase. Human recombinant myeloperoxidase was evaluated in a cell-free system for its inactivation properties on the replication of human immunodeficiency virus, HTLV-IIIB. In the presence of a hydrogen peroxide generating system (glucose and glucose oxidase) and sodium thiocyanate, the recombinant enzyme inhibited virus-induced syncytium formation and viral replication without causing any cytopathic effects on SupT1 reporter cells. In addition, U937 monocytoid cells, chronically infected with HIV1, were exposed to recombinant myeloperoxidase (10 U/ml) and monitored during 48 h for the accumulation of intracellular p24 viral antigen. Under these conditions, the recombinant enzyme significantly reduced intracellular viral replication without affecting cell viability.


Possible involvement of superoxide anion in the induction of cyanide-resistant respiration in Hansenula anomala. A chemiluminescence study showed that Qi site inhibitors such as antimycin A induce O2- generation in respiring cyanide-sensitive mitochondria from the yeast, Hansenula anomala. The O2- generation was suppressed by radical scavengers such as flavone, butylated hydroxyanisole, and Co0. Induction of cyanide-resistant respiration in H. anomala cells by Qi site inhibitors was also inhibited by these radical scavengers. Furthermore, antimycin A-induced synthesis of the mitochondrial 36-kDa protein, which is thought to be the alternative oxidase functional in the cyanide-resistant respiratory pathway, was abolished by the addition of flavone. These observations suggest that O2- is somehow involved in the induction of cyanide-resistant respiration.


Separation of fibronectin from a plasma gelatinase using immobilized metal affinity chromatography. Conventional preparations of plasma fibronectin are known to contain a co-purifying gelatinase [1986, J. Biol. Chem. 261, 4363-4366], but so far useful methods to remove the protease have not been available. In this study a number of different methods were tested in order to achieve separation of the two proteins. Immobilized metal affinity chromatography was found to be efficient for this purpose, and a convenient procedure to separate the two proteins under nondenaturing conditions on chelating Sepharose charged with Co2+, Ni2+, or Zn2+ is described. An alternative method employing pH gradient elution of an Fe3+ gel also resolved fibronectin from the gelatinase. The Fe3+ gel bound both proteins at pH 6.0 but not at pH 7.4, suggesting that the two proteins were phosphorylated. The described procedures will now allow studies of the functions of fibronectin in the absence of the contaminating protease.


TNF-mediated IL6 gene expression and cytotoxicity are co-inducible in TNF-resistant L929 cells. Interleukin (IL)-6 gene induction was studied in the murine cell line, L929r2, which is resistant to the cytotoxic action of tumor necrosis factor (TNF). Increasing concentrations of TNF slightly elevated the background levels of IL6 expression as compared to non-induced cells. Under conditions where the resistant cells are sensitive to TNF by combined TNF/IFN-gamma treatment, the IL6 levels were strongly induced. This induction could be further enhanced by the addition of lithium chloride, or reduced by inhibitors of cytotoxicity, such as dexamethasone. These results confirm our earlier conclusions regarding the close relationship between TNF-mediated IL6 gene expression and the pathway leading to cytotoxicity.


A novel ETA antagonist (BQ-123) inhibits endothelin-1-induced phosphoinositide breakdown and DNA synthesis in rat vascular smooth muscle cells. The effects of a novel cyclic pentapeptide (BQ-123), an endothelin (ET) antagonist selective for the ETA receptor subtype, on phosphoinositide breakdown and DNA synthesis stimulated by ET-1 were studied in cultured rat vascular smooth muscle cells (VSMC). BQ-123 competitively inhibited the binding of [125I]ET-1 to VSMC with the apparent Ki of 4 x 10(-9) M. BQ-123 dose-dependently inhibited formation of inositol-1,4,5-trisphosphate and [3H]thymidine uptake stimulated by ET-1. These data suggest that the ET-1-induced DNA synthesis in VSMC is mainly mediated by ETA receptor subtype.


Crystallization of human gelsolin.

Crystallization of human gelsolin. Human gelsolin has been crystallized by microdialysis techniques to give single crystals that diffract to 3.5 A resolution. The crystals belong to space group P42(1)2 and have cell dimensions a = 175.0 A, c = 151.6 A. They contain two gelsolin molecules in the asymmetric unit.


Evidence of cytosolic iron release during post-ischaemic reperfusion of isolated rat hearts. Influence on spin-trapping experiments with DMPO. Previous studies of oxygen-derived free radical generation based on spin-trapping methods have shown a signal formed of six bands (sextet) using electron spin resonance spectrometry (ESR) of coronary effluents collected during post-ischaemic reperfusion of isolated hearts perfused with 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The origin of this signal has recently become controversial. In the present study we show that, in the rat, this sextet and cytosolic iron release occur simultaneously, and that this signal can be inhibited by the iron chelator desferrioxamine. It also appears that the iron release is not protein bound, and could therefore have a marked catalytic activity. This may be responsible for the production of an artefactural signal observed as the sextet.


