Alpha 2-C10 adrenergic receptors expressed in rat 1 fibroblasts can regulate both adenylylcyclase and phospholipase D-mediated hydrolysis of phosphatidylcholine by interacting with pertussis toxin-sensitive guanine nucleotide-binding proteins. The alpha 2-C10 adrenergic receptor from human platelets was expressed permanently in Rat-1 fibroblasts. A series of clones that varied in expression of the receptor from 0 to 3.5 pmol/mg of membrane protein were isolated. We have demonstrated recently in cells of one of these clones (1C) that the alpha 2-C10 receptor interacts directly with two distinct pertussis toxin-sensitive G-proteins, Gi2 and Gi3 (Milligan, G., Carr, C., Gould, G. W., Mullaney, I., and Lavan, B.E. (1991) J. Biol. Chem. 266, 6447-6455). High affinity GTPase activity in membranes of cells from the various clones was stimulated by the addition of the alpha 2-adrenergic agonist UK14304, defining that the receptor coupled productively to the G-protein signaling system. Maximal stimulation of high affinity GTPase activity correlated with the levels of receptor expressed. Clones expressing the receptor also demonstrated agonist-mediated inhibition of adenylylcyclase. Futhermore, the alpha 2-C10 receptor in one clone (1C), but not other clones, promoted a marked stimulation in the generation of water-soluble products derived from phosphatidylcholine. The concentration of UK14304 required to produce half-maximal regulation of GTPase activity (20-30 nM), of forskolin-amplified adenylylcyclase activity (30-40 nM), and of choline generation (30-40 nM) were similar. Transphosphatidylation experiments with cells of clone 1C indicated that the receptor-mediated hydrolysis of phosphatidylcholine was via the action of a phospholipase D. All of these effects were attenuated by pretreatment of the cells with pertussis toxin. Dose-effect curves of pertussis toxin-treatment demonstrated similar effective concentrations of the toxin in causing endogenous ADP-ribosylation of both Gi2 and Gi3, inhibition of receptor-stimulated GTPase activity, and phospholipase D activity. Receptor activation of phospholipase D activity was not dependent upon prior phospholipase C-dependent activation of protein kinase C, as alpha 2-adrenergic stimulation of inositol phosphate production was negligible and the presence of the selective protein kinase C inhibitor RO-31-8220, at concentrations up to 10 microM, had no effect on UK14304-mediated production of phosphatidylbutanol. These results demonstrate that expression of the alpha 2-C10 receptor in a heterologous system can result in receptor regulation of signaling elements that appear not to be primary targets for the receptor in vivo. Such results are important in respect to recent observations that transfection of a single defined receptor into separate cell lines can lead to the regulation of distinct effector systems (Vallar, L., Muca, C., Magni, M., Albert, P., Bunzow, J., Meldolesi, J. and Civelli, O. (1990) J. Biol. Chem. 265, 10320-10326).(ABSTRACT TRUNCATED AT 400 WORDS)


Bacterial expression, purification, and functional mapping of the amyloid beta/A4 protein precursor. The secreted form of Alzheimer amyloid beta/A4 protein precursor (APP) has been shown to be involved in cell growth regulation (Saitoh, T., Sundsmo, M., Roch, J.-M., Kimura, N., Cole, G., Schubert, D., Oltersdorf, T., and Schenk, D.B. (1989) Cell 58, 615-622). Using a strong prokaryotic expression system, we expressed, in Escherichia coli, peptide fragments covering different regions of the secreted form of APP-695. The longest of these fragments (KB75, 572 amino acids from Val-20 to Ile-591), which contained neither the Kunitz-type protease inhibitor (KPI) domain nor the amyloid beta/A4-protein domain, was purified and shown to be biologically active in terms of growth regulation. Two other APP fragments (KB48, 316 amino acids from Val-20 to Met-335; and RB17, 150 amino acids from Thr-296 to Pro-445), overlapping by only 40 amino acids at a close site C-terminal to the KPI insertion site, were also active. Furthermore, a chemically synthesized 40-residue peptide corresponding to this region of overlap also stimulated the growth of A-1 fibroblasts. These results establish the presence of growth-promoting activity in the secreted form of APP-695 and suggest that the site of this activity of APP-695 lies within a 40-amino acid domain next to the KPI insertion site.


Genomic structure of keratinocyte transglutaminase. Recruitment of new exon for modified function. The gene for keratinocyte transglutaminase (TGK) spans 14 kilobase pairs and contains 15 exons. Many features of the TGK gene are very similar, if not identical, to those of the gene encoding the catalytic subunit of human clotting factor XIII: they have the same number of exons, corresponding introns always interrupt the coding region in the same phase of the codon, and most exons are of similar size (10 or 15 are exactly the same size). In these respects, the TGK and factor XIII catalytic subunit genes resemble each other more than either resembles the gene for erythrocyte band 4.2, a noncatalytic transglutaminase superfamily member. Exon II in both the TGK and factor XIII genes encodes an amino-terminal extension of nonhomologous sequence which in each protein confers a specialized function (membrane anchorage or activation of cross-linking, respectively). This suggests that the evolution of these genes included recruitment of a new exon to modify the enzyme action. Southern blots of genomic DNA reveal the presence of a TGK-like gene in birds, amphibians, and fish, but not in flies.


Glucose regulation of acetyl-CoA carboxylase in hepatoma and islet cells. The regulation of acetyl-CoA carboxylase (ACC) by glucose and other fuel molecules has been examined in Fao Reuber hepatoma cells and Syrian hamster insulin tumor (HIT) cells in order to determine whether lipogenic substrates acutely alter ACC activity and to examine the mechanism of such regulation. In Fao cells, preincubated in simple medium without substrates, glucose addition results in a rapid activation of ACC. This effect, mimicked by other fuels such as lactate, is characterized by an increase in enzyme Vmax and a decrease in the activation constant for citrate. Several lines of evidence indicate that this activation of ACC is due to enzyme dephosphorylation, including the kinetic changes observed, the persistence of enzyme activation through ACC isolation, the necessity of inclusion of sodium fluoride/EDTA in the cell lysis buffer for preservation of the glucose-induced change, and the direct demonstration of diminished 32P-labeling of ACC after glucose exposure. Identical effects of glucose are also observed in HIT cells, although the ACC activation is smaller in magnitude and less sensitive than that observed in Fao cells. Other insulin secretagogues such as glutamine, lactate, and isobutylmethylxanthine are also found to activate HIT ACC. Others have suggested that glucose-induced changes in malonyl-CoA in beta-cells may be linked to glucose-induced insulin secretion. However, studies conducted in late passage HIT cells, which fail to secrete insulin in response to glucose stimulation, reveal the same glucose-induced activation seen in early passages, secretion-competent HIT cells, suggesting that glucose-induced ACC activation is not by itself sufficient to provoke insulin secretion. Taken together, these findings indicate that glucose and other fuel molecules can play a major role in the rapid regulation of the fatty acid synthesis pathway. The activation of fatty acid synthesis by substrate-induced ACC dephosphorylation insures ultimate fuel storage of glucose-derived carbon as fatty acid, while substrate-induced increases in the ACC product, malonyl CoA, would serve to simultaneously limit the rate of fatty acid oxidation through its allosteric regulation of carnitine palmitoyltransferase I.


Evidence for an apical sorting signal on the ectodomain of human aminopeptidase N. In polarized epithelial cells aminopeptidase N is targeted to the apical membrane. The aim of this study was to determine whether a sorting signal is necessary for its correct transport to the apical membrane and, if so, to localize this sorting signal to one of the domains of the transmembrane protein. Anchor-minus aminopeptidase N, consisting of the hemagglutinin signal peptide including its cleavage site, and the ectoplasmic domain of human aminopeptidase N were stably expressed in Madin-Darby canine kidney cells cultured on polycarbonate filters. By measurement of the enzymatic activity it was found that the anchor-minus aminopeptidase N was secreted in a polarized manner to the apical side. As a reference the secretion of the secretory granule protein, cystatin C, was likewise studied. Cystatin C was found to be secreted in a nonpolarized manner to both domains. Our data thus show that human aminopeptidase N carries an apical sorting signal and that it is localized on the ectodomain of the enzyme.


Calcium ionophore treatment impairs the sterol-mediated suppression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, 3-hydroxy-3-methylglutaryl-coenzyme A synthase, and farnesyl diphosphate synthetase. We report that the sterol-mediated suppression of the mRNA levels of three cholesterogenic enzymes, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, HMG-CoA synthase, and farnesyl diphosphate (FPP) synthetase is partially overcome by the calcium ionophore A23187. Addition of A23187 to the human monocytic leukemia cell line THP-1 in the presence of fetal calf serum led to rapid increases in mRNA concentration of up to 40-fold for HMG-CoA synthase and 15-fold for HMG-CoA reductase with little or no change in FPP synthetase mRNA levels. Treatment of HepG2 cells with A23187 resulted in approximately 2-4-fold increases in the mRNA levels for these three enzymes. The increases in HMG-CoA synthase and HMG-CoA reductase mRNAs were maximal after treatment of THP-1 cells with 10 micrograms/ml A23187 for 3 h. The stimulation was blocked by actinomycin D but not by cycloheximide treatment. Ionophore treatment had no effect on the half-lives of the mRNAs for HMG-CoA reductase and HMG-CoA synthase. Surprisingly, the addition of A23187 to THP-1 cells incubated in the presence of 25-hydroxycholesterol and mevalonic acid also led to significant increases in the mRNA levels for HMG-CoA reductase and HMG-CoA synthase. Finally, the stimulation of these mRNA levels by A23187 was reduced in cells in which protein kinase C had been inactivated by preincubation of the cells with a phorbol ester. Taken together, these data suggest that A23187 treatment results in increased transcription of HMG-CoA reductase, HMG-CoA synthase, and, in some cell types, FPP synthetase by a mechanism that does not involve de novo protein synthesis. We speculate that A23187 treatment results in the modification of a trans-acting factor(s) which is common for the transcription of all these genes.


