Detection of JC virus DNA in peripheral lymphocytes from patients with and without progressive multifocal leukoencephalopathy. Progressive multifocal leukoencephalopathy (PML) results from lytic infection of oligodendrocytes by JC virus (JCV). Although JCV has been identified in mononuclear cells in bone marrow and hematogenous dissemination of the virus to the central nervous system has been suspected, JCV has never been clearly demonstrated in the peripheral circulation. Using polymerase chain reaction technology, we examined peripheral lymphocytes of 19 patients with brain biopsy-proven PML for the JCV genome. Two non-PML control groups, consisting of 26 patients seopositive for human immunodeficiency virus type 1 (HIV-1) and 30 immunocompetent patients with Parkinson's disease, were also examined for the presence of the JCV genome in lymphocytes. Cerebrospinal fluid from 10 patients with PML was examined for the presence of the JCV genome as well. The JCV genome was detected in the lymphocytes of 89% (17) of the patients with PML, 38% (10) of the HIV-1-seropositive patients without PML, and none of the patients with Parkinson's disease. Sequencing of the JCV regulatory region from the lymphocytes of three patients revealed the prototype MAD-1 strain of JCV in one patient with PML, a MAD-4 strain in a second patient with PML, and a slightly modified MAD-4 strain in an HIV-1-positive patient without PML. Only 3 of 10 patients with PML who had JCV detected in lymphocytes had the JCV genome in their cerebrospinal fluid. These results demonstrate that the JCV genome can be found in circulating lymphocytes from patients with PML and suggest that lymphocytes are an important vector for hematogenous dissemination of JCV to the central nervous system.(ABSTRACT TRUNCATED AT 250 WORDS)


9-(Phosphonoalkyl)guanine derivatives as substrates or inhibitors of guanylate kinase. Several 9-(phosphonoalkyl)guanines (Gua(CH2)nCH2-PO3H2; n = 4-6) and 9-(difluorophosphonoalkyl)guanines (Gua(CH2)nCF2PO3H2; n = 3-7) were studied as potential substrates and inhibitors of guanylate kinase. These compounds are inhibitors of the enzyme except 9-(5-phosphonopentyl)guanine (n = 4) which is a substrate with an efficiency of phosphorylation of about 0.3% that of GMP, as estimated from the Vmax/Km ratios. The phosphonate and difluorophosphonate derivatives with n = 5 produce optimal inhibition. These two compounds have similar affinity, both being competitive inhibitors with respect to GMP and noncompetitive inhibitors with respect to ATP. pH-dependence studies indicate that the dianionic rather than the monoanionic form of these compounds bind to the enzyme. The lack of phosphorylation of 9-(5,5-difluoro-5-phosphonopentyl)guanine by guanylate kinase is explained by the decreased nucleophilic character of the oxygen atoms of the phosphonate group rather than by inadequate binding to the GMP-binding site.


1H nuclear magnetic resonance study of the prosthetic group in sulfhemoglobin. The molecular and electronic structure of the modified prosthetic group of sulfhemoglobin (SHb) was investigated by 1H NMR for the low-spin ferric cyano-met and high-spin ferrous deoxy sulfhemoglobin complex. The 1H NMR resonances of the two subunits in the cyano-met SHb complex were differentiated on the basis of the differential stability toward regeneration of native subunits. The subunit origin for the two sets of resonances was established by formation of the sulfglobin protein for the isolated alpha-chain prior to assembling with the native beta-subunit to yield a tetramer with sulfhemin in the alpha-subunits. The subunit peak assignments establish that it is the beta-subunit of SHb which regenerates more rapidly to native protein. The hyperfine shifted sulfhemin peaks were assigned based on steady-state nuclear Overhauser effects which demonstrated that similarly hyperfine shifted peaks exhibit the same dipolar connectivities observed in the analogous sulfmyoglobin complex. Hence it is concluded that pyrrole B is the site of reaction in both hemoglobin and myoglobin. The initially formed SHb complex failed to equilibrate to yield a complex with a sulfhemin sufficiently stable to extraction as found previously for sulfmyoglobin. However, apoHb readily bound the green sulfhemin extracted from the terminal alkaline equilibration product of sulfmyoglobin. The inhibition on the equilibration to the alkaline form with the exocyclic thiolene ring is attributed to the interaction with Val FG5. The observations of the same dipolar connectivities among similarly hyperfine shifted peaks in the directly prepared and reconstituted SHb complexes further support the same structure for the sulfhemin in sulfmyoglobin and SHb. The strongly hyperfine shifted peaks in the deoxy form of both SHb complexes were found very similar to those of the analogous sulfmyoglobin complexes. The proximal His labile ring proton signal appears to experience a 5- to 10-ppm decrease upon conversion of a native globin to sulfglobin. This attenuation may provide a probe for differentiating chlorins and hemins in globin pockets.


Protein sulfhydryls are protected from irreversible oxidation by conversion to mixed disulfides. Protein mixed thioselenides formed by reaction of sarcoplasmic reticulum (SR) with diselenide biradical spin labels were quantified by ESR. Whereas the reaction of SR membranes with the diselenide spin label led to a large ESR signal of the unbound monoselenide at equilibrium, treatment of the reaction mixture with a few millimolar hydrogen peroxide converted all of the nitroxides to protein-bound thioselenides. This technique of spin-labeling protein thiols avoids the need to remove unreacted spin labels. The bound spin labels were removable by reduction with excess mercaptoethanol, indicating a specific and reversible labeling of protein thiols. SR that had been extensively labeled with the diselenide spin label was resistant to ATPase inactivation by potent oxidants that arise when myoglobin reacts with hydroperoxides. Unmodified SR lost all activity within 10 min of exposure to either 1 mM tert-butyl hydroperoxide in the presence of 200 microM equine myoglobin or to 100 mM hydrogen peroxide in the absence of myoglobin. In both cases the loss of activity could not be reversed by subsequent treatment with mercaptoethanol. On the other hand, membranes that had been extensively treated with the diselenide spin label and were then subjected to these peroxide treatments were fully active after mercaptoethanol-mediated cleavage of the thioselenides. ESR analysis of spin-labeled SR showed no detectable oxidative cleavage of the thioselenide bonds. Sodium dodecyl sulfate gel electrophoresis showed that peroxide-mediated crosslinking of ATPase observed in unmodified SR membranes did not occur in the diselenide-modified SR membranes. Only limited protection was observed when SR pretreated with glutathione disulfide was incubated with hydroperoxides. In this case, however, the degree of protection was greatly increased when the reaction with glutathione disulfide was carried out in the presence of the supernatant of centrifuged rat liver homogenate, consistent with an acceleration of mixed disulfide formation by a factor tentatively identified as thiol transferase. It is concluded that conversion of protein thiol residues to either thioselenides or mixed disulfides confers protection against irreversible peroxide-dependent oxidation. We suggest that mixed disulfide formation by thiol transferase activity may help protect protein thiols from irreversible oxidation by heme-activated hydroperoxides.


Inert gas enhancement of superoxide radical production. Two free radical generating systems, xanthine oxidase/hypoxanthine or phenazine methosulfate/NADH, were exposed to air plus He, N2, or Ar at partial pressures ranging from 0.2 to 6.0 MPa, and the rates of production of superoxide, hydroxyl, singlet O2, and H2O2 were measured. All three inert gases acted similarly to enhance the production of superoxide radicals by facilitating interactions between iron and H2O2, or O2 and organic radicals. These reactions occurred at quite low gas partial pressures, only 0.28 MPa, and hydrostatic pressures of up to 6.0 MPa had no effect on radical reactions. Enhanced radical production may be the basis for the inhibition of cellular growth mediated by inert gases, and inert gas enhancement of O2 toxicity.