Identification of individual amino acids in platelet-derived growth factor that contribute to the specificity towards the beta-type receptor. Platelet-derived growth factor constitutes a family of three isoforms (PDGF-AA, -AB, and -BB) composed of two homologous polypeptide chains (A and B). These isoforms interact with two types of receptors termed alpha or beta. Whereas PDGF-AA binds only to the alpha-receptor, PDGF-BB binds and activates both receptors with high affinity. To map regions that are specific for the beta-receptor, we introduced mutations into PDGF-AA located in previously identified epitopes [1991, Biochemistry 30, 3303-3309]. A single amino acid exchange in domain II of PDGF-AA (Ala67----Arg) was sufficient to bring about a reduced but significant activation of the beta-receptor. In domain I the exchange of residues Pro26----Arg together with Ser28----Asn switched the specificity towards the beta-receptor. These data indicate that parts of the exposed domains are indeed involved in receptor binding. Since these single mutations lead to mutant proteins which are about 100-fold less active than PDGF-BB, it is suggested that other amino acid residues also participate in the binding to the receptors.


A single step purification for recombinant proteins. Characterization of a microtubule associated protein (MAP 2) fragment which associates with the type II cAMP-dependent protein kinase. A 167 base pair DNA cassette has been constructed to facilitate the detection and purification of recombinant proteins. This cassette, kfc, encodes three distinct peptide units: a phosphorylation site for the cAMP-dependent protein kinase (PKA), called kemptide, a factor Xa cleavage site, and a calmodulin-binding peptide. Expressed kfc fusion proteins can be purified from bacterial lysates in one step by affinity chromatography on calmodulin-agarose using EGTA as eluant. As a test of this system, we describe the expression, purification and characterization of the PKA binding domain of the microtubule associated protein (MAP 2).


Expression of anchorin CII, a collagen-binding protein of the annexin family, in the developing chick embryo. Expression of anchorin CII, a collagen-binding protein of the annexin family, was followed in the developing chick embryo using Northern and in situ hybridization and Western blotting. During chick somite development, anchorin CII mRNA was detected by Northern blotting as early as stage 11. At stage 24, anchorin mRNA accumulated in the anterior part of the somite sclerotome near the resegmentation line, as shown by in situ hybridization. The presence of anchorin CII protein during stages 11 to 20 was confirmed by Western blotting. In situ hybridization identified anchorin CII also in the otic vesicle adjacent to the site of contact with the statoacoustic ganglion and in the mandibular mesenchyme. The level of anchorin CII mRNA in differentiated hyaline cartilage, exemplified by sternal cartilage, was lower than that in differentiating somites or cultured chondrocytes. These findings are consistent with our notion that anchorin CII may be involved in cell-matrix interactions preceding chondrogenic differentiation events in the chick embryo. A significant level of anchorin CII mRNA and protein synthesis was also found in cultured myoblasts, but less than that in chondroblasts. This distribution pattern is different from that reported for a related protein, p34, or calpactin, the major protein substrate for tyrosine kinase phosphorylation in chick chondrocytes and fibroblasts. The results confirm suggestions from previous sequencing studies that anchorin CII and p34 are different proteins of the annexin/calpactin family.


ACTH-like peptides in postimplantation mouse embryos: a possible role in myoblast proliferation and muscle histogenesis. ACTH and related peptides are mitogens for certain mesodermal cell types such as adrenocortical cells, T-lymphocytes, and skeletal myoblasts. In order to postulate a possible physiological role for these peptides in skeletal muscle histogenesis, it is necessary to establish whether they are present in muscle forming anlagens of postimplantation mouse embryos. By radioimmunoassay and immunofluorescence with antibodies specific for ACTH, we have detected these peptides in many areas of mouse embryos including neural tube, limb buds, eye lens, and myotomal muscles. During fetal development, immunoreactivity decreased in muscle tissue and appeared in visceral ganglia. Furthermore, primary myotubes or C2C12 myotubes, but not muscle or 3T3 fibroblasts, release significant levels of ACTH immunoreactive peptides into the culture medium. Using a microassay for mitogen production, primary myotubes or C2C12 myotubes, but not other mesodermal cells (with the exception of dermal fibroblasts) were shown to release factors into the medium which support myoblast proliferation. Neutralizing antibodies against ACTH inhibit myoblast but not fibroblast proliferation in a dose-dependent fashion. Based on these results, we propose that myotube-derived mitogens (including ACTH-like peptides) promote the proliferation of surrounding myoblast during muscle histogenesis in vivo.