Generation of signals activating neutrophil functions by leukocyte integrins: LFA-1 and gp150/95, but not CR3, are able to stimulate the respiratory burst of human neutrophils. To address the question whether leukocyte integrins are able to generate signals activating neutrophil functions, we investigated the capability of mAbs against the common beta chain (CD18), or the distinct alpha chains of CR3, LFA-1, or gp150/95, to activate neutrophil respiratory burst. These investigations were performed with mAbs bound to protein A immobilized to tissue culture polystyrene. Neutrophils plated in wells coated with the anti-CD18 mAbs IB4 and 60.3 released H2O2; H2O2 release did not occur when neutrophils were plated in wells coated with an irrelevant, isotype-matched mAb (OKDR), or with mAbs against other molecules (CD16, beta 2-microglobulin) expressed on the neutrophil surface at the same density of CD18. Four different mAbs, OKM1, OKM9, OKM10, 60.1, which recognize distinct epitopes of CR3 were unable to trigger H2O2 or O2- release from neutrophils. However, mAbs against LFA-1 or gp150/95 triggered both H2O2 and O2- release from neutrophils. Stimulation of neutrophils respiratory burst by both anti-CD18, and anti-LFA-1 or gp150/95 mAbs was totally inhibited by the microfilaments disrupting agent, cytochalasin B, and by a permeable cAMP analogue. While the capability to activate neutrophil respiratory burst was restricted to anti-LFA-1 and gp150/95 mAbs, we observed that mAbs against all members of leukocyte integrins, including CR3, were able to trigger neutrophil spreading. These findings indicate that, in neutrophils, all three leukocyte integrins can generate signals activating spreading, but only LFA-1 and gp150/95 can generate signals involved in activation of the respiratory burst. This observation can be relevant to understand the mechanisms responsible for the activation of neutrophil respiratory burst by tumor necrosis factor-alpha, which has been shown to be strictly dependent on expression of leukocyte integrins (Nathan, C., S. Srimal, C. Farber, E. Sanchez, L. Kabbash, A. Asch, J. Gailit, and S. Wright. 1989. J. Cell Biol. 109:13411349.


Epidermal growth factor and transforming growth factor-alpha decrease gamma interferon receptors and induction of intercellular adhesion molecule (ICAM-1) on cultured keratinocytes. The link between the epidermal keratinocytes of the skin and the activated T lymphocytes of the immune system is mediated by a variety of cytokines, including gamma interferon (IFN-gamma). We studied the influence of keratinocyte mitogens such as transforming growth factor-alpha (TGF-alpha), epidermal growth factor (EGF), and somatomedin-C (SM-C) on the ligand binding of 32P-labeled IFN-gamma to cultured keratinocytes derived from normal appearing adult human skin. Keratinocytes placed in a medium devoid of mitogens become growth arrested, and these quiescent cells expressed 2.4 times (28,900 versus 12,200 sites/cell) as many high affinity IFN-gamma receptors (Kd = 0.22 nM) compared to keratinocytes which were actively growing in medium containing TGF-alpha (25 ng/ml) or EGF (10 ng/ml). The reduction in IFN-gamma receptor sites by TGF-alpha/EGF was mitogen specific, as adding SM-C (500 ng/ml) did not have any effect on ligand binding, although it similarly stimulated keratinocyte growth. The reduction in IFN-gamma receptors was time dependent, occurring primarily after 24-48 hours of change in tissue culture conditions. The reduction in the number of high affinity IFN-gamma receptors by TGF-alpha/EGF had immunobiological consequences, because quiescent keratinocytes in basal medium had an increased expression of HLA-DR and intercellular adhesion molecule-1 (ICAM-1) induced by IFN-gamma, compared to actively growing TGF-alpha/EGF treated keratinocytes. These results suggest that rapidly proliferating keratinocytes exposed to TGF-alpha/EGF but not SM-C are capable of altering their response to IFN-gamma by decreasing their number of cell surface high affinity receptors for IFN-gamma.


TGF-beta-induced G2/M delay in proliferating rabbit articular chondrocytes is associated with an enhancement of replication rate and a cAMP decrease: possible involvement of pertussis toxin-sensitive pathway. This study was undertaken to gain more insight into the mechanism whereby TGF-beta influences the cell cycle progression of cultured rabbit articular chondrocytes. Using proliferating chondrocytes in fetal calf serum-containing medium, we have previously shown that TGF-beta induced a recruitment of cells at the end of the S phase (G2/M) observed 24 h after addition. The delayed cells may then be released, producing a proliferative effect at 48 h, provided a substantial amount of FCS (10%) is present in the medium. Otherwise, in low level of serum (2% FCS, for example), only inhibition of cell proliferation is observed. In chondrocytes synchronized in S phase by a thymidine block, we investigated here the time-course incorporation of [3H]-thymidine into DNA, the cell cycle traverse by flow cytofluorometric study of DNA content, the expression of PCNA (Proliferating Cell Nuclear Antigen), and cAMP levels. The data demonstrate that TGF-beta provoked a decrease of cAMP content (0.5-1 h) followed by an enhancement of the DNA synthesis rate (4 h) which was detectable through cytofluorometric analysis and [3H]-thymidine labeling and correlated with the PCNA expression. In contrast, addition of cAMP analogues to the cultures resulted in an inhibition of replication rate. We also showed that pertussis toxin produced a decrease of the DNA synthesis rate, in a transient manner and only in the presence of TGF-beta. All these results suggest that TGF-beta may accelerate the replication process of cyclized chondrocytes, making then accumulate at the G2/M boundary, via a mechanism that could involve the adenylate cyclase activity and a Gi-protein. The factor might be responsible for producing a pool of cells having already replicated their DNA and therefore capable of re-entering the cell cycle without delay. This cell population could serve as a tissue reserve able to induce a mitosis wave when necessary--for example, in the repair of tissue damage.


Differential expression of asialoglycoprotein receptor subunits in the endocytic compartment during liver regeneration. Asialoglycoprotein receptors, responsible for the removal of circulating asialoglycoproteins by the liver, are located in at least two different membrane locations in hepatocytes. Receptors on the cell surface account only for a minor proportion (20-36%), for the majority of receptors in the liver are located intracellularly, mainly in the endocytic membrane networks. An understanding of the basis of receptor distribution and the underlying trafficking of receptors between the hepatocyte's polarised cell surface and the endocytic compartment would be aided if biochemical differences between the receptors in these pools were established. We now show, using three antibodies that recognise the receptor subunits in rat liver (RHL-1, RHL-2 and RHL-3), that the asialoglycoprotein receptors located in the plasma membrane domains and the endocytic compartment differ in oligomeric composition, sialic acid content, and solubility in Triton X-114 using two-phase systems. It is well established that the expression of the asialoglycoprotein receptor is down-regulated in livers regenerating after a partial hepatectomy. We demonstrate that the levels of the receptor subtype that is located mainly in the endocytic compartment (RHL-1, 42 kDa) was elevated in regenerating liver by agents that regulate cAMP production, whereas the levels of the other receptor subtypes remained unchanged. The asialoglycoprotein receptor subtypes that are present in different subcellular locations are thus regulated independently.


Induction of proliferating cell nuclear antigen (PCNA) complex formation in quiescent fibroblasts from a xeroderma pigmentosum patient. Accumulated evidence indicates that proliferating cell nuclear antigen (PCNA) is an auxiliary protein of DNA polymerase delta and forms tight association with DNA replication sites during DNA replication or DNA repair synthesis. In this study, such PCNA complex formation was investigated by the indirect immunofluorescence method, using both normal human fibroblasts and those derived from a xeroderma pigmentosum group A (XP-A) patient. XP-A fibroblasts in both proliferating and quiescent states did not show any differences from normal fibroblasts in the properties of PCNA-staining in the untreated conditions. The PCNA complex formation was induced in quiescent normal fibroblasts by both ultraviolet light (UV)- and X-irradiation, whereas in XP-A fibroblasts it was induced by X-irradiation, but not by UV-irradiation. However, PCNA complex was induced in quiescent XP-A fibroblasts by UV-irradiation when the cells had previously incorporated 5-bromodeoxyuridine (BrdU). These observations indicate a close correlation of PCNA complex formation and unscheduled DNA synthesis (UDS). Thus, it was concluded that PCNA complex formation was commonly induced in at least three conditions to produce UDS in spite of different types of DNA damages and DNA repair mechanisms.


Immunological changes in cats with concurrent Toxoplasma gondii and feline immunodeficiency virus infections. To examine the immunological changes in cats concurrently infected with feline immunodeficiency virus (FIV) and Toxoplasma gondii, kittens (four per group) were inoculated with FIV, T. gondii, both agents, or no pathogens. Blood mononuclear cells and plasma were collected weekly for lymphocyte assays and serology. At week 14, spleen and lymph node cells were used for lymphocyte assays; brains and mesenteric lymph nodes were used for isolation of T. gondii. More T. gondii organisms were present in tissues of the dually infected cats than in tissues of cats with toxoplasmosis alone. Two dually infected cats and one cat infected with T. gondii developed chorioretinitis. Spleen, lymph node, and blood mononuclear cells from dually infected cats had the greatest reduction in mitogenic responses. By week 3, cats infected with FIV underwent a decrease in the number of CD4 cells that was not changed by concurrent T. gondii infection; the number of CD8 cells increased only in cats infected with T. gondii alone. For cats infected with T. gondii, the responses of lymphocytes to T. gondii antigen were not affected by FIV infection; the responses to FIV antigen were negligible in all groups. Overall, this study indicates that FIV infection favors T. gondii proliferation. Also, the establishment of toxoplasmosis may enhance FIV-induced immunodeficiency and is likely to cause a more rapid disease progression than that from infection with FIV alone.


Overview from the International Conference on Long-Term Tamoxifen Therapy for Breast Cancer. The development of tamoxifen therapy to treat selected patients, with all stages of breast cancer, has provided the clinical community with an efficacious and safe drug for long-term therapy. Issues of safety are under constant review, but justified concerns about high doses of tamoxifen acting as a promoter of liver cancer in rats or as a promoter of endometrial cancer in women have not, as yet, proved to be of clinical relevance. The situation will continue to be reviewed during the development of the prevention studies in Europe and the United States because an improvement in women's health is the ultimate goal of these programs. The hallmark for the successful development of tamoxifen has been the close cooperation between the laboratory and the clinic. The clinical strategy of long-term tamoxifen therapy is a direct application of a laboratory concept. Furthermore, potential problems in the clinic have been identified in the laboratory, and the clinical community has responded quickly to evaluate the real risks to the patient population. This close cooperation will continue. Issues of drug resistance, new antiestrogen development, and the application of the knowledge about steroid receptors to develop targeted gene therapies are being addressed so that additional treatment approaches for breast cancer will be in place by the turn of the century.


[Memory at the synaptic level].

[Memory at the synaptic level]. Ever since the discovery of the synapse at the end of the last century it has been surmised that elementary neural changes underlying learning and memory are located at the junctions between nerve cells. Experimental studies during the past 20 years have demonstrated the existence of several synaptic modification processes, the most prominent being long-term potentiation (LTP) in the hippocampus. Several links between LTP and learning/memory have been established. For example, memory impairment in older rats is well correlated, with increasing decline of LTP, and N-methyl-D-aspartate (NMDA) receptor antagonists give rise to a parallel blockade of LTP and of spatial task learning. Studies on rats in a natural learning situation have also demonstrated 'spontaneous' occurrence of LTP. LTP is induced as a consequence of coincident pre- and post-synaptic activity, and thus in conformity with a basic principle of learning theory known as Hebb's rule. Responsible for this associative induction is the NMDA-subtype of glutamate receptor channel with its unique property of being both transmitter and voltage controlled. Its opening allows calcium ions to enter the postsynaptic cell and to initiate biochemical processes leading to a lasting synaptic modification. The nature of the critical processes involved in establishing the modification(s) is uncertain, although the participation of calcium-activated protein kinases seems likely. There is still considerable controversy whether the actual change occurs postsynaptically, or presynaptically, triggered via a retrograde signal from the postsynaptic cell. LTP similar to that in the hippocampus has recently been described for various neocortical regions.