The effects of cadmium in vitro on adenosine triphosphatase system and protection by thiol reagents in rat brain microsomes. Cadmium (Cd) inhibited the activities of Na(+)-K+ ATPase (IC50 = 5.0 x 10(-5) M), K(+)-p-nitrophenyl phosphatase (PNPPase) (IC50 = 4.0 x 10(-5) M) and 3H-ouabain binding (IC50 = 7.5 x 10(-5) M) in rat brain microsomes. Monothiols (cysteine but not glutathione and D-penicillamine) and dithiols (dimercaprol, dimercaptosuccinic acid and dithiothreitol) offered varied levels of protection against Cd-inhibition of Na(+)-K+ ATPase. Protection of Na(+)-K+ ATPase by these sulfhydryl (SH) agents was higher at 7.5 as compared to 8.5 pH. The present data suggest that Cd-inhibited Na(+)-K+ ATPase, by interfering with phosphorylation of enzyme molecule and dephosphorylation of the enzyme-phosphoryl complex and exerts a similar effect to that of SH-blocking agents.


Production of angiotensin converting enzyme by rheumatoid synovial membrane. Vascular proliferation and mononuclear cell infiltration are prominent changes observed in synovium from actively inflamed joints of patients with rheumatoid arthritis. Angiotensin converting enzyme (ACE) is a halide activated peptidase produced mainly by endothelial cells and by activated monocytes. It has been proposed that levels of ACE activity in synovial fluid might reflect changes in membrane vascularity, the degree of monocyte infiltration, or the thickness of the lining layer. In this study, ACE activity in serum and synovial fluid samples from 18 patients with inflammatory arthritis was measured and compared with levels in 12 control subjects with non-inflammatory arthritis. Although serum levels were similar in the two groups, ACE activity in synovial fluid was significantly increased in the group with inflammatory arthritis compared with controls (mean (SE) 37 (5) v 19 (3)). Staining of synovial membranes from patients with rheumatoid arthritis with a monoclonal antibody to ACE localised ACE to the endothelium and to mononuclear cells of macrophage origin. ACE activity was then measured in supernatants of synovial membrane from patients with rheumatoid arthritis after one and seven days of culture. A significant increase in ACE activity was observed after seven days of culture (mean (SE) day 1, 17 (5) v day 7, 25 (3)). Levels of ACE activity, however, did not correlate with the lining layer thickness, with the number of macrophages per square millimetre, nor with the number of blood vessels per square millimetre of synovial tissue. No correlation was observed either between levels of ACE in the supernatant of synovial membrane and levels of interleukin 1 or interleukin 6. In conclusion, ACE is produced by the synovial membrane of patients with rheumatoid arthritis and is localised to monocytes and endothelial cells. Levels of activity do not directly reflect membrane vascularity, monocyte or macrophage number, or the thickness of the lining layer.


Neurogenic bladder due to peripheral neuropathy and a visual disturbance in an elderly man with systemic lupus erythematosus. A 63 year old man with central nervous system lupus with a neurogenic bladder and visual disturbance is described. The diagnosis of neurogenic bladder, attributed to peripheral neuropathy, was made on the basis of cystometrography and clinical symptoms. A brain magnetic resonance imaging scan showed gliosis along the cerebral vessels and the optic nerve. This case shows that systemic lupus erythematosus can be accompanied by a peripheral neurogenic bladder and visual disturbance, and that these symptoms may not improve despite the amelioration of other lupus symptoms on treatment with steroids.


Detection of cytomegalovirus antigens in phagocytosed serum complexes from a patient with rheumatoid arthritis, vasculitis, peripheral neuropathy, cutaneous ulceration, and digital gangrene. A patient with rheumatoid arthritis, vasculitis, peripheral neuropathy, cutaneous ulceration, and digital gangrene was studied. Circulating immune complexes were detected by C1q binding although serum complement levels were within the normal range. Immunofluorescent staining of buffy coat cells with specific antisera showed the presence of IgG and IgM in phagocytosed inclusions but complement C3 was not detected. A monoclonal antibody specific for cytomegalovirus detected antigens in phagocytosed inclusions on one occasion. These results may suggest that cytomegalovirus antigens are a hitherto unidentified component of serum complexes in patients with rheumatoid arthritis and may contribute to the pathogenesis of the vasculitic complications of rheumatoid arthritis by participating in immune complex formation.


[Follow-up of patients with localized germinal testicular tumors: 6 years' experience at the "Santa Creu i Sant Pau" Hospital]. From 1984 to 1989, 89 patients with stage I testicular carcinoma had been treated and followed at the Oncology Unit of the "Santa Creu i Sant Pau" Hospital. The histologic diagnosis was that of seminoma in 53 and nonseminomatous tumor in 36 patients. Treatment considered of inguinal orchidectomy and close clinical surveillance. Eight of the patients that had been diagnosed as having seminoma received adjuvant radiotherapy since follow-up could not be possible. Recurrence was observed in 5 of the 45 (11 por 100) patients with seminoma and 11 of the 36 (31 por 100) with nonseminomatous tumor that could be followed. All recurrences received chemotherapy (cisplatin and etoposide) which achieved complete remission. All of the patients are currently tumor-free after a mean follow-up of 34 months for the patients with seminoma, 39 months for those with nonseminomatous tumor and no recurrence, and 20 months for the nonseminomatous tumors that had recurred. Surveillance following orchidectomy for stage I testicular tumors is a valid approach if the patient can be followed correctly and achieves the same results as other more aggressive therapeutic strategies.


Salt-induced electrical epithelial responses of the frog (Rana catesbeiana) tongue and their relation to gustatory nerve activity in vivo. Stimulation of the frog tongue with various salts produced changes in epithelial potential of the tongue, accompanied by changes in gustatory nerve activity. Both changes varied similarly according to the stimulus. The results indicate that cation transport in the lingual epithelium is involved in gustation.


[Infection of C type hepatitis virus in patients with alcoholism. Studied by serum HCV antibody of C100-3 and 2nd generation]. To investigate the infectious situation and causes of hepatitis C virus (HCV) in patients with alcoholism, C100-3 antibody (C100-3) and HCV antibody of the 2nd generation (HCV-2nd) were examined on admission, and the factors that affected the infection of HCV were studied in 226 alcoholics. The positive rate of anti-HCV was as high as 16.4% by C100-3 and much higher by HCV-2nd as 33.2%. The incidence of cases with the history for blood transfusion, drug addict and/or tattoos in alcoholics was as high as 39.4% (89/226). To clarify the influence of the background such as blood transfusion, drug addict and/or tattoos on HCV infection, the positive rate of anti-HCV was compared between those with the background and without the background. In alcoholics without the backgrounds, the positive rate of C100-3 and HCV-2nd were significantly (p less than 0.001) higher than patients without the backgrounds, namely 27.0% vs 9.5%, 56.2% vs 18.2%, respectively. In alcoholics without the backgrounds, the positive rate of anti-HCV became higher in proportion as the age of the patients grows older from 20 to 60 years. These results suggest that a high incidence of HCV infection in alcoholics may resulted from increased the backgrounds that can provide chances of HCV infection and that in those without the backgrounds, alcohol and aging may lead the tolerance to HCV infection.