Sperm-induced currents at fertilization in sea urchin eggs injected with EGTA and neomycin. Membrane currents were measured in single voltage-clamped sea urchin eggs (Lytechinus pictus and Lytechinus variegatus) that were injected with either EGTA or neomycin and inseminated. Although egg activation and the fertilization calcium wave were prevented by injection of either of these compounds, sperm attached and still elicited inward currents. Sperm-induced currents in EGTA-injected eggs had an abrupt onset, quickly reached a maximum, and then slowly declined in amplitude. Sperm incorporation occurred readily in EGTA-injected eggs. Similar results were obtained with another calcium chelator, BAPTA. In neomycin-injected eggs, sperm-induced currents generally had an abrupt onset and, in contrast to EGTA-injected eggs, the currents usually cut off rapidly. Sperm failed to enter the neomycin-injected eggs and the duration of sperm-induced currents in neomycin-injected eggs was markedly dependent upon the voltage-clamp holding potential, with shorter duration currents occurring at -70 than at -20 mV. The lability of the initial interaction between sperm and egg at negative holding potentials may explain why activation often fails when the egg membrane is voltage clamped at these potentials (Lynn et al., Dev. Biol. 128, 305-323, 1988).


Transgenic mice expressing a constitutively active retinoic acid receptor in the lens exhibit ocular defects. Retinoic acid receptors (RARs) modulate gene expression following association with retinoic acid (RA). In transient transfection, an RAR alpha-beta-galactosidase fusion protein (RAR-LacZ) was able to transactivate expression in the absence of RA. When expressed in the ocular lens of transgenic mice, this constitutively active RAR-LacZ fusion gene resulted in founder and progeny animals that exhibited cataracts and microphthalmia, both being characteristics of retinoid-induced teratogenesis. The transgenic phenotypes indicate that retinoid teratogenesis can be mimicked by expression of a constitutively active RAR-LacZ fusion protein in retinoid-sensitive tissues.


A tier approach for evaluating the respiratory allergenicity of low molecular weight chemicals. A multi-level approach for evaluating low molecular weight chemicals as respiratory sensitizers is proposed. The approach involves four levels of testing that utilize both in vitro and in vivo methods. Tier 1 evaluates structure-activity information to determine if the chemical can covalently modify carrier molecules. It also includes a literature search to determine if the compound belongs to a family of chemicals that has been reported to induce hypersensitivity. Tier 2 tests the chemical's potential to haptenate carrier molecules (i.e., protein) under in vitro conditions. Positive results in Tiers 1 and 2 lead to testing in a guinea pig injection model to assess chemical immunogenicity (Tier 3). A positive result at this level leads to testing in a guinea pig inhalation model to address questions about relevant routes of chemical exposure and allergenicity (Tier 4). Tier 4 results are used in determining safe chemical exposure levels. We have evaluated three chemicals using this scheme: phthalic anhydride, reactive black b dye, and toluene diisocyanate. All three have reactive groups and haptenate protein in vitro. They induce a humoral immune response when injected into guinea pigs at equimolar concentrations, and they sensitize animals via inhalation exposure. The severity of the response (antibody titer and respiratory reactivity) can be used to rank-order the chemicals in terms of allergenic "potency." Our data indicate that this approach can detect chemical allergens and can be used to characterize them as moderate or strong respiratory sensitizers.


Acute toxic effects of oxamyl in the rat. The effects of single acute oral doses of 1, 2.1, and 3.5 mg/kg oxamyl (a carbamate insecticide) on selected biochemical parameters in male Sprague-Dawley rats were investigated. The animals exhibited significantly decreased weight gain when compared to control animals. The compound inhibited brain and blood acetylcholinesterase significantly in the first few hours of exposure. Liver glucose-6-phosphatase was inhibited substantially after 7 and 4 days at the levels of 2.1 and 3.5 mg/kg, respectively. Maximum inhibition of liver succinic acid dehydrogenase was noted after 1 day at the level of 1 mg/kg and after 6 hr at the level of 2.1 and 3.5 mg/kg. Significant changes in serum total lipids and glucose were observed when oxamyl was given at 2.1 and 3.5 mg/kg, but serum protein was not affected at any dose level. However, the absence of statistically significant effects between Days 7 and 14 in most of the investigated parameters is indicative of an overall moderate degree of toxicity of oxamyl following acute oral administration of the selected doses.