Comparison of artemether and chloroquine for severe malaria in Gambian children. Artemether is an oil-soluble methyl ether of artemesinin (qinghaosu). It has been studied extensively in China, where it has been shown to be rapidly effective in severe falciparum malaria. Nearly all the patients studied previously were adults. We have investigated the efficacy of artemether in children with moderate or severe falciparum malaria. In the preliminary study of moderately severe malaria, 30 Gambian children were randomised in pairs to receive either intramuscular artemether (4 mg/kg loading dose followed by 2 mg/kg daily) or intramuscular chloroquine ('Nivaquine') 3.5 mg base/kg every 6 h. Both drugs were well tolerated and rapidly effective. The times to parasite clearance were significantly shorter in the artemether recipients (mean 36.7 [SD 11.3] vs 48.4 [16.8] h, p less than 0.05). 43 children with severe malaria were then randomised to receive intramuscular treatment with the same regimens of artemether (n = 21) or chloroquine (n = 22) as used in the preliminary study. 8 children (19%) died. There were no significant differences between the two groups in the clinical, haematological, biochemical, or parasitological measures of therapeutic response in survivors and there was no evidence of local or systemic toxicity. Despite similar parasite counts on admission, clearance times overall were longer in severe malaria than in moderate malaria. Artemether is a well tolerated and rapidly effective parenteral treatment for severe malaria in children, and would be especially valuable in areas with chloroquine-resistant P falciparum.


Lymphocytic sialadenitis of Sjögren's syndrome associated with chronic hepatitis C virus liver disease. Viral infection has often been suggested as a possible cause of Sjögren's syndrome or chronic lymphocytic sialadenitis, and Epstein-Barr virus has been found in the salivary glands of patients with this condition. After we had noted Sjögren's syndrome in several patients infected with hepatitis C virus (HCV), a virus also excreted in saliva, we set up a prospective study to investigate the association of chronic lymphocytic sialadenitis, with or without symptoms, to chronic HCV liver disease. The histological appearances of labial salivary glands in patients with proven HCV hepatitis or cirrhosis were compared with those in dead controls. Histological changes characteristic of Sjögren's syndrome were significantly more common in HCV-infected patients (16 of 28, 57%) compared with controls (1 of 20, 5%). Focal lymphocytic sialadenitis characteristic of Sjögren's syndrome (though only 10 patients had xerostomia and none complained of xerophthalmia) appears to be common in patients with chronic HCV liver disease; if this association is confirmed, identification of the underlying mechanism may improve our understanding of both disorders.


Trial of cyclosporin in corticosteroid-dependent chronic severe asthma. The treatment of chronic severe asthma is unsatisfactory for many patients. In a randomised, double-blind, placebo-controlled, crossover trial we have tested whether cyclosporin, which is thought to act primarily by inhibition of T lymphocyte activation, improves lung function in corticosteroid-dependent asthmatics. After a 4-week run-in period, 33 patients with longstanding asthma (mean duration 27 years), and who had required continuous oral corticosteroids for a mean of 9.3 years, were randomised to receive either cyclosporin (initial dose 5 mg/kg per day) or placebo for 12 weeks, crossing over after a 2-week washout period. Mean baseline forced expiratory volume in 1 s (FEV1) was 60.1% of the predicted value. 2 patients failed to complete the protocol and 1 withdrew because of hypertrichosis. Cyclosporin therapy resulted in a mean increase above placebo of 12.0% in morning peak expiratory flow rate (PEFR; p less than 0.004) and 17.6% in FEV1 (p less than 0.001). The frequency of disease exacerbations requiring an increased prednisolone dose was reduced by 48% in patients on cyclosporin compared with placebo (p less than 0.02). Diurnal variation in PEFR decreased by a mean of 27.6% (p = 0.04). Cyclosporin for 12 weeks was well tolerated by this group of chronic asthmatics, in whom the mean whole-blood trough concentration was 152 micrograms/l. These findings provide further evidence of a role for activated T lymphocytes in the pathogenesis of asthma. Specific pharmacological targeting of this cell could form the basis of a novel approach to the treatment of asthma.


Enzyme-linked immunosorbent assay for diagnosis of acute sporadic hepatitis E in Egyptian children. Hepatitis E virus (HEV) is thought to be a cause of enterically transmitted non-A, non-B (ET-NANB) hepatitis. Waterborne epidemics have been recorded in many developing countries, mainly affecting young-to-middle-aged adults; sporadic infection and overt illness in children are rare. However, a convenient and sensitive diagnostic test for HEV infection is not yet available. We now report the use of a solid-phase enzyme-linked immunoassay (ELISA) that detects IgM and IgG antibody to HEV. In a prospective study of endemic acute hepatitis during 1986 in rural Benha, Egypt, 15 (42%) of 36 children with NANB hepatitis (from whom convalescent-phase sera were available every 3 months to 9 or 12 months) were positive for anti-HEV-IgG by ELISA. Of 20 sera from healthy Benha children (controls), 5 (25%) were also positive for anti-HEV-IgG. When evaluated for anti-HEV-IgM, 6 of the 15 IgG-positive children, but none of the controls, were IgM positive and were thus regarded as having confirmed acute HEV infections. These 6 cases together with 2 presumptive cases (IgM negative, IgG seroconversion from positive to negative) presented sporadically over 9 months. This ELISA is a convenient method for the diagnosis of HEV infection; we have shown that the disease is present in Egypt, that it can occur endemically as sporadic cases, and that children do have overt infection.


Balloon dilatation of the arterial duct in congenital heart disease. The systemic circulation of newborn infants with congenital left-heart obstruction is supplied from the right ventricle via a patent arterial duct between the pulmonary artery and descending aorta. The duct closes during the first few days of life, but infusion of prostaglandin E2 can prevent closure in some cases. We report four newborn infants (aged 3-8 days) with intractable heart failure due to severe obstruction of the left heart in the presence of a closing arterial duct. Infusion of prostaglandin E2 did not improve their clinical condition. Cardiac catheterisation and balloon dilatation of their arterial ducts resulted in a dramatic improvement in the babies' clinical condition; during subsequent surgical repair of the infants' hearts, the arterial ducts were found to be widely patent. Balloon dilatation gives immediate and sustained wide patency of the arterial duct in infants who do not respond adequately to prostaglandin E2.


Toxicity of clindamycin as prophylaxis for AIDS-associated toxoplasmic encephalitis. Community Programs for Clinical Research on AIDS. A double-blind, placebo-controlled trial was set up to compare clindamycin and pyrimethamine as prophylaxis for toxoplasmic encephalitis (TE) in HIV-infected patients at risk of the disorder. Interim analysis showed that clindamycin-treated patients were 4.4 (95% confidence interval 1.3-15.2) times more likely to experience an adverse effect that necessitated withdrawal of the study drug than those who received placebo. Diarrhoea and rash were reported in 16 (31%) and 11 (21%), respectively, of 52 patients treated with clindamycin (300 mg twice daily) compared with 2 (6%; p = 0.06) and none (p = 0.01) of the 32 placebo-treated patients. The clindamycin arm of the trial was prematurely terminated, although recruitment to the pyrimethamine arm continues.


Dermatan sulphate in haemodialysis.

Dermatan sulphate in haemodialysis. Experimental work suggests that dermatan sulphate has potential as an antithrombotic agent: it can inhibit venous thrombi yet has less effect upon bleeding than heparin. While heparin functions as an anticoagulant primarily by its ability to accelerate the action of the plasma protein inhibitor antithrombin III, dermatan sulphate acts selectively through a structurally related inhibitor, heparin co-factor II, to inhibit thrombin. We have done a series of dose-finding studies of the use of dermatan sulphate as an anticoagulant/antithrombotic agent in patients on maintenance haemodialysis. Dermatan sulphate proved to be an effective anticoagulant in this setting.


Clinical value of polysomnography.

Clinical value of polysomnography. Polysomnography is used increasingly to investigate patients with possible sleep apnoea/hypopnoea syndrome (SAHS), but it has not been assessed critically. We thus examined prospectively the value of electrophysiological and respiratory monitoring in 200 consecutive adults (163 men, 37 women; mean [SD] age 50 [13] years) having polysomnography. At polysomnography, 91 patients had SAHS (greater than 15 apnoeas + hypopnoeas [A + H] per h asleep) and 11 had periodic limb-movement disorder. Recording sleep electrophysiologically was of no diagnostic value and SAHS could be as accurately defined by A + H per time in bed as by A + H per time asleep. 66% of patients with SAHS could be diagnosed with oximetry alone, but many of the undiagnosed patients had moderately severe SAHS and benefited from treatment. Neurophysiological sleep recording is unnecessary and oximetry alone is of limited value in the overnight investigation of patients suspected of having SAHS.


Beta-adrenergic agonists and hypertrophy of skeletal muscles. Chronic administration of some beta-adrenergic agonists markedly stimulates hypertrophy of skeletal muscles. It appears that type II fibers are more responsive to beta-adrenergic agonists than type I fibers. The hypertrophic effect of beta-adrenergic agonists is transient, with the effect diminishing during prolonged treatment. Similarly, some cellular responses including the increase in RNA concentration and the decrease in calpain I activity are also short-lived. Recent evidence suggests that the temporal response is associated with decreased beta-adrenoceptor density. Both increased rate of protein synthesis and/or decreased protein degradation have been suggested as the mechanism of action of these compounds on hypertrophy of skeletal muscles. It is important to consider the temporal nature of cellular responses to chronic treatment of beta-adrenergic agonists as well as the differential effects of these compounds on protein metabolism among skeletal muscle fiber types when investigating the mechanism(s) of action of these compounds.


Ethanol inhibits mitogen-induced calcium mobilization in mouse splenocytes. Ethanol inhibited the mitogen-induced initial increase in cytoplasmic free-calcium [Ca2+]i in mouse splenocytes. This effect was concentration-dependent, reversible, and observed at pharmacologically relevant concentrations (24-166mM). Other short-chain alcohols such as propanol, butanol, and pentanol also inhibited this mitogen-induced increase in [Ca2+]i. The potencies of these alcohols to produce this effect were highly correlated (r = 0.98, p less than 0.001) with their membrane/buffer partition coefficients. Analysis of mouse splenocyte subpopulations demonstrated that this effect was manifest in both B and T lymphocytes. Within T lymphocyte subpopulations, both CD4+ and CD8+ T cells were affected. These results suggest that the inhibition of [Ca2+]i increase may be an early event mediating ethanol-induced immunosuppression and that this may be a predisposing factor to infection and malignancies associated with alcoholism.