Heliotropium europaeum poisoning of sheep with low liver copper concentrations and the preventive efficacy of cobalt and antimethanogen. In a field experiment in the Mallee district of Victoria, Merlno xBorder Leicester ewes and wethers grazed Heliotropium europaeum (heliotrope) over periods of 3 to 4 months in 4 successive years. By the end of the second year 12% (14 of 120) of the sheep had died; after 4 years the loss attributable to heliotrope was between 18% and 35%. Mortality was not affected by intraruminal treatment with cobalt or antimethanogen. At the end of the experiment the highest concentration of copper in the liver was 1.95 mmol/kg wet weight (approximately 413 micrograms/g dry weight). The relatively low mortality from primary heliotrope poisoning and the low concentration of copper in the liver of sheep grazing the plant are discussed in relation to the contrasting situation that prevails in the Riverina area of New South Wales. The importance of local environmental factors in the management of heliotrope grazing by sheep is emphasised, particularly in relation to the number of seasons in which the plant may be a major component of the diet.


Urinary bladder carcinoma as apparent cause of antepartum haemorrhage in pregnancy. A 34-year-old multipara presented with painless profuse genital bleeding during the third trimester of pregnancy in a state of hemorrhagic shock. Haematuria because of urinary bladder carcinoma was found to be the cause of bleeding. Cystoscopy was useful for accurate diagnosis as well as for achieving haemostasis. This case emphasizes the possibility and importance of haematuria when dealing with a patient with antepartum haemorrhage.


Effects of BIM-22015, an analog of ACTH4-10, on functional recovery after frontal cortex injury. Male rats, 90-100 days old, with frontal cortex lesions were given either subcutaneous sterile water (SW) as a vehicle control or 1, 10, or 100 micrograms of BIM-22015 every other day for 20 days. Brain-injured subjects tested in the Morris water maze with either 10 micrograms BIM-22015 or SW took significantly more trials than sham-operated rats to locate a submerged platform eight consecutive times within 60 s. The animals given 1 or 100 micrograms BIM-22015 took significantly fewer trials to reach criterion than brain-injured animals in the other drug treatment groups. On a percentage of savings, measured 8 days after reaching criterion, the brain-injured subjects given 1, 10, or 100 micrograms BIM-22015 did not differ from sham-operated rats. In contrast, the brain-injured animals given SW took longer to find the submerged platform than they did during the initial training. To assess long-term effects of the ACTH analog treatment, rats were trained on a delayed spatial alternation task 30 days after receiving the last injection. On this task, brain-injured rats treated with the 10-micrograms dose performed significantly better than those given sterile water. Acetylcholinesterase (AChE)-labeled neurons counted in the nucleus basalis magnocellularis indicated that rats with frontal cortex damage given the 10-micrograms treatment did not differ from the sham controls and had significantly more AChE-positive neurons than injured counterparts treated with SW or 100 micrograms.


Control of transcription of Drosophila retrotransposons. Studies of transcriptional control sequences responsible for regulated and basal-level RNA synthesis from promoters of Drosophila melanogaster retrotransposons reveal novel aspects of gene regulation and lead to identification of trans-acting factors that can be involved in RNA polymerase II transcription not only of retrotransposons, but of many other cellular genes. Comparisons between promoters of retrotransposons and some other Drosophila genes demonstrate that there is a greater variety in basal promoter structure than previously thought and that many promoters may contain essential sequences downstream from the RNA start site.


Visualization of neurotransmitter receptors and their mRNAs in the human brain. The study of neurotransmitter receptor localization at the microscopic level has been influenced by progress in biochemistry and molecular biology of these proteins and by the increasing development of new and more selective ligands. Receptor autoradiography is a well established technique in widespread use for the visualization and characterization of receptors at the light microscopic level. This technique can be readily used to examine receptors in control and diseased human brain tissues obtained post mortem. The applications of these techniques in human brain pharmacology and neuropathology as well as its limitations are discussed. The recent cloning of the genes coding for many neurotransmitter receptors has allowed the use of in situ hybridization histochemistry to demonstrate cells expressing mRNA coding for specific receptor subtypes. This technique is now being applied to human brain post mortem tissues. The results provided by the combination of these techniques will significantly modify our understanding of the chemical neuroanatomy of the human brain in health and disease.


GABAergic synaptic transmission. Regulation by drugs. Beside the gamma-aminobutyric acid (GABA)-transporter and the GABAB-autoreceptor, the subsynaptic GABAA-receptor is therapeutically the most relevant target for drug actions influencing GABAergic synaptic transmission. New strategies in drug development focus on partial agonists acting at the benzodiazepine receptor. Since these compounds display less of the undesirable effects associated with the presently used full agonists, a major therapeutic advance is to be expected in the treatment of anxiety disorders and epilepsy. In addition, the extensive structural heterogeneity of GABAA-receptors, derived from a family of more than 15 subunits, may point to an unexpected functional heterogeneity of the receptor which may be exploited pharmacologically. The potential diversity of GABAA-receptor function is presently being analyzed using recombinant GABAA-receptors, which consist of various subunit combinations. These studies point not only to variations in the affinity of GABA, depending on the type of subunit combination, but also to differences in the affinities and intrinsic efficacies of benzodiazepine receptor ligands. Provided these distinctions can be confirmed at GABAA-receptors in situ, a new picture of the physiological and pharmacological regulation of the subsynaptic actions of GABA will emerge.


GABAB receptors as targets for drug action. Data obtained from studies in molecular biology indicate that there may be at least 500 forms of the receptor for the amino acid neurotransmitter gamma-aminobutyric acid (GABA), which are coupled to Cl- channels in mammalian neurones. In addition to this apparent subtyping, the receptors for GABA can be further differentiated on pharmacological grounds into GABAA and GABAB subclasses. GABAA receptors are coupled to Cl- channels, possess allosteric sites for benzodiazepines, barbiturates and neuroactive steroids and mediate fast synaptic inhibition, while GABAB receptors are coupled through G-proteins to neuronal K+ or Ca++ channels. Activation of these receptors increases K+ or decreases Ca++ conductances and mediates slow synaptic inhibition. Inhibition and potentiation of stimulated adenylyl cyclase activity can be attributed to GABAB site activation. The clinically effective muscle relaxant (-)baclofen is a selective agonist for the GABAB site but the therapeutical potential for antagonists of the receptor has yet to be examined. The present article reviews the background to GABAB receptor research and considers the future of drugs targetting the receptor.


5-Hydroxytryptamine receptors. An example for the complexity of chemical transmission of information in the brain. Since serotoninergic axon terminals (the cell bodies of which are mainly located in the raphe nuclei of the brain stem) innervate virtually all brain regions, it is intelligible that the serotoninergic system is involved in many physiological and pathological cerebral functions. The marked heterogeneity of the serotonin (5-hydroxytryptamine, 5-HT)receptors and the differences in distribution of the various 5-HT receptor types and subtypes within the brain makes serotoninergic neurotransmission in the brain very complex. This complexity of chemical transmission of information is a prerequisite for the development of drugs which may specifically influence certain cerebral functions, but which do not mimic or block other effects of 5-HT, thus making it possible to reduce or even avoid undesired side effects. The current classification scheme of 5-HT receptors is based on the integration of results of pharmacological, biochemical, electrophysiological and molecular investigations. The multiplicity of 5-HT receptors can be incorporated into the scheme of two main receptor families, i.e. 1. the G protein receptor family, comprising, among the 5-HT receptors, the 5-HT1 and 5-HT2 subtypes and the 5-HT4 type, and 2. the ligand gated ion channels to which (at least according to the current knowledge) only one 5-HT receptor, the 5-HT3 type, belongs. The three 5-HT1 subtypes and the two 5-HT2 subtypes of the current classification scheme, but not the 5-HT3 and 5-HT4 receptors, have already been cloned.(ABSTRACT TRUNCATED AT 250 WORDS)