Effects of diet type on incidence of spontaneous and 2-acetylaminofluorene-induced liver and bladder tumors in BALB/c mice fed AIN-76A diet versus NIH-07 diet. Diet is a major influence on the responses of experimental animals to drugs, toxins, and carcinogens. Two diets used widely in toxicological and/or nutritional studies, and considered to be nutritionally adequate, were compared with respect to their influence on growth, body weight, lifespan, spontaneous neoplasia, and neoplastic responses to 2-acetylaminofluorene (2-AAF). Both sexes of weanling BALB/c mice were fed either a purified diet (AIN-76A) or a nonpurified, natural ingredient diet (NIH-07), with or without 2-AAF for up to 2 years. Dosages of 2-AAF were administered to males at 0, 20, 40, or 60 ppm in each diet and to females at 0, 100, 125, or 150 ppm. Each group consisted of 96 mice. In most instances, males and females fed purified diet (AIN-fed) gained weight more rapidly, attained higher maximum body weights, and died earlier than their non-purified diet (NIH-fed) counterparts. 2-AAF inhibited weight gain significantly only in AIN-fed females. Thus, females receiving 150 ppm 2-AAF gained little more than their NIH-fed counterparts. At the dosages used in males, 2-AAF did not induce liver neoplasia but the AIN diet was clearly associated with a higher spontaneous frequency of liver neoplasia than the NIH diet. Although 2-AAF induced liver tumors in females fed either diet at all dosages, a higher frequency and earlier appearance of liver tumors among AIN-fed females than their NIH-fed counterparts was apparent mainly at the lowest dosage. 2-AAF induced bladder neoplasia in both sexes.(ABSTRACT TRUNCATED AT 250 WORDS)


Neurotoxic effects of the anti-varicella zoster virus agent 2'-valeryl-6-methoxypurine arabinoside in monkeys and rats dosed orally for 90 days. The 2'-valerate ester of 6-methoxypurine arabinoside (170U88), a nucleoside analog with anti-varicella zoster virus (VZV) activity, was given to monkeys and rats. In subchronic preclinical toxicity studies, dosing was by gavage to monkeys (distilled water vehicle) and rats (0.5% methylcellulose vehicle) for 90 days. Groups of 5 male and 5 female monkeys (Macaca fascicularis) were given 170U88 at 0, 25, 50, or 100 mg/kg/day. The daily dose was given in two equal portions with 6 hr between doses. Monkeys in the high-dose group lost weight. Food consumption was decreased for mid- and high-dose monkeys and for low-dose female monkeys. Slightly decreased values for erythrocyte and leukocyte counts at the mid- and high dose were fully reversed during an 8-week recovery period. Two high-dose male monkeys and a middose female monkey developed signs of central nervous system toxicity and were necropsied before dosing was complete. These signs were first observed in the fifth week of dosing and included body tremors, incoordination, reduced activity, sleepiness, stupor, and lack of eye tracking. Axonal lesions were observed in histologic sections of sciatic nerve in monkeys at all dose levels. Neither the signs of central nervous system toxicity nor the axonal lesions reversed during the 8-week recovery period. Groups of 14 male and 14 female CD rats (Sprague-Dawley derived) were given single daily doses of 170U88 at 0, 150, 300, or 600 mg/kg. Body weights were decreased at all dose levels and food consumption was decreased for mid- and high-dose rats.(ABSTRACT TRUNCATED AT 250 WORDS)


Beta-adrenergic receptors in catfish liver membranes: characterization and coupling to adenylate cyclase. beta-Adrenergic binding sites in catfish liver membranes have been characterized by centrifugal assay, using a beta-adrenergic receptor antagonist, (-)-[3H]dihydroalprenolol ([3H]DHA). Binding of the radioligand was saturable and reversible. At 22 degrees equilibrium conditions were established in 15 min and the half-time for dissociation of bound [3H]DHA was approximately 4 min. Analysis of binding data was compatible with the existence of two classes of binding sites: a low-affinity site had a Kd of 62.3 nM and a Bmax of 452.0 fmol/mg protein, while the high-affinity site had a Kd of 2.04 nM and a Bmax of 46.7 fmol/mg protein. The dissociation constant of (-)-alprenolol for the beta-adrenergic receptors was about 2 nM as determined independently by direct kinetic studies and by inhibition of isoproterenol-stimulated adenylate cyclase activity. Phenylephrine was as potent as other catecholamines in inhibiting [3H]DHA binding, indicating that fish adrenoceptor subtyping is different from that of mammals.


Synthesis and degradation of polyphosphate in the fission yeast Schizosaccharomyces pombe: mutations in phosphatase genes do not affect polyphosphate metabolism. The fission yeast Schizosaccharomyces pombe was found to accumulate large amounts of polyphosphate, particularly when grown on arginine as the nitrogen source. Upon transfer to a medium without phosphate, polyphosphate was degraded and served as an endogenous phosphate reserve. When phosphate was added again after a prolonged period of phosphate starvation, fission yeast cells synthesized more polyphosphate than they had contained before starvation, a phenomenon known as over-compensation. Strains carrying mutated structural genes for three different phosphatases, pho1, pho2 or pho3, degraded polyphosphate at the same rate as the wild-type strain during phosphate starvation and showed the same type of over-compensation when phosphate was added again.