A genetic comparison of human and wildlife isolates of Echinococcus granulosus in Queensland: public health implications. To test the hypothesis that the hydatid parasite infecting macropods and dingoes in Queensland is a sylvatic strain of Echinococcus granulosus, distinct from the domestic strain which produces cysts in sheep and humans. Molecular biological techniques were used to compare DNA isolated from hydatid cysts from humans, local macropods and sheep from New South Wales and the United Kingdom, as well as from adult tapeworms in dingoes. The human cysts were surgically resected from two patients seen with hydatidosis in Brisbane teaching hospitals over a one-year period. Neither patient had had previous contact with sheep farms. Macropods and dingoes were shot randomly in the localities where the patients presumably acquired their infections. Sheep liver cysts were obtained from abattoirs. Studies comprised extraction of DNA from cysts, digestion by a series of restriction endonucleases, slab gel electrophoresis. Southern blotting and then hybridisation with defined DNA probes. Polymerase chain reaction, in combination with direct DNA sequencing, was used to compare DNA from cysts and adult worms from dingoes. The restriction fragment length polymorphism (RFLP) patterns of DNA from all cysts and a defined mitochondrial DNA sequence from all sources were indistinguishable. This finding is significant as both techniques can clearly distinguish between genetically distinct, well characterised strains of E. granulosus. Hydatid cysts are prevalent in some macropod populations and adult worms are common in dingoes. Since there are relatively few sheep-rearing areas in Queensland, contact with wild animals may be the main source of human hydatid infection in this State. The strain of E. granulosus in both patients was genetically indistinguishable from that found in macropods, dingoes and sheep from New South Wales and the United Kingdom. This strongly suggests that the domestic strain of E. granulosus, or a form very close genetically, freely infects Australian wildlife, and argues against the existence of a distinct sylvatic strain. The implications for public health are considerable.


Different conformations for the same polypeptide bound to chaperones DnaK and GroEL. The proteins DnaK (hsp70) and GroEL (cpn60) from Escherichia coli are prototypes of two classes of molecular chaperones conserved throughout evolution. The analysis of transferred nuclear Overhauser effects in two-dimensional NMR spectra is ideally suited to determine chaperone-bound conformations of peptides. The peptide vsv-C (amino-acid sequence KLIGVLSSLFRPK) stimulates the ATPase of BiP and Hsc70 (ref. 3) and the intrinsic ATPase of DnaK. The affinity of the vsv-C peptide for DnaK is greatly reduced in the presence of ATP. Here we analyse transferred nuclear Overhauser effects and show that the peptide is in an extended conformation while bound to DnaK but is helical when bound to GroEL. NMR also indicates that the mobility of the peptide backbone is reduced more by binding to DnaK than by binding to GroEL, whereas the side chains are less mobile when bound to GroEL.


Nearly ubiquitous tissue distribution of the scrapie agent precursor protein. The "modified host protein" model of scrapie proposes that the transmissible agent is composed of the degradation-resistant protein, Sp33-37, and that clinical and pathologic signs result from neurotoxic accumulations of this protein. Sp33-37 is an abnormal, amyloidogenic isoform of the normally occurring cellular protein Cp33-37. This study investigated the tissue distribution of Cp33-37 in hamster. In brain, Cp33-37 was most concentrated in the hippocampal formation. Immunohistochemical studies localized Cp33-37 to neurons and surrounding neuropil in hippocampus; septal, caudate, and thalamic nuclei; dorsal root ganglia cells; and large-diameter dorsal root axons. In non-neuronal hamster tissues, Cp33-37 was detected in circulating leukocytes, heart, skeletal muscle, lung, intestinal tract, spleen, testis, ovary, and some other organs. The presence of Cp33-37 in extracerebral tissues indicates that its function is not unique to brain. These results indicate that the molecular substrate for the production of Sp33-37, the scrapie agent, and scrapie amyloid is present in a variety of cerebral and extracerebral sites.


T-cell receptor alpha chain germline gene polymorphisms in multiple sclerosis. It has been proposed that genetic predisposition to multiple sclerosis (MS) and other diseases with autoimmune features is conferred by T-cell receptor (TcR) genes in addition to HLA class II genes. Although a family study has suggested linkage of susceptibility to MS to TcR genes, reports of disease associations with restriction fragment length polymorphism (RFLP)-defined alleles of TcR genes have been difficult to confirm, including a report of association of MS with TcR beta-chain gene RFLPs. We report here the distribution of three sets of RFLPs of the TcR alpha chain genes. Similar frequencies in patients and controls were observed for TaqI and BglII RFLPs of the TcR alpha constant segment (C alpha), the latter earlier reported to be associated with MS. A previously reported MS-associated PssI RFLP of the V alpha 12 and C alpha gene segments could not be confirmed. Our results indicate that these seemingly polymorphic restriction fragments are possibly the results of incomplete enzymatic cleavage of DNA in the RFLP analysis. We conclude that no convincing evidence exists for the association of MS with RFLPs of the TcR alpha or beta chain genes.


[Prevention of carcinoid tumor crisis].

[Prevention of carcinoid tumor crisis]. A case of the carcinoid tumour of ileum causing hormone producing multiple hepatic metastases was described. Sometimes after feeding and drinking of beer the "flush" and the diarrhoea appeared. Multiple hepatic metastases were established by ultrasound. Two and a half years ago the patient already was examined and treated by another hospital. In this time the origin of the primaer carcinoid tumour was not found and the superselective embolisation of the right lobe of the liver was made which caused a carcinoid crisis. Later the complaints were renewed and once more the patient was examined. The origin of the illness was proved in the lower ileum by CT (computer tomography), angiography and I131 MIBG (metajod-benzyl-guanidin) scintigraphy. Another embolisation of the liver caused a newer carcinoid crisis. The operation of primaer carcinoid tumour was decided because of the danger of carcinoid crisis and ileus. In the perioperative period the patient was protected against carcinoid crisis by Sandostatin (made in SANDOZ, Basel), because the preoperative therapy, the anaesthetics and the surgical manipulation could have caused a carcinoid crisis. In Hungary the authors used for the first time somatostatin in perioperative period to protect the patient against carcinoid crisis.


Glomerulopathy induced by graft-versus-host reaction in the rat. Requirement of donor CD4+ T lymphocytes and MHC class II incompatibility at the lymphoid compartment. Graft-versus-host reactions (GVHR) can be associated with several autoimmune phenomena involving the kidney as a target organ. By transferring lymphocytes of AO rats into complete Freund's adjuvant-pretreated (AO x BN)F1 hybrids, a dose-dependent GVHR with glomerulopathy was experimentally induced. IgM, IgG1, and IgG2a were deposited in the mesangial area and along the glomerular basement membrane. Eluted immunoglobulins from diseased kidneys bound to normal basement membranes and especially to laminin. Anti-laminin reactivity was also present in sera from F1 recipients with GVHR. Parental CD4+ T lymphocytes were required and sufficient to induce GVHR and glomerulopathy in sublethally irradiated F1 hybrids. Using various F1 hybrids, MHC class II incompatibility was shown to be required for the induction of GVHR-associated glomerulopathy. Across MHC class I incompatibility, GVHR without glomerulopathy could be induced, provided that both CD4+ and CD8+ donor T lymphocytes were administered. Finally, MHC incompatibility between donor T lymphocytes and the recipient non-lymphoid compartment was found to be sufficient for the induction of GVHR, but not for GVHR-associated glomerulopathy. The results indicate that alloreactive donor CD4+ T lymphocytes have to interact directly with MHC class II alloantigen bearing host B lymphocytes in order to stimulate the latter to produce (auto-)antibodies. GVHR-induced glomerulopathy shares several immunopathological features with HgCl2-induced autoimmune glomerulopathy in the rat.


Modulation of activity of the promoter of the human MDR1 gene by Ras and p53. Drug resistance in human cancer is associated with overexpression of the multidrug resistance (MDR1) gene, which confers cross-resistance to hydrophobic natural product cytotoxic drugs. Expression of the MDR1 gene can occur de novo in human cancers in the absence of drug treatment. The promoter of the human MDR1 gene was shown to be a target for the c-Ha-Ras-1 oncogene and the p53 tumor suppressor gene products, both of which are associated with tumor progression. The stimulatory effect of c-Ha-Ras-1 was not specific for the MDR1 promoter alone, whereas a mutant p53 specifically stimulated the MDR1 promoter and wild-type p53 exerted specific repression. These results imply that the MDR1 gene could be activated during tumor progression associated with mutations in Ras and p53.


High probability opening of NMDA receptor channels by L-glutamate. Synaptic plasticity can be triggered by calcium flux into neurons through synaptically activated N-methyl-D-aspartate (NMDA) receptor channels. The amplitude and time course of the resulting intracellular calcium transient depend on the number of open NMDA receptor channels and the kinetics of their activation. Short applications of L-glutamate to outside-out patches from hippocampal neurons in the presence and absence of MK-801 revealed that about 30 percent of L-glutamate-bound channels are open at the peak of the current. This high probability of opening suggests that very few channels are required to guarantee a large, localized postsynaptic calcium transient.


Familial recurrence-pattern analysis of cleft lip with or without cleft palate. Cleft lip with or without cleft palate (CL/P) is a common congenital malformation with an incidence in European white populations of about 1/1,000. The familial clustering of CL/P has been extensively characterized, and epidemiological studies have proposed monogenic models (with reduced penetrance), multifactorial/threshold models, and mixed major-gene/multifactorial models to explain its inheritance. The recognition of an association between two RFLPs at the transforming growth factor alpha (TGFA) locus and CL/P supports a major-gene component to the etiology of CL/P. Risch has shown that the recurrence risk ratio lambda R (risk to relatives, vs. population prevalence) is a useful pointer to the mode of inheritance. Here we further develop the use of lambda R to analyze recurrence-risk data for CL/P. Recurrence risks for first-, second-, and third-degree relatives equate well with oligogenic models with as few as four loci. A monogenic/additive model is strongly rejected. The limited available twin data are also consistent with this model. A "major gene" interacting epistatically with an oligogenic background is shown to be a plausible alternative. Power calculations for a linkage study to map the CL/P major-risk locus suggest that a sample of 50 affected sib pairs will be adequate, but linkage to minor-risk loci will require very much larger samples.