Neuropeptides. Function and clinical applications. Neuropeptides are the most abundant chemical messengers in the brain and their major role seems to be the modulation of amine and amino acid neurotransmission. This appears to be achieved at many sites by the co-release of peptide with the primary transmitter. The presynaptic biochemistry and physiology of neuropeptides ensure that neuromodulation is highly plastic with almost infinite adaptive potential. The recent development of novel drugs (termed peptoids) that mimic or block neuropeptide function have opened up new clinical approaches to a number of conditions. Thus high efficacy kappa opioid-receptor agonists such as CI-977 (enadoline) have potential for the treatment of pain and stroke whilst the development of highly selective and bioavailable cholecystokinin B (CCK-B) antagonists such as CI-988 ([R-(R*,R*)]-4-[[2-[[3-(1H-indol-3-yl)-2-methyl-1-ox6-2- [[tricyclo[3.3.1.1.3.1]dec-2-yloxy)carbonyl]amino]propyl]ami no]-1-phenethyl]amino-4-oxobutanoic acid) have offered new insights into the mechanisms underlying and the treatment of anxiety disorders and drug abuse. In general it appears that peptoids may offer a greater selectivity of drug action when compared to amino acid/amine based compounds. Peptoid antagonists appear to be relatively free of side effects possibly because neuropeptide systems are only activated under very selective conditions. Peptoid agonists on the other hand can exert extremely powerful actions on brain function and this may be related to the key position neuropeptide receptors occupy in the hierarchy of chemical communication in the brain.


Positron emission tomography--examination of chemical transmission in the living human brain. Development of radioligands. The imaging technique Positron Emission Tomography (PET) allows examination of chemical neurotransmission in brain. Of key importance for PET-research on neuroreceptors is the development of suitable radiolabelled tracers (ligands). This paper illustrates the multidisciplinary research activities necessary for ligand development. The selective D1- and D2-dopamine receptor antagonists SCH 23390 and raclopride (CAS 84225-95-6), respectively, were labelled with [3H] and characterized in biochemical studies in vitro on human brain homogenates and in autoradiographic studies on cryosections from human hemispheres. The experimental information was used to interpret and support the PET-findings with [11C]-labelled SCH 23390 and raclopride in vivo in humans. In conclusion, these ligands can be used to quantitatively examine dopamine receptors in the human basal ganglia in vivo. An applied study for PET-determination of D1- and D2-dopamine receptor occupancy during antipsychotic drug treatment indicates that the D2-dopamine receptor and possibly also the D1-dopamine receptor are targets for neuroleptic drug action.


Toxicity of a family from vacuumed mercury. A family of four developed toxic blood levels of mercury after the mother vacuumed a spilled jar of liquid mercury from a closet in their apartment. The youngest son developed severe thrombocytopenia which was initially diagnosed as idiopathic thrombocytopenic purpura secondary to viral illness. A possible association between acute mercury toxicity and idiopathic thrombocytopenic purpura has not been previously reported. Chelation therapy with penicillamine for the older child was administered soon after toxic blood mercury levels were known by the physician. Because thrombocytopenia has been reported to occur in up to 5% of patients receiving penicillamine therapy, the younger child was treated with dimercaptosuccinic acid. The mother was also treated with dimercaptosuccinic acid. The father received dimercaprol therapy. The toxic effects and rationale for now outdated therapeutic uses of mercury are discussed.


Genetic linkage between copper accumulation and hepatitis/hepatoma development in LEC rats. The concentration of copper in the livers of Long-Evans rats with cinnamon-like coat color (LEC), in which hepatitis and then hepatomas develop spontaneously, was recently found to be abnormally high. Therefore, we examined the copper concentrations in the livers of LEC F1 backcrosses (LEC F1 x LEC) to determine the linkage of copper accumulation with development of hepatitis. Consistent with a previously reported ratio of rats with hepatitis to rats without hepatitis of about 1:1, hepatitis developed in 14 of 30 F1 backcrosses. The copper concentrations in the livers of all LEC F1 backcrosses with hepatitis were abnormally high and comparable to those of LEC rats. In contrast, the concentrations in all backcrosses without hepatitis were similar to those in normal Long-Evans with agouti coat color or Brown-Norway rats. Copper accumulation was shown to be closely linked with the development of hepatitis in LEC rats and appeared to be a possible cause of hepatitis. The concentrations of copper in the livers of Fischer 344 rats after carbon tetrachloride treatment were in the range for normal liver, indicating that a high copper concentration in the liver is specific to LEC rats and not a specific characteristic of hepatitis. Furthermore, we found that the size and level of ceruloplasmin mRNA in the livers of LEC rats were the same as those in LEA rats and that the size and level of ceruloplasmin polypeptide in their livers and plasma were almost the same as those in LEA rats. Therefore, these results suggest that the copper accumulation is not due to alteration of expression or to gross alteration of the ceruloplasmin gene.


Induction of CYP1A1 gene expression in H4-II-E rat hepatoma cells by benzo[e]pyrene. In the rat, expression of the CYP1A1 gene is closely associated with arylhydrocarbon hydroxylase (AHH) enzyme activity. AHH is an inducile enzyme activity known to play an important role in the bioactivation of polycyclic aromatic hydrocarbons (PAHs) to mutagenic and carcinogenic metabolites. PAH-induced expression of the CYP1A1 gene appears to be regulated by several trans-acting factors, including the Ah receptor and the 4S PAH-binding protein. In this study, we used the PAH isomers benzo[a]pyrene (BaP) and benzo[e]pyrene (BeP) to further evaluate the role of the 4S PAH-binding protein in induction of the CYP1A1 gene in H4-II-E rat hepatoma cells. Although BaP is believed to bind to both the Ah receptor and the 4S protein, BeP has been reported to bind exclusively to the 4S protein. The results of the study presented here indicate that BaP and BeP induce the expression of the CYP1A1 gene, as measured by ethoxyresorufin O-deethylase (EROD) activity, in a concentration-dependent manner. However, BaP is about 25 times as potent as BeP in inducing EROD activity in these cells. Slot-blot analysis of total RNA isolated from these cells indicated that BeP, BaP, and 3-methylcholanthrene increased the level of CYP1A1 mRNA expression. Sucrose-gradient analysis of BeP binding activity indicated that BeP bound with high affinity to the 4S PAH-binding protein, but not to the Ah receptor. These results suggest that the 4S protein may play a role in the PAH-induced expression of the CYP1A1 gene in rat H4-II-E cells.


Catalytic properties of Escherichia coli polyphosphate kinase: an enzyme for ATP regeneration. Catalytic properties of Escherichia coli polyphosphate kinase (EC 2.7.4.1), a promising enzyme for use in ATP regeneration (Hoffman, et al., 1988, Biotechnol. Appl. Biochem. 10, 107-117), are reported here. E. coli polyphosphate kinase (PPK) is broadly active in the pH range 5.5 to 8.5, having an optimal Vmax at pH 7.2. The Km values for the substrates, ADP and polyphosphate (Pn), change little in the same pH range. The optimal concentration range for the Mg2+ activator is 1-20 mM, with an activity maximum at 10 mM Mg2+. In addition to Mg2+, Mn2+ and Co2+ can serve as activators of E. coli PPK, whereas Zn2+ and Cu2+ are highly inhibitory. E. coli PPK is most active with Pn substrates of chain length greater than 132 phosphoryl units. The enzyme activity decreases with decreasing Pn chain length and approaches zero (less than 1%) at a chain length less than or equal to 5. Equilibrium yields of ATP of greater than 85% are readily attained at substrate concentrations below 1 mM. An operational equilibrium constant for the PPK reaction, defined as [ATP]/[ADP][Pn], was determined to be 7.5 (+/- 3.4) x 10(5) M-1. The data presented here serve as a base of information from which assessments of the suitability of E. coli PPK for specific ATP regeneration applications can be made.