Depression and neurological disease. Their distinction and association. Depression and neurological disease often coexist but sometimes are difficult to distinguish. By analyzing eight patients, all seen by psychiatrists and the same neurologist, and by reviewing the pertinent literature, we will explore the complex differential features, coexistence, and interaction between depression and neurological disease. Neurological disease may lead to changes in mood and behavior, and these patients may present to psychiatrists with depression. In addition, depression may present with or exaggerate neurological signs and symptoms. Awareness of these interactions can lead to appropriate evaluation and treatment of both these disorders.


Analysis of promoter activity by transformation of Acremonium chrysogenum. Promoter activity was examined in the beta-lactam-producing fungus, Acremonium chrysogenum, by assessment of the properties of transformant isolates. Transformation was achieved using plasmid constructs specifying hygromycin B resistance (HyR) linked to the promoter elements of gpdA (the glucose-6-phosphate dehydrogenase-encoding gene of Aspergillus nidulans), and pcbC [the gene encoding the isopenicillin N synthetase (IPNS) enzyme of A. chrysogenum]. Transformation frequency, HyR levels, and Hy phosphotransferase (HPT) levels suggested that the transformants of constructs using the gpdA promoter showed a higher level of expression of the HyR gene than in transformants obtained using the pcbC promoter. The patterns of integration of the transforming DNA also differed in that pcbC promoter construct transformants appeared to have tandem repeats. All integrations of plasmid DNA occurred on a single chromosome which was different in four out of five transformants studied. Multiple copy transformants of constructs using the pcbC promoter did not show the regulated pattern of expression of HPT activity observed with IPNS in untransformed strains.


Genomic typing of hepatitis C viruses present in China. Hepatitis C virus (HCV) genomic clones were obtained from the serum of Chinese HCV carriers using a polymerase chain reaction-based approach. Consensus sequences were derived from (1) the structural region (nt 1-1543) for one carrier, (2) the hypervariable region V (nt 1156-1233) from four carriers and (3) region V3 from four carriers. Region V3, located in the nonstructural domain NS5 (nt 7066-7137), has been previously shown to be a particularly good marker for the genomic typing of HCV isolates [Inchauspe et al., Proc. Natl. Acad. Sci. USA 88 (1991) 10292-10296]. Comparison of these sequences with sequences from geographically distinct HCV isolates indicates that Chinese HCV strains are closely related to, though distinguishable from, Japanese prototype strains. One amino acid motif, GGAA, located in region V, was found to be conserved only among Chinese isolates. This may define a new subgroup among HCV isolates.


The cis-acting 3'-element of rubella virus RNA has DNA promoter activity. The 3'-terminal region of the rubella virus (RV) positive-strand RNA, referred to here as the cis-acting element (CAE), is implicated in the initiation of negative-strand RNA synthesis. Sequence analysis of the 3'-CAE shows that there is a putative TATA box which is surrounded by G + C-rich sequences. To determine whether this element, in a DNA form, has the capability to initiate transcription, a 3'-end 165-bp NarI-EcoRI fragment from the RV cDNA was cloned upstream from a cat reporter gene. The level of CAT activity was dependent on the presence of the 3'-CAE and the SV40 enhancer. Primer extension analysis of the CAT mRNA showed that the transcription start point is in the RV 3'-CAE, 34 bp downstream from the putative TATA box. DNA-gel shift analysis revealed that three nucleoprotein-specific complexes were formed with the 3'-CAE and the binding sites for these proteins were between bp -64 to -108. The possible promoter function of the RV 3'-CAE is discussed in context to RV persistence.


[Role of metalloproteases of extracellular matrix in the premature rupture of fetal membranes: a novel physiopathogenic model]. Experimental proofs are presented, that involve the participation of the metalloproteinases family, of extracellular matrix in the genesis of premature rupture of membranes. The expression of this group of enzymes, which presents in normal conditions of labor appears in the RPM without relation with gestational age. This phenomenon is not presented as an isolated example of participation of metalloproteinases in events related to labor and delivery, as is very well known its participation in maturation of uterine cervix preceding product expulsion, and that consists in similar mechanisms to the ones now chosen for the maturation of fetal membranes. The working hypothesis in pathologic conditions, implies, then, the activation of a normal system in an inadequate moment that results in premature rupture of membranes.