Isolation and characterization of new highly polymorphic DNA markers from the Huntington disease region. The defect causing Huntington disease (HD) has been mapped to 4p16.3, distal to the DNA marker D4S10. Subsequently, additional polymorphic markers closer to the HD gene have been isolated, which has led to the establishment of predictive testing programs for individuals at risk for HD. Approximately 17% of persons presenting to the Canadian collaborative study for predictive testing for HD have not received any modification of risk, in part because of limited informativeness of currently available DNA markers. Therefore, more highly polymorphic DNA markers are needed, which will further increase the accuracy and availability of predictive testing, specifically for families with complex or incomplete pedigree structures. In addition, new markers are urgently needed in order to refine the breakpoints in the few known recombinant HD chromosomes, which could allow a more accurate localization of the HD gene within 4p16.3 and, therefore, accelerate the cloning of the disease gene. In this study we present the identification and characterization of nine new polymorphic DNA markers, including three markers which detect highly informative multiallelic VNTR-like polymorphisms with PIC values of up to .84. These markers have been isolated from a cloned region of DNA which has been previously mapped approximately 1,000 kb from the 4p telomere.


Factor IXMadrid 2: a deletion/insertion in factor IX gene which abolishes the sequence of the donor junction at the exon IV-intron d splice site. DNA from a patient with severe hemophilia B was evaluated by RFLP analysis, producing results which suggested the existence of a partial deletion within the factor IX gene. The deletion was further localized and characterized by PCR amplification and sequencing. The altered allele has a 4,442-bp deletion which removes both the donor splice site located at the 5' end of intron d and the two last coding nucleotides located at the 3' end of exon IV in the normal factor IX gene; this fragment has been replaced by a 47-bp sequence from the normal factor IX gene, although this fragment has been inserted in inverted orientation. Two homologous sequences have been discovered at the ends of the deleted DNA fragment.


Integrated treatment of panic disorder.

Integrated treatment of panic disorder. The purpose of this article is to provide the nonpsychiatric physician with a practical approach to the treatment of panic disorder by reviewing the literature on treatment efficacy. The author reviews studies and uncontrolled case series on pharmacologic and nonpharmacologic treatment of panic disorder and presents a clinical approach based on these. Setting the stage for treatment by educating patients and obtaining a detailed description of their particular clinical syndrome is a vital, but frequently neglected, precursor to treatment. Treatment selection is based on a risk-benefit analysis of the individual treatment and the particular exigencies of the patient's clinical presentation. Both pharmacologic and nonpharmacologic therapies are highly effective for acute treatment. Because panic disorder is a chronic illness, long-term treatment or reinitiation of acute treatment at a later date is often required.


Alcohol abuse, substance abuse, and panic disorder. The purpose of this article is to review the literature concerning the interaction of alcohol and/or substance abuse with panic disorder, the comorbidity of these disorders, possible causal relationships, biologic relationships, and the recognition and treatment of dually disordered patients. A number of studies suggest significant comorbidity between panic disorder and alcoholism or abuse of drugs, especially cocaine and sedatives. Panic may lead to drinking or sedative use and also result from prolonged use or withdrawal of alcohol or other drugs. Possible biologic relationships involve the gamma-aminobutyric acid (GABA)-benzodiazepine receptor complex and the central noradrenergic system. Although treatment of panic in substance abusers has not been studied specifically, guidelines for recognition and management of these patients, including use of antipanic medication, are discussed.


Lack of association between dopamine D1 and D2 receptor genes and bipolar affective disorder. Fifty-six patients with bipolar affective disorder and 69 healthy control subjects were tested for association of restriction fragment length polymorphism alleles at the dopamine D1 and D2 receptor loci. No significant associations were found; thus, the hypothesis that a single mutant form of either receptor gene is responsible for the phenotype of patients with bipolar affective disorder was not supported.


Cardiovascular function during induced hypotension by fenoldopam or sodium nitroprusside in anesthetized dogs. Fenoldopam, a selective dopamine1 receptor agonist, has been recommended for induced hypotension because it effectively lowers arterial blood pressure and improves renal perfusion. We examined cardiovascular functions during hypotension induced by fenoldopam or sodium nitroprusside. In eight halothane-anesthetized dogs, the left ventricle (LV) was instrumented with pressure and ultrasonic dimension transducers for the assessment of LV contractility using the analysis of the pressure-diameter relationship. Blood flow distribution was measured by radioactive microspheres. Doses of fenoldopam and nitroprusside were titrated to reduce mean arterial blood pressure to 60 mm Hg. After 40 min of hypotension, fenoldopam and nitroprusside caused similar increases in heart rate (17% +/- 4% vs 19% +/- 10%, respectively) and decreases in systemic vascular resistance (-24% +/- 5% vs -27% +/- 4%). Hypotension induced by fenoldopam was associated with higher LV end-diastolic pressure (4.4 +/- 0.6 vs 2.5 +/- 1.1 mm Hg) and end-systolic meridional wall stress (33.0 +/- 4.3 vs 17.8 +/- 2.1 g/cm2) when compared with nitroprusside. There were no significant changes in cardiac output and cardiac contractility as expressed by the slope (Ees) of the LV end-systolic pressure-diameter relationship, velocity of shortening of the diameter, and percentage of wall thickening of the LV. In contrast to nitroprusside, which decreased renal blood flow from 197 +/- 19 to 163 +/- 15 mL/min, renal blood flow increased during fenoldopam-induced hypotension from 187 +/- 20 to 239 +/- 18 mL/min. The increase in renal perfusion was similar in upper, middle, and lower regions of the kidney; however, it was more in the medulla compared with the cortex (37% +/- 17% vs 25% +/- 7%).(ABSTRACT TRUNCATED AT 250 WORDS)


Expression and amplification of neu oncogene in pleomorphic adenomas of salivary gland. Amplification of the neu oncogene and overexpression of its product was found to be a potential marker for aggressive biological behavior in breast cancer. However, the expression of the neu oncoprotein in normal breast ducts and myoepithelial cells has also been demonstrated. Hence, we examined normal salivary gland tissue and 15 cases of pleomorphic adenoma for the expression of the neu oncoprotein by immunohistochemistry using two polyclonal antibodies and 12 cases for the amplification of the neu oncogene using slot blot hybridization. Immunohistochemistry in the normal salivary gland revealed positive staining of all ductal cells. In the pleomorphic adenomas all cellular elements stained to a variable degree. The positive staining was seen in the ductal cells, in the solid sheets, and in chondroid, myxoid, and metaplastic foci. The normal salivary gland and 11 of 12 cases of pleomorphic adenoma showed no increase in copy number of the neu oncogene, whereas one case showed threefold amplification. These results indicate that the neu oncoprotein is expressed but the neu copy number is not increased in normal salivary gland epithelium and in most pleomorphic adenomas. The threefold amplification of the gene in one case may indicate an aggressive biological behavior.


Somatostatin in the management of gastrointestinal fistulas. A multicenter trial. To evaluate the effectiveness of treatment with total parenteral nutrition (TPN) alone (group A) or combined with continuous intravenous infusion of somatostatin (group B) in postoperative gastrointestinal fistulas, a multicenter, controlled and prospective randomized trial was designed. We present the results obtained after the evaluation of 40 cases (group A, n = 20; group B, n = 20). No significant differences among these treatment schedules were observed in the percentage of closure of fistulas (group A, 81.25%; group B, 85%), but patients treated with total parenteral nutrition plus somatostatin had the fistulas close within a significantly shorter period of time. Moreover, this treatment was associated with a significantly lower morbidity. These preliminary results indicate that somatostatin is a useful therapeutic complement in the conservative treatment of patients with gastrointestinal fistulas.


Congenitally defective aldosterone biosynthesis in humans: the involvement of point mutations of the P-450C18 gene (CYP11B2) in CMO II deficient patients. The gene for steroid 18-hydroxylase (P-450C18) has been recently assigned to encode corticosterone methyl oxidases Type I and Type II which were previously postulated to catalyze the final two steps in the biosynthesis of aldosterone in humans. Molecular genetic analysis of the P-450C18 gene is three patients from three different families affected with CMO II deficiency has indicated that a point mutation of CGG----TGG (181Arg----Trp) in exon 3 and one of GTG----GCG (386Val----Ala) in exon 7 occur exclusively in the gene of the patients. Analysis of PCR products by restriction enzymes (HapII and HphI) has indicated that the patients are homozygous and the unaffected parent is heterozygous for both mutations, in accordance with the established concept that CMO II deficiency is inherited in an autosomal recessive manner. These data clearly provide the molecular genetic basis for the characteristic biochemical phenotype of CMO II clinical variants.


Rational design and pre-clinical pharmacology of drugs for reversing multidrug resistance. Drugs that interfere with the action of P-glycoprotein (P-gp), the membrane efflux pump responsible for multidrug resistance (MDR), should be valuable in the treatment of patients with drug-resistant cancer. We have used one class of drug, the phenothiazines, to study the structural features required for optimum interference with the function of P-gp. The structure-activity relationships revealed three important components including the hydrophobicity of the tricyclic ring, the length of the alkyl bridge and the charge on the terminal amino group. Trans-flupenthixol is a lead compound that conforms to these structural requirements and demonstrates significant activity as a sensitizer of MDR cell lines to drugs affected by the MDR phenotype. Based on these data, we have proposed a model for the binding of modulators to P-gp and have speculated on the structure of the drug-binding domain. We have developed pre-clinical models of MDR that may help predict clinical activity of chemo-modulators. L1210/VMDRC.06 is a murine lymphocytic leukemia line transformed by a retroviral expression vector containing a full-length cDNA for the human mdr1 gene. K562/VBL1-3 are clones of human myeloid blast cells that were transformed with the same vector. Resistance in these lines is not complicated by changes in the cellular content of glutathione or alterations in topoisomerase II. The transformed L1210 line grows in mice as a slowly proliferating non-metastatic peritoneal implant. Both MDR lines are restored to sensitivity by cyclosporin A or trans-flupenthixol, and the K562 clones are induced to differentiate by hemin. These lines should provide simple, sensitive screens for new drugs for use against cancers expressing P-gp. We have proposed a model to explain how the pumping activity of P-gp is activated in response to toxic drugs. In this schema, basal activity of P-gp is modulated through phosphorylation/dephosphorylation reactions mediated by protein kinase C (PKC) and calcium sensitive phosphatases. In response to the activation of phospholipase C by toxic drugs and the local production of 1,2-diacylglycerol, PKC is translocated to the cell membrane where it phosphorylates P-gp. Following the extrusion of drug from the cell membrane, phospholipase C activity returns to baseline, diacylglycerol is metabolized, PKC returns to the cytosol and serine/threonine phosphatases dephosphorylate P-gp returning it to the basal state.


Cyclosporins as drug resistance modifiers. Cyclosporin A (CsA), a cyclic peptide of 11 amino acids isolated from the fungus Tolypoclodium inflatum Gams, is the principle drug used for immunosuppression in organ transplant patients. It is known to have a very specific effect on T-cell proliferation although the precise mechanism remains unclear. Following internalization, CsA binds to a cytosolic protein, cyclophilin, which has been shown to possess peptidyl-prolyl cis-trans isomerase activity. CsA is an effective modifier of multidrug resistance in human and rodent cells at doses in the range of 1 to 5 micrograms/mL. Although it reverses the drug accumulation deficit associated with multidrug resistance in some cell types, this is not always the case. CsA has P-glycoprotein binding activity but less specific membrane effects and inhibition of protein kinase C may also be involved in its resistance modifier action. A number of non-immunosuppressive analogues of CsA have been shown to have resistance modifier activity and some are more potent than the parent compound. One analogue from Sandoz, PSC-833, has been shown to be approximately 10-fold more potent than CsA and is expected to enter clinical trial in the near future. The use of such agents may allow a full test of the hypothesis that reversal of multidrug resistance will prove a useful clinical strategy.