Superoxide anion release from blood monocytes and alveolar macrophages in patients with diffuse lung fibrosis. Superoxide anion release (O2-) after stimulation with phorbol myristate acetate was measured in alveolar macrophages (AM) obtained by bronchoalveolar lavage and in blood monocytes from 47 patients with diffuse interstitial lung disease: idiopathic pulmonary fibrosis (N = 15), hypersensitivity pneumonitis (N = 7), pneumoconiosis (N = 6) and sarcoidosis (N = 19). Differential cell counts demonstrated a lymphocyte predominance in patients with hypersensitivity pneumonitis (HP) and sarcoidosis while the other groups had neutrophil predominance. No correlation between O2- activity in alveolar macrophages (AM) or blood monocytes (BM) compared to lung function (VC and diffusing capacity) could be demonstrated. Smoking pneumoconiotics had significantly decreased BM O2- release (1.25 +/- 0.25 (SEM) nmol/min/10(6) cells) and significantly increased AM/BM O2- ratios (2.04 +/- 0.26) compared to smokers with idiopathic pulmonary fibrosis (IPF) who had the following mean values: BM O2- release = 2.58 +/- 0.25 and AM/BM O2- ratio = 0.86 +/- 0.23. When matched for sex and smoking, a significantly increased AM/BM O2- ratio was seen among patients with HP (2.19 +/- 0.98) in comparison with patients who had sarcoidosis (0.40 +/- 0.18). Patients with either HP or pneumoconiosis had generally elevated AM O2- release and reduced BM O2- release. These results suggest that environmentally related interstitial lung disorders (HP and pneumoconiosis) may be associated with elevated AM O2- release relative to BM O2- release in comparison to non-environmentally related disorders (IPF or sarcoidosis).


Agrin released by motor neurons induces the aggregation of acetylcholine receptors at neuromuscular junctions. To test the hypothesis that agrin mediates motor neuron-induced aggregation of acetylcholine receptors (AChRs) in skeletal muscle fibers and to determine whether the agrin active in this process is released by motor neurons, we raised polyclonal antibodies to purified ray agrin that blocked its receptor aggregating activity. When the antibodies were applied to chick motor neuron--chick myotube cocultures, they inhibited the formation of AChR aggregates at and near neuromuscular contacts, demonstrating that agrin plays a role in the induction of the aggregates. Rat motor neurons, like chick motor neurons, induce AChR aggregates on chick myotubes. This effect was not inhibited by our antibodies, indicating that, although the antibodies inhibited the activity of chick agrin, they did not have a similar effect on rat agrin. We conclude that agrin released by rat motor neurons induced the chick myotubes to aggregate AChRs.


Two- and three-dimensional distributions of opioid receptors on NG108-15 cells visualized with the aid of fluorescence confocal microscopy and anti-idiotypic antibodies. Polyclonal anti-idiotypic antibodies previously shown to be specific for mu- and delta-opioid receptor subclasses on rat brain membrane preparations using radioreceptor assays have been labeled with fluorescent conjugates of avidin. The resulting complexes have been used to study the distribution and properties of opioid receptors on living and fixed NG108-15 cells using normal fluorescence, and confocal fluorescence, microscopy. From the confocal data three-dimensional views of immunofluorescent distributions have been obtained using a computer transformation of the data. From experiments involving co-incubation with media containing naloxone, selective opioid peptides and stereoisomeric synthetic opioid analogs as blocking agents, the specificity and stereospecificity of immunofluorescent binding is reported. The effects on immunofluorescent binding by co-incubation with media containing a sulfhydryl reagent, 100 mM-NaCl and sodium azide are also reported. The results have implications concerning the use of immunoprecipitation in the biochemical isolation of the receptors. Finally, the confocal two-dimensional sections and two-dimensional reconstructions obtained from fixed cells show internal immunofluorescence in the form of cytoplasmic clusters. The reagents and methods should be applicable to visualizing native opioid receptor distributions on neurons.


Recruitment of Ca2+ channels by protein kinase C during rapid formation of putative neuropeptide release sites in isolated Aplysia neurons. Activation of protein kinase C (PKC) in Aplysia bag cell neurons causes the recruitment of voltage-dependent calcium channels. Using imaging techniques on isolated cells, we have now found that an activator of PKC, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), promotes the rapid appearance of new sites of calcium influx associated with a change in the morphology of neurite endings. In untreated cells, calcium influx triggered by action potentials occurs along neurites and in the central region of growth cones, but does not usually occur at the leading edge of lamellipodia. TPA produces extension of the lamellipodium, and action potentials now trigger calcium influx at the distal edge of the newly extended endings. Cotreatment with TPA and a cyclic AMP analog promotes movement of secretory organelles toward the new sites of calcium influx. Our results suggest that these second messenger systems promote the rapid formation of morphological structures that contribute to the potentiation of peptide release.


Mutations in an S4 segment of the adult skeletal muscle sodium channel cause paramyotonia congenita. The periodic paralyses are a group of autosomal dominant muscle diseases sharing a common feature of episodic paralysis. In one form, paramyotonia congenita (PC), the paralysis usually occurs with muscle cooling. Electrophysiologic studies of muscle from PC patients have revealed temperature-dependent alterations in sodium channel (NaCh) function. This observation led to demonstration of genetic linkage of a skeletal muscle NaCh gene to a PC disease allele. We now report the use of the single-strand conformation polymorphism technique to define alleles specific to PC patients from three families. Sequencing of these alleles defined base pair changes within the same codon, which resulted in two distinct amino acid substitutions for a highly conserved arginine residue in the S4 helix of domain 4 in the adult skeletal muscle NaCh. These data establish the chromosome 17q NaCh locus as the PC gene and represent two mutations causing the distinctive, temperature-sensitive PC phenotype.


A two-motif isoform of the major calcium channel subunit in skeletal muscle. Evidence is presented that two isoforms of the voltage-dependent, dihydropyridine-sensitive calcium channel alpha 1 subunit are present in newborn and adult skeletal muscle and that expression of these isoforms is developmentally regulated. A voltage-dependent calcium channel alpha 1 cDNA from newborn muscle was cloned and found to be identical to that published from the adult, except that it was 2 kb shorter owing to an internal deletion. Nucleotide sequences, Northern blots, reverse-transcriptase PCR experiments, and sequencing of the PCR product confirmed that a segment corresponding to the inner two repeats of the structural prototype four homologous motifs is missing from the immature isoform. Immunological studies using antisera raised against synthetic peptides that correspond to sequences in the two isoforms show that the abbreviated transcript is predominant in newborn muscle, whereas the four-repeat isoform is the major species in the adult.