Interactions of 1,10-phenanthroline and its copper complex with Ehrlich cells. Mechanistic details of the interaction of 1,10-phenanthroline and its copper complex with Ehrlich ascites tumor cells were examined, using inhibition of cell proliferation, DNA breakage, and increased membrane permeability as indices of cellular damage. The metal chelating agent, 1,10-phenanthroline (OP), the 1:0.5 complex of 1,10-phenanthroline and CuCl2 [(OP)2Cu], and CuCl2 inhibited growth of Ehrlich ascites tumor cell monolayers during 48-h treatments by 50% at about 3.5, 2, and 70 nmol/10(5) cells/mL, respectively. (OP)2Cu at 10 nmol/10(5) cells also enhanced uptake of trypan blue dye during 6 h of treatment, while dye uptake in OP- and CuCl2-treated cells remained similar to controls. DNA breakage, measured by DNA alkaline elution, was produced during 1-h treatments with (OP)2Cu at drug/cell ratios similar to those producing growth inhibition. Copper uptake was similar for both (OP)2Cu and CuCl2. Electron spin resonance (ESR) spectroscopy suggested that cellular ligands bind copper added as (OP)2Cu or CuCl2 and then undergo time-dependent reductions of Cu(II) to Cu(I) for both forms. Inhibition of (OP)2Cu-induced single-strand scission and trypan blue uptake by scavengers of activated oxygen is consistent with participation of superoxide and H2O2 in both processes. In contrast, superoxide dismutase (SOD) did not reduce the magnitude of the fraction of cellular DNA appearing in lysis fractions prior to alkaline elution of (OP)2Cu-treated cells. Dimethyl sulfoxide (DMSO) inhibited uptake of trypan blue dye but did not inhibit DNA strand scission produced by (OP)2Cu. Thus, multiple mechanisms for generation of oxidative damage occur in (OP)2Cu-treated cells. Growth inhibition produced by OP or (OP)2Cu, as well as the low levels of strand scission produced by OP, was not reversed by scavengers.


Methemoglobin formation from butylated hydroxyanisole and oxyhemoglobin. Comparison with butylated hydroxytoluene and p-hydroxyanisole. The widely used food additives butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) react with oxyhemoglobin, thereby forming methemoglobin. The reaction rates were measured using visible spectroscopy, and second order rate constants were established for BHA and compared with p-hydroxyanisole. Using ESR we investigated the involvement of free radical reaction intermediates. The expected one-electron oxidation product of BHA and BHT, the phenoxyl radical, could only be detected with pure 3-t-butyl-4-hydroxyanisole and oxyhemoglobin. With the commercial mixture of 2- and 3-t-butyl-4-hydroxyanisole a very strong ESR signal of a secondary free radical species was observed, similar to the one observed earlier with p-hydroxyanisole and dependent on the presence of free thiol groups, so that we assumed the intermediate existence of a perferryl species, the MetHb-H2O2 adduct. In a second series of experiments we investigated the reactivity of this postulated intermediate with BHA and BHT, starting with a pure MetHb/H2O2-phenol mixture in a stopped-flow apparatus linked to the ESR spectrometer, detecting the expected phenoxyl radicals from BHA and p-hydroxyanisole. Due to the low solubility and decreased reactivity of BHT only traces of phenoxyl type radical were found together with a high concentration of unreacted perferryl species. The reactivity of BHA, BHT and p-hydroxyanisole with free thiol groups is demonstrated by an increased reaction rate in the presence of the thiol group blocking substance NEM.


Measurement of free radicals from smoke inhalation and oxygen exposure by spin trapping and ESR spectroscopy. Research in smoke inhalation has established that free radicals are produced from gases released during combustion and these species impair lung function. Using spin traps and their adducts in an animal model free radicals were measured. Various hyperbaric oxygen regimens were tested in an attempt to attenuate pulmonary damage caused by free radical reactions. Our data demonstrated that persistent oxygen- and carbon-centered free radicals are detectable in intravascular fluids after smoke inhalation. The smoke inhalation model showed however, clearing of spin trap adducts one hour after smoke exposure. Other researchers have found that when 100% oxygen is given at 1 atmosphere absolute (ATA) for 1 h, free radicals were not detectable. However, oxygen given at 2.5 ATA does produce detectable free radicals. With continued exposure at this pressure, the levels of free radicals increase for up to 60 min. This study suggests that the level of free radical induced oxygen toxicity may be a function of oxygen pressure and duration of oxygen exposure.


Xanthine oxidase/dehydrogenase activity in intact cultured cells (in situ analysis). The measured ratio of xanthine oxidase activity to the total activity of xanthine oxidase and dehydrogenase showed higher values in intact cells than when similar cells were homogenized. The total activity was the same for both systems. The xanthine oxidase ratio was 90, 60, 50, 50, 60% in V79, RIF/Ha3, SCC7, KHT intact cells and freshly extracted murine peritoneal macrophages respectively while the corresponding ratios measured were 25, 40, 38, 35, 22% when the cells were lysed by homogenization. Superoxide radical O2-. production by addition of xanthine to intact or homogenized cells to activate intracellular xanthine oxidase was higher in intact than homogenized cells. Homogenization of cells and tissues in the presence of dithioerythritol (DTE) can evidently lead to a considerable under-estimation of the xanthine oxidase ratio. The effect of hypoxia on cells has also been examined.