Biochemical and genetic characterization of the multidrug resistance phenotype in murine macrophage-like J774.2 cells. The development of multidrug resistance (MDR) in malignant tumors is a major obstacle to the treatment of many cancers. MDR sublines have been derived from the J774.2 mouse macrophage-like cell line and utilized to characterize the phenotype at the biochemical and genetic level. Two isoforms of the drug resistance-associated P-glycoprotein are present and distinguishable both electrophoretically and pharmacologically. Genetic analysis has revealed the presence of a three-member gene family; expression of two of these genes, mdr1a and mdr1b, is associated with MDR whereas the expression of the third, mdr2, is not. Studies of these three genes have revealed similarities and differences in the manner in which they are regulated at the transcriptional level, and have suggested that post-transcriptional effects may also be important.


Photoaffinity substrates for P-glycoprotein. A variety of compounds can inhibit the function of P-glycoprotein (Pgp) by binding to it and preventing the efflux of anticancer drug substrates. While the molecular architecture of the drug binding site(s) in Pgp is not known, it is clear that modulators in general appear to conform to some general physical-chemical rules. In this paper, we discuss the basic concepts of drug recognition by Pgp as currently understood. We also examine the compounds used to photoaffinity label this protein and discuss their utility in identifying drug binding sites. Finally, we show that a photoaffinity analog of daunorubicin, [3H]azidobenzoyl-daunorubicin ([3H]AB-DNR), is a good affinity labeling reagent for Pgp. A finding of interest is that vinblastine and verapamil compete more effectively than daunorubicin for [3H]AB-DNR binding to Pgp, suggesting that vinblastine and verapamil have similar structural features not shared by daunorubicin.


The role of the MDR1 (P-glycoprotein) gene in multidrug resistance in vitro and in vivo. This review describes the studies that address the role of the MDR1 (P-glycoprotein) gene in multidrug resistance in cell lines selected in vitro and in clinical cancer. Molecular genetic studies have demonstrated that expression of P-glycoprotein, an efflux pump acting at diverse lipophilic compounds, is sufficient to provide resistance to a large number of lipophilic drugs in tissue culture. The MDR1 gene is expressed in several normal human tissues associated with secretory or barrier functions and in some bone marrow and blood cells, including hematopoietic progenitor cells. MDR1 expression in clinical cancer is often found in untreated tumors of different types. Several studies showed a correlation between MDR1 expression and tumor resistance to combination chemotherapy. MDR1 expression in untreated tumors may reflect their origin from MDR1-positive normal cells or cellular changes associated with neoplastic transformation or progression. MDR1 expression in some types of cancer may be a marker of a more aggressive subpopulation of tumor cells, possessing multiple mechanisms for resistance to treatment.


Linkage between rheumatoid arthritis susceptibility and the presence of HLA-DR4 and DR beta allelic third hypervariable region sequences in southern Chinese persons. To analyze HLA-DR and DQ associations with rheumatoid arthritis (RA) in patients from southern China. In 66 patients and 45 controls, restriction fragment length polymorphism studies were performed using DRB, DQA, and DQB probes, and DRB allele-specific typing of polymerase chain reaction-amplified DRB DNA: The frequency of HLA-DR4 was significantly increased among RA patients (42.4% versus 17.8%). Increased frequencies of the DQA3 allele (77.8% versus 48.9%) and the DQB1*0302 allele (71.0% versus 46.3%), which are in linkage disequilibrium with DR4, were also found. Oligonucleotide typing showed that the amino acid sequence LLEQRRAA, spanning amino acid positions 67-74 of the DR beta molecule, was found in 19 of 49 patients and 5 of 32 controls. The main DR4 allelic subtypes found in the population were DRB1*0404 and DRB1*0405, both of which carried the sequence. There was no difference in subtype distribution between patients and controls. Chinese RA patients have an increased frequency of HLA-DR4 alleles which possess the same DRB third allelic hypervariable sequence shown to be associated with susceptibility in Caucasian RA patients.


Lack of superiority of steroids plus plasma exchange to steroids alone in the treatment of polyarteritis nodosa and Churg-Strauss syndrome. A prospective, randomized trial in 78 patients. To define the most effective treatment for polyarteritis nodosa (PAN) and Churg-Strauss syndrome (CSS). We conducted a prospective, randomized, multicenter trial in which 78 patients were randomly assigned to receive either prednisone and plasma exchange (group A; n = 36) or prednisone alone (group B; n = 42) as first-line treatment of PAN and CSS. Patients with hepatitis B virus-related PAN were not included in this study. The end point of the study was control of the disease (recovery and remission) or death. Clinical symptoms and laboratory findings did not differ statistically in the 2 groups at study entry. Initial control of the disease was similar in both groups. The assigned treatment was stopped in 16 patients because of lack of efficacy. Oral cyclophosphamide or dapsone therapy reversed the disease evolution in 7 of these 10 group A patients and in 4 of these 6 group B patients. At 7 years of followup, 56 patients had completely recovered (27 in group A, 29 in group B), 7 patients were in clinical remission, and 15 patients had died (19.2%; 6 group A patients and 9 group B patients). The prednisone-plasma exchange combination was no more beneficial than corticosteroids alone in preventing relapses over the long term. There was no significant difference in the 7-year cumulative survival rates of the two groups (83% and 79%, respectively). Based on our data, we conclude that combined treatment with prednisone and plasma exchange is not superior to treatment with prednisone alone and must not be systematically employed for initial treatment of PAN and CSS. In most cases, cyclophosphamide as second-line treatment is effective and well tolerated.


Amplification of reproducible allele markers for amplified fragment length polymorphism analysis. A procedure for amplification by PCR of reproducible allele markers for amplified fragment length polymorphism (Amp-FLP) analysis is presented. We have prepared markers for the allelic products of the VNTR loci D1S80 (MCT118) and D17S30 (YNZ22) and for the hypervariable VNTR locus close to the 3' end of the apolipoprotein B gene (apoB) by re-amplifying a mixture of PCR products from individuals with known alleles. These allele markers allow precise and discrete determination of the VNTR alleles at these loci using the Amp-FLP technique that should prove suitable in forensic analyses, paternity testing and population studies.


Substitution of glutamic acid 109 by aspartic acid alters the substrate specificity and catalytic activity of the beta-subunit in the tryptophan synthase bienzyme complex from Salmonella typhimurium. In an effort to understand the catalytic mechanism of the tryptophan synthase beta-subunit from Salmonella typhimurium, possible functional active site residues have been identified (on the basis of the 3-D crystal structure of the bienzyme complex) and targeted for analysis utilizing site-directed mutagenesis. The chromophoric properties of the pyridoxal 5'-phosphate cofactor provide a particularly convenient and sensitive spectral probe to directly investigate changes in catalytic events which occur upon modification of the beta-subunit. Substitution of Asp for Glu 109 in the beta-subunit was found to alter both the catalytic activity and the substrate specificity of the beta-reaction. Steady-state kinetic data reveal that the beta-reaction catalyzed by the beta E109D alpha 2 beta 2 mutant enzyme complex is reduced 27-fold compared to the wild-type enzyme. Rapid-scanning stopped-flow (RSSF) UV-visible spectroscopy shows that the mutation does not seriously affect the pre-steady-state reaction of the beta E109D mutant with L-serine to form the alpha-aminoacrylate intermediate, E(A-A). Binding of the alpha-subunit specific ligand, alpha-glycerol phosphate (GP) to the alpha 2 beta 2 complex exerts the same allosteric effects on the beta-subunit as observed with the wild-type enzyme. However, the pre-steady-state spectral changes for the reaction of indole with E(A-A) show that the formation of the L-tryptophan quinonoid, E(Q3), is drastically altered. Discrimination against E(Q3) formation is also observed for the binding of L-tryptophan to the mutant alpha 2 beta 2 complex in the reverse reaction. In contrast, substitution of Asp for Glu 109 increases the apparent affinity of the beta E109D alpha-aminoacrylate complex for the indole analogue indoline and results in the increased rate of synthesis of the amino acid product dihydroiso-L-tryptophan. Thus, the mutation affects the covalent bond forming addition reactions and the nucleophile specificity of the beta-reaction catalyzed by the bienzyme complex.


Double blind dose-response study of zidovudine in AIDS and advanced HIV infection. Nordic Medical Research Councils' HIV Therapy Group. To compare the efficacy and side effects of 400 mg, 800 mg, and 1200 mg zidovudine daily in patients with AIDS or advanced HIV infection. Randomised, double blind, parallel group multicentre study. Hospital departments of infectious diseases and dermatology in Denmark, Sweden, Norway, Finland, and Iceland. 474 patients: 126 (27%) with AIDS; 248 (52%) with HIV related symptoms; 100 (21%) with low CD4+ cell counts. Zidovudine 400 mg (160 patients), 800 mg (158), or 1200 mg (156) daily. All patients received one capsule from each of three bottles four times daily. Survival; incidence of new HIV related events; CD4+ cell count; quality of life; incidence of haematological side effects. 460 (97%) of the 474 patients had not received zidovudine previously. The median follow up period was 19 months, during which the death rates in the three treatment groups were 23% (36/160 patients), 23% (36/158), and 19% (30/156) respectively (p = 0.49; log rank test). One year after the trial was terminated the death rates were 38% (61/160), 41% (64/158), and 44% (68/156) respectively (p = 0.54). There was no significant difference between the groups in time to a new AIDS defining event or death, average number of events per patient, decline in CD4+ cell counts, wellbeing (visual analogue scale), or Karnofsky score. Zidovudine was withdrawn in 132 (28%) patients, mainly because of side effects (71 cases; 15%). The incidences of anaemia and leucopenia, time to first dose reduction, and numbers of patients withdrawn were all dose related. Zidovudine should be limited to 400-600 mg daily in patients with AIDS or advanced HIV infection.