Inhibition of N- and L-type calcium channels by muscarinic receptor activation in rat sympathetic neurons. Modulation of N- and L-type Ca2+ channels by oxotremorine-M (oxo-M) acting on muscarinic receptors and norepinephrine (NE) acting on alpha-adrenergic receptors was studied in superior cervical ganglion neurons. Oxo-M depresses dihydropyridine-augmented tail currents in whole-cell recordings, whereas NE does not. This modulation of L-type Ca2+ channels by oxo-M is abolished by adding 20 mM BAPTA to the pipette solution. Oxo-M, acting via a diffusible messenger, reduces the probability of opening of single N- and L-type channels recorded in cell-attached patches. We conclude that a diffusible messenger signaling pathway activated by oxo-M inhibits both N- and L-type Ca2+ channels, whereas a membrane-delimited pathway activated by oxo-M and NE inhibits only N-type Ca2+ channels.


A chimera between platelet-derived growth factor beta-receptor and fibroblast growth factor receptor-1 stimulates pancreatic beta-cell DNA synthesis in the presence of PDGF-BB. This study was undertaken to characterize the expression of a chimeric growth factor receptor composed of the extracellular and transmembrane domains of the platelet-derived growth factor (PDGF) beta-receptor (PDGFR-beta) fused to the intracellular domain of the fibroblast growth factor receptor-1 (FGFR-1) and to assess its effect on the growth potential of pancreatic islet cells. For this purpose rat pancreatic islets or monolayers of pancreatic islet cells were transfected with recombinant DNA constructs coding for the PDGF B-chain, the PDGFR-beta, the FGFR-1 and the chimera between PDGFR-beta and FGFR-1. DNA synthesis, monitored as the percentage of labelled nuclei and [3H]thymidine incorporation, was stimulated in pancreatic islet cells cotransfected with the constructs coding for the PDGF B-chain and the PDGFR-beta or the chimeric PDGFR-beta/FGFR-1 as compared with that determined after transfection with control plasmid. PDGF-BB stimulated DNA synthesis when islet cells had been transfected with PDGFR-beta or PDGFR-beta/FGFR-1. Cotransfection of the PDGFR-beta and the chimeric PDGFR-beta/FGFR-1 constructs attenuated the stimulation of DNA synthesis in response to PDGF-BB. Receptor binding studies showed binding with a Kd of 0.7 nM to the chimeric receptor. The present findings show that when the chimeric PDGFR-beta/FGFR-1 construct is expressed in beta-cells it is efficient in increasing DNA synthesis when stimulated with ligand.


Trends in childhood and adolescent cancer survival in Sweden 1960 through 1984. The temporal changes in childhood and adolescent cancer survival in Sweden 1960-1984 were analyzed. Complete follow-up through 1986 of 6,262 patients younger than 20 years at diagnosis revealed that the overall 5-year survival rates increased from 36.1 to 65.7% in males and from 43.6 to 73.6% in females. The temporal trends differed markedly between age groups and tumour sites and types. Over the study period, 5-years, survival for testicular cancer increased from 46.9 to 87.2%, kidney cancer, predominantly Wilms' tumour from 35.5 to 77.1% (with a higher rate of 89.1% in 1975-1979), Hodgkin's disease from 61.2 to 91.9%, non-Hodgkin's lymphoma from 32.5 to 76.6%, and all leukemias from 8.9 to 58.7%. Only a moderate improvement was noted for tumours of the bone, muscle and connective tissue, and survival rates for tumours of the nervous system remained largely unchanged. Our data reflect the remarkable therapeutic improvements that have occurred for cancer in the young and indicate that these improvements have rapidly become available in Sweden.


Quality of life during chemotherapy for small cell lung cancer. II. A longitudinal study of the EORTC Core Quality of Life Questionnaire and comparison with the Sickness Impact Profile. Sixty-two patients with small cell lung cancer, 36-80 years of age, who were receiving chemotherapy during a maximum of one year, were consecutively included in a study of quality of life during treatment. An interim version (C-36) of the EORTC Core Quality of Life Questionnaire (QLQ) was applied for quality of life assessment prior to treatment and every third month during the treatment period. The assessments were related to clinical variables (performance status and tumour response), and compared with results from assessment with the Sickness Impact Profile (SIP). The QLQ was sensitive to differences in clinical status and responded to clinical change over time. In general, the pattern of correlations with SIP lends support to the construct validity of the QLQ. However, some questions arose from the comparison with SIP: QLQ emotional functioning did not change in concordance with SIP, and assessment of social functioning was not optimal prior to treatment. The questionnaire was well accepted by the patients. The EORTC QLQ C-36 constitutes a promising step in the development of a feasible standard instrument for quality of life assessment in cancer clinical trials.


Intracellular localization and translocation of 1 alpha, 25-dihydroxyvitamin D3 receptor in osteoblasts. The intracellular localization and translocation of the 1 alpha,25-dihydroxyvitamin D3 receptor (1 alpha,25(OH)2D3 receptor) in osteoblasts of mouse parietal bone and MC3T3-E1 cells were examined immunocytochemically using a rat monoclonal antibody to 1 alpha,25(OH)2D3 receptor. In osteoblasts of parietal bones in vivo, immunoreactivity for 1 alpha,25(OH)2D3 receptor was detected not only in the nuclei but also in lysosomal structures, and also sparsely in the cytoplasmic matrix. The transport of 1 alpha,25(OH)2D3 receptor was investigated immunocytochemically after incubation with 1 alpha,25(OH)2D3. In osteoblasts of parietal bones, after 1 min incubation with 10(-8) M 1 alpha,25(OH)2D3, the perinuclear cytoplasm showed intense immunoreactivity for 1 alpha,25(OH)2D3 receptor. After 10 min incubation, immunoreactivity was intense in the nuclei, especially in the heterochromatin. In MC3T3-E1 cells, after 1 min incubation with 1 alpha,25(OH)2D3, immunoreactivity for 1 alpha,25(OH)2D3 receptor was found in the form of a fibrillar structure extending radially to the periphery of the cells. The immunostaining pattern of anti-1 alpha,25(OH)2D3 receptor was similar to the distribution of microtubules stained with anti-beta-tubulin antibody. After 10 min incubation, the nuclei showed intense immunoreactivity for 1 alpha,25(OH)2D3 receptor. Incubation with colchicine dissociated the fibrillar structures and inhibited the intranuclear localization of the 1 alpha,25(OH)2D3 receptor. These findings suggest that the 1 alpha,25(OH)2D3 receptor is located in the nuclei, in lysosomal structures and also sparsely in the cytoplasmic matrix of osteoblasts in vivo, and that the receptor is transported to the perinuclear cytoplasm via microtubules to be then translocated into the nucleus, especially into the heterochromatin.


Synovial sarcoma. A clinicopathological study of 31 cases. Thirty-one surgically treated cases of synovial sarcoma were reviewed. The mean age of the patients at primary surgery was 37 years (range 10-78 years). Twenty-nine of the tumors were of the biphasic type and two were monophasic. Currently a monophasic synovial sarcoma can be considered as a specific entity. In contrast to other soft-tissue sarcomas, synovial sarcomas present the characteristics of a carcinosarcoma. The 5-year survival rate in this study was 55% and the 6-year survival rate 50%; after 6 years there were no recurrences. The primary treatment should follow the same guidelines that are currently given for other soft-tissue sarcomas: wide and radical excisional margins should be aimed at. Excisional treatment even of repeated pulmonary recurrences may be rewarding.