ESR spin trapping analysis of gamma induced radicals in sucrose: II. Radicals induced by gamma-irradiation of sucrose, in the solid state at different temperatures and in aqueous solution, have been investigated by the spin trapping method. Electron spin resonance (ESR) combined with high performance liquid chromatography (HPLC), followed by spectral analysis with a simulation program (Voyons) revealed seven main radical species. A comparative study of the ESR signals from spin trapped gamma-induced radicals in some glycosides, disaccharides, 13C specifically labelled carbohydrates, as well as in several deoxysucroses and fructans, led to the assignment of a chemical structure to five out of the seven sucrose-nitroxide adducts previously evidenced. Sucrose is shown to be a conceivable model for the study of fructans gamma-radiolysis mechanism in aqueous solution.


Essential oil phenyl propanoids. Useful as .OH scavengers? In order to search for radical scavengers which could be used as raw materials for cosmetics, phenyl propanoids (eugenol, isoeugenol, dehydrodieugenol, dehydrodieugenol B and coniferyl aldehyde) were examined for their hydroxyl radical (.OH) scavenging ability. A Fenton system was used to produce .OH. In order to see scavenging by these phenyl propanoids, competition reactions between a spin trap, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), and these phenyl propanoids for .OH were studied. The relative yield of the spin adduct of .OH (DMPO-OH) was measured by electron spin resonance spectroscopy. The approximate rate constants of the reactions between these phenyl propanoids and .OH estimated by measuring the reduced height of the ESR signals of DMPO-OH were found to be at least in the order of 10(9) M-1 s-1 (diffusion-controlled). Also, using the TBA tests, the reactions between .OH and several compounds reactive with .OH were investigated in the presence of the phenyl propanoids and it was found that the phenyl propanoids compete with such reactive compounds for .OH. These results indicate that these phenyl propanoids can be used as antioxidants for skin damage perhaps caused by .OH generated by UV-light.


Metastatic gestational trophoblastic disease: a comparison of prognostic classification systems. The records of 53 consecutive patients with metastatic gestational trophoblastic disease (MGTD) treated at the University of Southern California/Los Angeles County Medical Center since 1970 were analyzed. Forty-eight were evaluable for this study. Treatment during the study period was based predominantly on the NIH good-prognosis-poor-prognosis system, employing single-agent therapy (methotrexate or actinomycin D) for the good-prognosis patients and methotrexate, actinomycin D, cyclophosphamide (MAC) for the poor-prognosis patients. The overall survival rate was 83.3%. The study patients were retrospectively classified according to the FIGO, NIH, and WHO systems to test each system's accuracy in predicting outcome and the appropriateness of single-agent or multiagent chemotherapy as the initial treatment in each category. None of the systems as currently used is clearly superior to the others. Analysis of the WHO scoring system showed that 21 of the 25 (84.0%) study patients with a point score less than 8 were treated primarily with a single-agent regimen. All of 21 of these patients achieved a complete sustained remission although 3 (14.3%) required multiagent chemotherapy. The 4 patients in this point category whose initial therapy was a multidrug regimen were also cured. The 23 patients in the WHO high-risk category (greater than 7) had treatment initiated with combination chemotherapy. There were no deaths among the 11 patients in the 8-12 point group, although 3 (27.3%) were salvaged by alternate multiagent chemotherapy after failing on MAC. There were 8 deaths in the 12-patient greater than 12 point WHO category (66.7%). On the basis of this analysis we recommend that the WHO scoring system be utilized for reporting results of treatment for MGTD, but the risk categories should be redefined: low, less than 8 points; medium, 8-12 points; high, greater than 12 points.


Secretion of prorenin by a virilizing ovarian tumor. A 53-year-old normotensive, normokalemic female presented with a 6-month history of virilization. Estradiol, LH, FSH, urinary-free cortisol, and DHEA-S levels were normal. Pelvic ultrasound and computerized tomography were also within normal limits. Her serum testosterone (551 ng/dl; nl, 20-70) and plasma prorenin (124 ng AI/ml/hr; nl, less than 50) levels were elevated. At surgery, a lipoid/steroid cell tumor of the right ovary was removed. Postoperative testosterone and prorenin levels were normal. Ovarian tumor cells, in culture, produced large amounts of prorenin. Immunohistochemistry localized prorenin and/or renin to tumor cells. Determining plasma prorenin levels may be a useful adjunct in diagnosing or following patients with nonepithelial ovarian tumors. A larger clinical study of prorenin levels in patients with such tumors is needed.