Aminergic neurons in the brain of blowflies and Drosophila: dopamine- and tyrosine hydroxylase-immunoreactive neurons and their relationship with putative histaminergic neurons. The distribution and morphology of neurons reacting with antisera against dopamine (DA), tyrosine hydroxylase (TH) and histamine (HA) were analyzed in the blowflies Calliphora erythrocephala and Phormia terraenovae. TH-immunoreactive (THIR) and HA-immunoreactive (HAIR) neurons were also mapped in the fruitfly Drosophila melanogaster. The antisera against DA and TH specifically labeled the same neurons in the blowflies. About 300 neurons displayed DA immunoreactivity (DAIR) and THIR in the brain and subesophageal ganglion of the blowflies. Most of these neurons were located in bilateral clusters; some were distributed as bilateral pairs, and two ventral unpaired median (VUM) neurons were seen in the subesophageal ganglion. Immunoreactive processes were found in all compartments of the mushroom bodies except the calyces, in all divisions of the central body complex, in the medulla, lobula and lobula plate of the optic lobe, and in non-glomerular neuropil of protocerebrum, tritocerebrum and the subesophageal ganglion. No DA or TH immunoreactivity was seen in the antennal lobes. In Drosophila, neurons homologous to the blowfly neurons were detected with the TH antiserum. In Phormia and Drosophila, 18 HA-immunoreactive neurons were located in the protocerebrum and 2 in the subesophageal ganglion. The HAIR neurons arborized extensively, but except for processes in the lobula, all HAIR processes were seen in non-glomerular neuropil. The deuto- and tritocerebrum was devoid of HAIR processes. Double labeling experiments demonstrated that TH and HA immunoreactivity was not colocalized in any neuron. In some regions there was, however, substantial superposition between the two systems. The morphology of the extensively arborizing aminergic neurons described suggests that they have modulatory functions in the brain and subesophageal ganglion.


Light- and electron-microscopic studies of the somatostatin-immunoreactive plexus in the cat retina. Two monoclonal antibodies directed against somatostatin 14 were used to study immunoreactive neurons, their processes and their synapses in the cat retina. In retinal whole-mounts, a sparse population of wide-field displaced amacrine cells was observed predominantly in the ventral retina and near the retinal margin. Processes of these cells ramified mainly in two distinct strata within the inner plexiform layer: one near the inner nuclear layer (INL), and the other near the ganglion cell layer (GCL). The length of immunoreactive fibres within each plexus was measured: 232 +/- 32 mm/mm2 near the INL and 230 +/- 74 mm/mm2 near the GCL in all retinal regions. The immunoreactive processes were studied using electron-microscopic techniques; conventional and some ribbon-containing synapses ("dyads") were found. Immunolabelled processes received input synapses from other amacrine cell processes. These investigations provide further evidence that this cell population has a diffuse, regulatory or modulatory role for visual-information processing in the inner plexiform layer.


Dose-dependent modulation of the cardiac sodium channel by sea anemone toxin ATXII. The effects of sea anemone toxin ATXII on single sodium channels were studied in cell-attached patches on rabbit ventricular myocytes at 20-22 degrees C. Exposure of patches to 1,000 nM ATXII induced long-lasting bursts of openings, which were more dramatically different from control at -20 mV than at -50 mV. Mean open duration, which had a biphasic dependence on voltage in control patches, was monotonically dependent on voltage in toxin-exposed patches, being 3.5 times longer than control at -20 mV and 4.5 times longer at -10 mV. Multiple mean open durations were detected at depolarized potentials. To test whether the multiple mean open durations resulted from a mixture of modified and unmodified openings, histograms of late openings (when unmodified channels would be inactivated) were constructed. Because in most cases these fit a single exponential with a mean open duration like that of modified channels, we conclude that voltage-dependent toxin unbinding produced a mixed population of unmodified and modified openings. Consistent with this hypothesis, lower concentrations of toxin most often produced open-duration histograms best fit with two exponentials. Ensembles revealed complex decay kinetics, which could be interpreted within the context of the toxin-induced increase in mean open duration and burst duration and the summation of modified and unmodified events. Analysis of the numbers of early versus late events at -20 mV for patches exposed to 20 nM, 100 nM, and 1,000 nM ATXII predicted the ED50 for ATXII block to be 285 nM at this potential. Using a five-state Markovian model, the action of ATXII could be explained as a reduction of the open-to-inactivated rate constant without effect on inactivation from closed states or other rate transitions.


The effects of acute and chronic cocaine use on the heart. It is clear that cocaine has cardiotoxic effects. Acute doses of cocaine suppress myocardial contractility, reduce coronary caliber and coronary blood flow, induce electrical abnormalities in the heart, and in conscious preparations increase heart rate and blood pressure. These effects will decrease myocardial oxygen supply and may increase demand (if heart rate and blood pressure rise). Thus, myocardial ischemia and/or infarction may occur, the latter leading to large areas of confluent necrosis. Increased platelet aggregability may contribute to ischemia and/or infarction. Young patients who present with acute myocardial infarction, especially without other risk factors, should be questioned regarding use of cocaine. As recently pointed out by Cregler, cocaine is a new and sometimes unrecognized risk factor for heart disease. Acute depression of LV function by cocaine may lead to the presence of a transient cardiomyopathic presentation. Chronic cocaine use can lead to the above problems as well as to acceleration of atherosclerosis. Direct toxic effects on the myocardium have been suggested, including scattered foci of myocyte necrosis (and in some but not all studies, contraction band necrosis), myocarditis, and foci of myocyte fibrosis. These abnormalities may lead to cases of cardiomyopathy. Left ventricular hypertrophy associated with chronic cocaine recently has been described. Arrhythmias and sudden death may be observed in acute or chronic use of cocaine. Miscellaneous cardiovascular abnormalities include ruptured aorta and endocarditis. Most of the cardiac toxicity with cocaine can be traced to two basic mechanisms: one is its ability to block sodium channels, leading to a local anesthetic or membrane-stabilizing effect; the second is its ability to block reuptake of catecholamines in the presynaptic neurons in the central and peripheral nervous system, resulting in increased sympathetic output and increased catecholamines. Other potential mechanisms of cocaine cardiotoxicity include a possible direct calcium effect leading to contraction of vessels and contraction bands in myocytes, hypersensitivity, and increased platelet aggregation (which may be related to increased catecholamine). The correct therapy for cocaine cardiotoxicity is not known. Calcium blockers, alpha-blockers, nitrates, and thrombolytic therapy show some promise for acute toxicity. Beta-Blockade is controversial and may worsen coronary blood flow. In patients who develop cardiomyopathy, the usual therapy for this entity is appropriate.


Elevated endothelin-1 in heart failure and loss of normal response to postural change. The possible contribution of endothelin-1, a potent endothelium-derived vasoconstrictor peptide, to neurohumoral compensation for hemodynamic stress was examined in nine normal volunteers and six patients with severe congestive heart failure. Plasma levels of endothelin-1 were measured with a sensitive and specific radioimmunoassay. Venous blood samples were obtained after 90 minutes of supine rest and serially during 30 minutes of 60 degrees upright tilt. Endothelin-1 levels were compared with those of known neurohumoral mediators of compensation. In normal subjects, the resting levels of endothelin-1 were low (0.74 +/- 0.11 pg/ml), and there was a rapid increase to 1.37 +/- 0.07 pg/ml at 5 minutes of upright tilting (p less than 0.05). This increase was not sustained at 10 and 15 minutes of tilt, but there was a trend toward a second rise at 30 minutes (1.14 +/- 0.17 pg/ml; p = 0.06). This biphasic pattern of response was shared by dopamine and reflected the response of systemic blood pressure to postural change. In contrast, slower and more sustained increases in circulating levels were observed for norepinephrine, epinephrine, aldosterone, plasma renin activity, and vasopressin, whereas atrial natriuretic peptide tended to decrease progressively. Patients with congestive heart failure had markedly higher basal levels of circulating endothelin-1 than normal subjects (3.7 +/- 0.5 pg/ml; p less than 0.01), and there was no further increase on postural change. Similar patterns were observed for the other neurohumoral mediators measured, with the degree of blunting of the response to upright tilting in heart failure being inversely related to the magnitude of increase in basal levels. Alterations in plasma levels of endothelin in congestive heart failure and in response to postural change were qualitatively and quantitatively similar to the alterations of known mediators of neurohumoral compensation. In addition, the increase in plasma endothelin-1 during upright tilting in normal subjects preceded the increases in circulating levels of the other vasoconstrictor mediators, consistent with a role of endothelin-1 in neurohumoral compensation for hemodynamic stress.


Effect of xamoterol in Shy-Drager syndrome. Xamoterol, a cardioselective beta 1-adrenoceptor partial agonist, has been reported to be effective on postural hypotension. We investigated the effect of xamoterol in five patients with Shy-Drager syndrome (SDS) in relation to their prevailing sympathetic nerve activity and sensitivity of beta-adrenoceptors and the change in circadian variation of blood pressure. Ambulatory blood pressure over 24 hours was monitored by noninvasive sphygmomanometer (model 5200, Spacelab). Plasma norepinephrine levels of SDS patients were significantly lower than that of normal subjects (n = 5) both at rest (54 +/- 15 versus 178 +/- 83 pg/ml) and after 10-minute standing (74 +/- 24 versus 318 +/- 143 pg/ml). Infusion of isoproterenol (0.02 micrograms/kg/min) produced a mild rise of systolic blood pressure and tachycardia in normal subjects but resulted in marked hypotension and tachycardia in SDS subjects. After xamoterol administration (200 mg b.i.d.), systolic blood pressure and heart rate were significantly increased in the averages during the day; however, increases were more pronounced at night. In two of the five patients, the improvement in dizziness was large enough to enable them to increase their daily activities. Our observations suggest that 1) beta 1-selective, high intrinsic sympathomimetic activity of xamoterol increases blood pressure and heart rate in patients with SDS as a consequence of their prevailing beta 1-adrenoceptor hypersensitive state, and 2) blood pressure monitoring over 24 hours appears to have important advantages in evaluating the therapeutic effects on postural hypotension.


Study of CYP2D6 gene in children with autoimmune hepatitis and P450 IID6 autoantibodies. Cytochrome P450 IID6 is an autoantigen recognized by the sera of children affected with a subtype of autoimmune hepatitis. It was hypothesized that a mutation in the CYP2D6 gene could explain the autoimmune response in these patients. To examine this question, genomic DNA from peripheral lymphocytes (n = 9) and liver (n = 1) of 10 patients with anti-LKM-1 antibody was analysed by Southern blot for genetic association studies between a particular CYP2D6 haplotype and autoimmune hepatitis. In addition, a region of CYP2D6, from the same genomic DNA, was amplified by polymerase chain reaction (PCR) and digested by BstNI, in a search for the most prevalent 29B mutation, described in subjects who do not express the P450 IID6. Total RNA and proteins, prepared from the liver of an anti-LKM-1+ patient, were analysed by Northern and Western (immunoblot) blots respectively. Our results do not reveal any major structural change in the DNA of this patient at the CYP2D6 locus that could explain their autoimmune response. Corroborating this observation, no changes were noted either in P450 IID6 mRNA size or in the corresponding protein. However, these data do not exclude the possibility of subtle changes in the protein due to point mutations in critical regions that might trigger an autoimmune response.