Fibrous dysplasia with locally aggressive malignant change. This is a case report of a tumour which showed all the histological features of fibrous dysplasia without any features of high-grade malignancy, yet had become locally aggressive, causing cortical erosion and extension into soft tissue. Fibrous dysplasia is a well-recognised entity that encompasses monostotic lesions, polyostotic involvement and Albright's syndrome [6, 8]. Lesions in bone usually spare the epiphysis before puberty, but often involve the epiphyseal area after maturity and can progress during adult life [3]. Unless cystic [6, 10] or malignant change [7, 10, 11] occurs, fibrous dysplasia usually remains contained within bone.


A study of ethacrynic acid as a potential modifier of melphalan and cisplatin sensitivity in human lung cancer parental and drug-resistant cell lines. We have studied alterations in glutathione (GSH) levels and glutathione-S-transferase (GST) activity in a series of in vitro derived multidrug resistant and cisplatin resistant sublines of the human lung cancer lines NCI-H69 (small cell), COR-L23 (large cell) and MOR (adenocarcinoma). We have also investigated the effects of ethacrynic acid, a putative inhibitor of GSTs, on levels of GSH and GST activity and on cellular sensitivity to melphalan and to cisplatin. Neither GSH content nor GST activity were significantly greater in the resistant sublines compared with their respective parental lines. The only effects of treating with ethacrynic acid at doses of 1 microgram ml-1 and 3 micrograms ml-1 for 2 h were a reduction in GSH content in the cisplatin resistant subline H69/CPR at the 3 micrograms ml-1 dose, and an increase to over 140% of control at 1 microgram ml-1 and 3 micrograms ml-1 in the MOR parental line (MOR/P) and at 1 microgram ml-1 in the multidrug resistant subline MOR/R. Exposure of parental line COR-L23/P to 3 micrograms ml-1 and 6 micrograms ml-1 of ethacrynic acid for 24 h, however, increased the GSH content to over 300% and 500% of control respectively. Variable effects of ethacrynic acid on GST activity were seen in these cell lines. Doses of 1 microgram ml-1 and 3 micrograms ml-1 reduced activity to 59% and 48% of control respectively in multidrug resistant subline H69/LX4. On the other hand, activity was increased in the cisplatin resistant subline H69/CPR (to 146% and 218% of control) and in MOR/P (to 117% and 137% of control) by 1 microgram ml-1 and 3 micrograms ml-1 respectively of ethacrynic acid. Addition of ethacrynic acid (3 micrograms ml-1) to treatment of the cell lines with melphalan or with cisplatin did not alter the dose-response curves to these agents.


Clinical evaluation of a new serum tumour marker CA 242 in pancreatic carcinoma. The aim of this study was to evaluate the new monoclonal tumour marker CA 242 in the diagnosis of pancreatic carcinoma and to compare it with the established markers CA 50 and CEA. Serum concentrations were determined in 113 patients with jaundice, in 20 patients with laboratory values suggesting cholestasis, and in 60 patients with a suspicion to have chronic pancreatitis. Twenty-four of these 193 patients had pancreatic carcinoma and two patients had carcinoma of papilla of Vater. The sensitivities of CA 242, CA 50 and CEA were 80.7%, 96.1%, and 92.3%, respectively. The specificities were 79.0%, 58.0%, and 59.2%. The sensitivities of combinations of CA 50 and CEA with CA 242 did not exceed the sensitivity of CA 50 alone. The specificity of CA 242 was improved by combining it with CEA (92.2%). The serum marker CA 242 seems to be less sensitive than CEA and CA 50 in the detection of pancreatic carcinoma, but it may prove useful because of its high specificity.


Clinical efficacy and toxicity of standard dose adriamycin in hyperbilirubinaemic patients with hepatocellular carcinoma: relation to liver tests and pharmacokinetic parameters. A standard dose of Adriamycin (60 mg m-2) was administered to 30 patients with inoperable hepatocellular carcinoma, 16 of whom were hyperbilirubinaemic (18-37 mumol l-1). The hyperbilirubinaemic patients experienced marked myelosuppression, but only minor symptomatic side-effects. The degree of neutropenia was directly related to the serum bilirubin concentration, but not to any other standard liver test, presence or absence of cirrhosis, or any pharmacokinetic parameter studied including the area under the Adriamycin or adriamycinol concentration-time curve to 48 h or infinity, or the terminal half-life of Adriamycin. The area under the log concentration-time curve was significantly greater for both Adriamycin and adriamycinol in patients who were hyperbilirubinaemic compared to those with normal bilirubin. Whilst hyperbilirubinaemic patients may tolerate a full dose of Adriamycin, we found no evidence that this was associated with a better response rate, which was disappointingly low at only 18%.


Increased incidence of germ cell testicular cancer in New Zealand Maoris. A higher incidence of germ cell testicular cancer was found in Maoris (6.84/100,000) compared with non-Maoris (5.26/100,000) in New Zealand from 1975 to 1986, especially in the 15-49 year age group (Maoris 12.30/100,000, non-Maoris 9.47/100,000; P = 0.04). Previous studies have shown Whites to have the highest incidence of this malignancy. Possible reasons for this and some other epidemiological features are discussed.


Modified cisplatin, etoposide (or vinblastine) and ifosfamide salvage therapy for male germ-cell tumors. Long-term results. Between 1985 and 1989, 36 consecutive male patients with advanced germ-cell tumors, who had failed to be cured with either the cisplatin, vinblastine, bleomycin (PVB) or the cisplatin, etoposide, bleomycin (PEB) combinations, entered either of two modified salvage therapy regimens consisting of cisplatin, etoposide, and ifosfamide (PEI) or cisplatin, vinblastine and ifosfamide (PVI). All patients had evidence of active disease. Ifosfamide was given at the dosage of 2.5 gr/m2 (with mesna protection) on days 1 and 2; etoposide and cisplatin were given at the dosage of 100 mg/m2 and 40 mg/m2, respectively, on days 3 to 5. In the PVI schedule, vinblastine 6 mg/m2 was given on day 3. Overall, 20 (56%, C.I. 39 to 72) patients entered complete response (CR) or achieved disease-free status (NED) with post-chemotherapy surgery. After a follow-up of 2 to 7 years, 15 patients (42%, C.I. 24 to 58) remain alive and free of disease. None of the 9 patients unresponsive to the first-line therapy and/or with extragonadal primaries entered CR or achieved the NED status, versus 20 (74%, C.I. 58 to 91) of the 27 patients with primary testicular tumors who were responsive to the first-line therapy (p less than 0.001). PEI was used in 20 of these 27 patients, with excellent results (90% CR and 70% continuously NED) independently of primary therapy, PVB or PEB. By contrast, only 2 of the 7 patients treated with PVI following PEB entered CR. Toxicity was not life-threatening. Nine (25%) patients suffered granulocytopenic fever and 3 (8%) required platelet transfusions.(ABSTRACT TRUNCATED AT 250 WORDS)


Invertebrate epithelial Na+ channels: amiloride-induced current-noise in crab gill. Epithelial sheets (including cuticle) from posterior gills of the freshwater-adapted euryhaline crab Eriocheir sinensis were obtained according to the method of Schwarz and Graszynski ((1989) Comp. Biochem. Physiol. 92A, 601-604; (1989) Verh. Dtsch. Zool. Ges. 82, 211 and (1989) Arch. Int. Physiol. Biochim. 97, C45). With external NaCl-saline, the outward-directed short-circuit current (Isc) could hardly be influenced by external amiloride up to 100 mumol/l but was, on the contrary, strictly dependent on apical Cl- (Onken, Graszynski and Zeiske (1991) J. Comp. Physiol. B 161, 293-301). In absence of external chloride an inward-directed, amiloride-inhibitable Isc was observed which depended on external Na+ (thus, Isc approximately INa) in a two-step, saturating mode. The Isc-block by amiloride obeyed saturation kinetics (half-maximal at less than or equal to 1 mumol/l, suggesting apical Na(+)-channels). Only for Na+ concentrations below 100 mmol/l we found an indication for a competitive interaction between Na+ and amiloride at the channel. Current fluctuation analysis revealed the presence of an amiloride-induced relaxation (Lorentzian) component in the Isc-noise (so-called 'blocker-noise'). The Lorentzian parameter-shifts with increasing amiloride concentration indicate first-order kinetics of the blocker with its apical receptor. Using a 'two-state' blocking model we calculated, for amiloride concentrations between 2 and 5 mumol/l, a mean single-channel current of 0.46 pA and a mean channel density of 250.10(6) cm-2.