Primary vaginal adenocarcinoma of the intestinal type associated with adenosis. Primary vaginal adenocarcinoma unrelated to intrauterine hormone exposure is very uncommon. We report a case of a 52-year-old woman who presented with primary vaginal adenocarcinoma that showed intestinal differentiation with prominent papillary formations and numerous papillary and villous features with prominent goblet cells. There was vaginal adenosis in the immediate vicinity of the tumor. To our knowledge, this is the first reported case of a primary vaginal adenocarcinoma of the intestinal type associated with adenosis in a patient unexposed to prenatal diethylstilbesterol.


Trisomy 12 and 4 in a thecoma of the ovary. Cytogenetic analysis of short-term tissue culture from a thecoma of the ovary demonstrated the presence of trisomies of chromosomes 12 and 4 in all analyzed cells. Our finding confirms the consistency with which trisomy 12 is observed in benign sex cord/stromal tumors and suggests that trisomy 4 may be a second event in tumorigenesis of thecoma.


Ceruloplasmin serum level in post-menopausal women treated with oral estrogens administered at different times. The liver is an estrogen-responsive organ and the administration of estrogens in humans increases the hepatic synthesis of many proteins. The existence of a circadian rhythm of estrogen receptors in the liver has been proved by different authors. We studied the presence of a different responsiveness of the human liver to the estrogens in two groups of post-menopausal women by evaluating the changes in ceruloplasmin serum level. Conjugated equine estrogens were administered at different times (A: 8 a.m. and B: 8 p.m.). The replacement therapy increased ceruloplasmin serum levels both in group A and B, but the increase was higher in group B than in group A. These data reflect indirectly the presence of a circadian rhythm of hepatic responsiveness to the estrogens.


Predicting cocaine use among methadone patients: analysis of findings from a national study. Findings from a large-scale national study of clients admitted to publicly funded drug treatment programs between 1979 and 1981 were used to determine whether cocaine use by current and former methadone patients could be predicted. The sample for this analysis comprised 526 daily or weekly heroin users admitted to 17 methadone maintenance programs. The study found that cocaine use by both current and former methadone patients showed an overall decline during the follow-up year; that patients who stopped using heroin after entering treatment were much more likely to quit using cocaine than were their heroin-using counterparts; and that the odds of initiating cocaine use after admission to a methadone program were much higher among patients who continued using heroin. These findings suggest that methadone programs may be able to reduce cocaine use among some patients by improving their effectiveness in reducing heroin use.


Discovery of a genetic polymorphism of human plasma protein C inhibitor (PCI): genetic survey utilizing isoelectric focusing followed by immunoblotting, immunological and biochemical characterization. The objectives of this study were to determine the genetic basis of the electrophoretic differences of human plasma protein C inhibitors (PCI) from 977 individuals. Three discrete antibodies were produced against the PCI purified from human plasma and peptides that corresponded to the N-terminal 15 amino acid residues and the C-terminal 15 residues of human PCI, the chemical structures of which were determined by cDNA sequence analysis. The combined techniques of polyacrylamide gel isoelectric focusing and immunoblotting with these three different antibodies resolved the plasma PCI into several isoprotein bands, with a pH range of 6-7. These PCI isoproteins, however, were not stained by anti-human kallikrein, anti-human protein C or anti-human urokinase antibodies. Therefore, each of the PCI bands, which were detected by immunoblotting with the anti-PCI antibody and the two different anti-peptide antibodies, were derived from free PCI, and not an inactive PCI species. Two common phenotypes, designated PCI 1 and 1-2, were recognized, and family studies showed that they represented homozygosity or heterozygosity for two autosomal codominant alleles, PCI*1 and PCI*2. A population study of plasma samples collected from 977 Japanese individuals indicated that the frequencies of the PCI*1 and PCI*2 alleles were 0.988 and 0.012, respectively.


No evidence for constitutional chromosome instability in testicular cancer. Chromosome aberrations in 20 lymphocytes of 20 patients with testicular germ cell tumors (TGCT) treated with surgery alone were compared with those of 20 cells from 20 healthy controls using standard G-banding technique. No increase in structural aberrations was found in the cancer group. An unexpected finding was that of more cells with losses of chromosomes being present in the control group. These losses predominantly affected small chromosomes in the control group, whereas the pattern of chromosome loss was different in the cancer group. The literature claiming increased chromosome instability in TGCT patients is reviewed. Point estimates and 95% confidence intervals to exclude such a hypothesis based on our results were calculated.


Correlation between antibodies to mannophosphoinositides & detection of antigen in circulating immune complexes in patients with pulmonary tuberculosis. Enzyme linked immunosorbent assays were used to detect the concentration of mannophosphoinositides in circulating immune complexes (CICs) and antibodies to these antigens in the sera of patients with pulmonary tuberculosis. Serum samples from 235 tuberculosis patients at various levels of disease progression were analysed. Multiple regression analysis showed a direct correlation between antigen levels in CICs and serum antibodies to mannophosphoinositides.