Substitution of metered-dose inhalers for hand-held nebulizers. Success and cost savings in a large, acute-care hospital. Administration of beta-agonist bronchodilators by metered-dose inhaler (MDI) is as effective as administration by hand-held nebulizer (NEB). Recent studies have suggested that MDI therapy is less costly to administer and that routine substitution of MDI for NEB would result in considerable savings to patients and to hospitals. To our knowledge, the actual extent to which MDI therapy would replace NEB therapy or the cost savings realized has not been reported previously. We examined the success and impact on hospital costs of the routine substitution of MDI for NEB therapy in a large tertiary-care hospital. Following introduction of this strategy, more than 60 percent of all aerosol therapy was actually given as MDI. The mean amount of time spent by therapists to provide aerosol therapy was significantly reduced by MDI substitution, falling from 1,576 +/- 131 h/mo, to 992 +/- 116 h/mo (p less than 0.002). The total cost to deliver aerosol therapy fell from $27,600 +/- $2,277/mo to $20,618 +/- $2,086/mo (p = 0.008). Potential cost savings of $83,000 annually were achieved by the hospital, and charges to patients were lowered by approximately $300,000 per year. Routine substitution of MDI therapy for NEB therapy can be accomplished with considerable, but not total, success. This approach results in significant reductions in the cost of health care provision.


Indomethacin accelerates clearance of labeled cells and increases DNA synthesis in gastrointestinal mucosa of the rat. Sprague-Dawley rats treated with placebo, parenteral indomethacin, or oral prostaglandin E2 for six days were given an intraperitoneal injection of [3H] methyl-thymidine and killed at 45 min and 96 hr after labeling. Treatments were continued until death. The dpm/DNA index was determined in mucosal scrapings of the stomach, duodenum, and jejunum and used to estimate DNA synthesis (45 min) and the clearance of labeled cells (96 hr). Indomethacin increased the DNA synthesis in both the duodenal and jejunal mucosa (P less than 0.05). In comparison to the controls, the clearance of labeled cells from the antral, duodenal, and jejunal mucosa was accelerated by indomethacin treatment, whereas the elimination of labeled cells from the antral and jejunal mucosa was slowed by PGE2 treatment (P less than 0.05). DNA synthesis of the antral mucosa was significantly reduced by PGE2 (P less than 0.05). The cyclooxygenase blocker did not affect the cell kinetic parameters of the oxyntic mucosa. The plasma levels of somatostatin were significantly higher both in PGE2- and indomethacin-treated rats than in controls (P less than 0.05). It is concluded that indomethacin treatment increases the cell losses from the epithelial surface, which in turn trigger a compensatory trophic reaction. It is suggested that an important physiological action of endogenous prostaglandins is to regulate the outflow of cells from the superficial zones of the epithelium. Finally, this study disclosed the presence of hitherto unknown regulatory mechanisms that promote cell proliferation in the gastrointestinal mucosa despite inhibition of the synthesis of endogenous prostaglandins.


Helicobacter pylori infection induces a decrease in immunoreactive-somatostatin concentrations of human stomach. Immunoreactive-somatostatin (ir-somatostatin) concentrations of the gastric mucosa and gastric juice with Helicobacter pylori infection were measured in the human stomach. One hundred seventy-one patients (106 males, 65 females; mean age, 52.0; range, 19-84 years) were registered. Gastric juice and mucosa were obtained with the usual endoscopy procedure. Somatostatin concentration was measured by radioimmunoassay. The ir-somatostatin concentrations in the H. pylori-negative group were significantly higher than in the positive group gastric mucosa, whereas its levels in gastric juice tended to decrease with H. pylori infection. There was an inverse correlation between luminal ammonia levels and ir-somatostatin concentrations of the gastric mucosa. On the other hand, ir-somatostatin concentrations of the gastric mucosa significantly decreased with chronic and active inflammatory change. This decrease was not correlated with the grade of active inflammation, which was in close relation to H. pylori infection, but with the grade of chronic inflammation. These results indicate that H. pylori may reduce ir-somatostatin concentrations of the human stomach and that its effect is partly mediated via luminal ammonia produced by H. pylori.


Identification of carrot cDNA clones encoding a second putative proliferating cell-nuclear antigen, DNA polymerase delta auxiliary protein. The proliferating cell-nuclear antigen (PCNA) plays a key role in the control of eukaryotic DNA replication. We have isolated two cross-hybridizing groups of cDNA encoding carrot homologs of PCNA. Sequence analysis and Southern-blot experiments showed that the cDNA were derived from two distinct genes. One corresponded to the typical PCNA, which is known to be highly conserved in eukaryotes from yeast to man; its mRNA is 1.2 kb in size and the calculated molecular mass of the protein is 29 kDa. The other encoded a larger PCNA homolog which has not previously been reported; the mRNA is 1.5 kb in size, the N-terminal three quarters (calculated molecular mass, 29 kDa) of the protein product is 88% identical at the amino acid level to the typical PCNA, but the protein has an extra C-terminal domain of 11 kDa. Both PCNA homologs were apparently coexpressed concomitant with somatic embryogenesis. The mRNA level of the novel homolog is 10-20% that of the typical PCNA in the embryos. The presence of the second putative PCNA may provide new insight into studies on the mechanism of DNA replication in eukaryotes.


An oxidized analog of alpha-human atrial natriuretic polypeptide is a selective agonist for the atrial-natriuretic-polypeptide clearance receptor which lacks a guanylate cyclase. The differences in biological functions between alpha-human atrial natriuretic polypeptide (alpha-hANP) and its oxidized analog, MetSO-alpha-hANP, have been investigated. Analysis of the ANP receptor subtypes by affinity labeling has shown that a bovine pulmonary aortic endothelial cell line (CPAE cells) primarily expresses ANP-R1 (R, receptor) coupled to particulate guanylate cyclase, while Hela cells from human cervical carcinoma predominantly express ANP-R2, which lacks a guanylate cyclase. alpha-hANP could bind to both ANP receptor subtypes with high affinity, while MetSO-alpha-hANP showed more selective binding to ANP-R2 than to ANP-R1. The activity of MetSO-alpha-hANP for stimulation of guanylate cyclase coupled to ANP-R1 was about 520-fold less than that of alpha-hANP (median effective dose = 2.5 nM for alpha-hANP, 1.3 microM for MetSO-alpha-hANP), indicating that MetSO-alpha-hANP was a partial agonist for this receptor. While this oxidized analog could inhibit the cAMP production through ANP-R2, with 0.15 times the activity of alpha-hANP (median concentration = 0.31 nM for alpha-hANP, 2.0 nM for MetSO-alpha-hANP). In in vivo studies, the diuretic activity of MetSO-alpha-hANP was 25-100-fold less than that of alpha-hANP. In addition, MetSO-alpha-hANP could potentiate the diuretic activity of alpha-hANP that was also caused by C-ANF4-23, a specific agonist for ANP-R2. These results demonstrate that MetSO-alpha-hANP can act as an agonist more selective for ANP-R2 than for ANP-R1, both in vivo and in vitro. The relationship between receptor selectivities and the conformation of alpha-hANP or MetSO-alpha-hANP was also discussed.


Diurnal rhythm of phosphorylation of rat liver acetyl-CoA carboxylase by the AMP-activated protein kinase, demonstrated using freeze-clamping. Effects of high fat diets. 1. Acetyl-CoA carboxylase was purified to homogeneity, in the presence of protein phosphatase inhibitors, from rat liver sampled without freeze-clamping. The enzyme was in a highly phosphorylated state (4.8 mol/subunit) of low specific activity, and could be dramatically reactivated by treatment with protein phosphatase-2A. Amino acid sequencing and fast-atom-bombardment mass spectrometry showed that the enzyme was phosphorylated in Ser79, Ser1200 and Ser1215, the three sites known to be phosphorylated in cell-free assays by the AMP-activated protein kinase. 2. The inactive enzyme could also be completely reactivated using a limited treatment with trypsin, which removes the N-terminal segment containing Ser79 and reduces the phosphate content to 3.5 mol/subunit. These results strengthen previous findings that it is phosphorylation at Ser79 by the AMP-activated protein kinase that is responsible for the inactivation, and not the phosphorylation of the 220-kDa core fragment (which contains Ser1200 and Ser1215). 3. Analysis of the phosphorylation state of Ser79 in acetyl-CoA carboxylase from rat liver showed that phosphorylation occurs post mortem if freeze-clamping is not used. The higher phosphorylation observed in extracts made without freeze-clamping correlates with a large increase in AMP and decrease in ATP (presumably caused by hypoxia during removal of the liver), and with increased activity of the AMP-activated protein kinase. These results provide a rational explanation for the post mortem phosphorylation events, and re-emphasize the point that rapid cooling of cells and tissues is essential when measuring the expressed activity of acetyl-CoA carboxylase (as well as 3-hydroxy-3-methylglutaryl-CoA reductase). 4. Using the freeze-clamping procedure, the ratio of 'expressed' activity (measured in the presence of protein phosphatase inhibitors) to 'total' activity (measured after complete dephosphorylation) of rat liver acetyl-CoA carboxylase showed a marked diurnal rhythm, changing from 50% in the active form in the middle of the dark period to less than 10% active in the middle of the light period. The very low activity in the light period was associated with a high level of phosphorylation in Ser79. This diurnal rhythm is very similar to that previously described for the phosphorylation of 3-hydroxy-3-methylglutaryl-CoA reductase, another substrate for the AMP-activated protein kinase. Neither the activity of the AMP-activated protein kinase nor the content of AMP, ADP or ATP changed between the dark or light periods.(ABSTRACT TRUNCATED AT 400 WORDS)


Developmental changes in the activity of membrane-bound gamma-glutamyl transpeptidase and in the sialylation of synaptosomal membranes from the chick embryonic brain. gamma-Glutamyl transpeptidase (GGT) is a membrane-bound sialoglycoprotein. The developmental changes in GGT activity and in sialic acid content were determined in a crude synaptosomal membrane fraction from the cerebral hemispheres of the chick embryo between days 11 and 19 of incubation. The GGT activity increased almost eightfold during the examined developmental period, while sialic acid content rose significantly only between days 11 and 15. Cortical administered on day 13 significantly increased GGT activity. On the other hand, the content of membrane bound sialic acid was not substantially affected. The value of the GGT apparent Michaelis constant (Kmapp) for gamma-glutamyl-p-nitroanilide in the presence of 20 mmol.l-1 glycylglycine was 1.5 mmol.l-1 and cortisol did not influence it. However, Vmax was increased by this hormone. The affinity of GGT to concanavalin A (ConA) did not change during development. Neither the administration of cortisol nor neuroaminidase treatment had any effect on the interaction of GGT with ConA. Desialylation of crude synaptosomal fraction did not change GGT activity. The results presented here suggest no developmental nor functional relationship between the activity of GGT and the level of sialylation in synaptosomal membranes from the cerebral hemispheres of the chick embryo.