Patterns of Epstein-Barr virus infection in non-neoplastic lymphoid tissue. Taking advantage of the abundant expression of the small Epstein-Barr virus (EBV)-encoded RNAs (EBERs) in latently infected cells, we have analyzed 72 normal and hyperplastic lymph nodes and three tonsils of acute infectious mononucleosis (IM) for the presence and distribution of EBV+ cells using EBER-specific in situ hybridization, in some cases combined with immunohistologic demonstration of cell type-characteristic antigens. In IM, large numbers of EBV+ lymphoid B blasts were detectable in extrafollicular areas, whereas germinal centers were generally free of EBV+ cells. In reactive lymph nodes, the frequency of EBV+ cells varied with the degree of lymphoid hyperplasia and underlying immune status. The lowest numbers of EBV+ cells were detected in nonactivated lymph nodes and highest in human immunodeficiency virus-associated lymphadenopathy. If present in these lymph nodes, EBV+ cells were almost exclusively localized to extrafollicular areas, as also observed in IM. However, in contrast to IM, these cells were mainly small lymphocytes. Furthermore, in some instances, occasional scattered EBV+ cells were seen within germinal centers, and in two cases diffuse expansions of EBV+ cells occurred within a single germinal center each, indicating that under certain circumstances EBV+ B lymphocytes may participate in physiologic germinal center reactions. These findings reflect the interference of EBV with physiologic lymphoid differentiation pathways and provide a link to EBV-associated malignant lymphomas with a postulated origin from germinal center cells.


An approach for the analysis of relapse and marrow reconstitution after autologous marrow transplantation using retrovirus-mediated gene transfer. Autologous bone marrow transplantation (ABMT) is widely used as treatment for malignant disease. Although the major cause of treatment failure is relapse, it is unknown if this arises entirely because of residual disease in the patient or whether contaminating cells in the rescuing marrow contribute. Attempts to purge marrow of its putative residual malignant cells may delay hematopoietic reconstitution and are of uncertain efficacy. We now describe how retrovirus-mediated gene transfer may be used to elucidate the source of relapse after ABMT for acute myeloid leukemia and to evaluate the efficacy of purging. Clonogenic myeloid leukemic blast cells in patient marrow can be transduced with the NeoR gene-containing helper-free retrovirus, LNL6, with an efficacy of 0% to 23.5% (mean, 10.5%). Transduced colonies grow in selective media and the presence of the marker gene can be confirmed in individual malignant colonies by polymerase chain reaction. If such malignant cells remain in harvested "remission" marrow, they will therefore be marked after exposure to LNL6. Detection of the marker gene in the malignant cells present at any later relapse would be firm evidence that residual disease contributed to disease recurrence, and would permit rapid subsequent evaluation of purging techniques. The technique also marks normal marrow progenitors from patients with acute myeloblastic leukemia. These colony-forming cells can be detected in long-term marrow cultures at a frequency of 1% to 18% for up to 10 weeks after exposure to the vector. Animal models and analysis of probability tables both suggest that these levels of marking in vitro are sufficient to provide information about the mechanisms of relapse and the biology of marrow regeneration in vivo. These preclinical data form part of the basis for current clinical studies of gene transfer into marrow before ABMT.


Onset of erythropoietin response in murine erythroid colony-forming units: assignment to early S-phase in a specific cell generation. Murine erythroid colony-forming units (CFU-E) representing successive cell generations in a six-generation long in vitro maturation sequence were tested for their response to erythropoietin (Epo) by measurement of Epo-exposure times necessary to stimulate heme biosynthesis. Generation I CFU-E, which produce mainly 32-cell erythroid colonies, were isolated in 82% average purity from spleens of thiamphenicol-treated anemic animals via differential centrifugation. Generation II CFU-E, which produce mainly 16-cell colonies, were similarly isolated in 51% average purity. Although both types of CFU-E had equivalent dose sensitivity to and affinity for Epo, generation II CFU-E responded to shorter pulses of Epo than did generation I. Correlations between DNA cell-cycle profiles and 59Fe-heme biosynthesis resulting from pulsed exposures established that appreciable Epo response only begins when CFU-E attain early S-phase of generation II. Because CFU-E did not require Epo or other serum factors to pass from generation I to II and because the onset of Epo responsiveness coincided with the beginning of DNA replication in generation II, we suppose that differentiation has reprogrammed one or more of the events associated with generation II S-phase in CFU-E and that these alterations allow Epo to act. Further comparisons between CFU-E from generation I and II may allow us to identify the alterations in question and the nature of their interaction with Epo.


Detection of functional interferon alpha receptors in human neuroendocrine tumor cell lines using a new monoclonal antibody. While first described as antiviral agents, interferons (IFNs) exhibit significant antiproliferative and antitumor effects as well. IFN alpha has been successfully used in clinical trials to treat several malignancies, including leukemias and certain solid tumors. While many cell types have been studied for IFN alpha receptor expression, very little is known about receptor expression on human neuroendocrine cells. Using a novel anti-IFN alpha receptor monoclonal antibody, we examined IFN alpha receptor expression in 10 human cell lines derived from tumors of neuroendocrine origin, including neuroblastoma, neuroepithelioma and small cell lung carcinoma. All cell lines studied displayed a similar pattern of IFN alpha receptor expression and 5 of 8 cell lines demonstrated reduced thymidine incorporation following IFN alpha treatment. Addition of exogenous IFN alpha caused a decrease in IFN alpha receptor expression, while differentiating agents, such as phorbol esters and retinoic acid, induced an increase in receptor number without altering receptor affinity.


Genes for beta-thromboglobulin and platelet factor 4 are closely linked and form part of a cluster of related genes on chromosome 4. The small inducible gene (SIG) family encodes related proteins that are involved in the overlapping processes of coagulation, inflammation, immune response, and wound repair. This family contains two branches, termed CXC and CC, which are distinguished by whether or not the first two of four conserved cysteine residues are separated by an additional amino acid residue. All of the CXC SIGs map to chromosome 4, including those encoding beta-thromboglobulin (beta TG) and platelet factor 4 (PF4), both of which are expressed by megakaryocytes in a tissue-specific fashion. Both of these latter two genes have been previously reported to be duplicated, there being a PF4 and a PF4alt gene, and a beta TG1 and beta TG2 gene. We now show by pulse-field gel electrophoresis (PFGE) that the beta TG genes are closely linked to the PF4 genes and to other previously mapped CXC SIGs, namely IL8 (encoding interleukin-8), GRO1 (encoding a cytokine also called melanoma growth-stimulatory activity), and two related genes GRO2 and GRO3, on a single 700-kb Sfil fragment localized to chromosome bands 4q12-q13. The only CXC SIG not linked to this cluster is that encoding gamma-interferon-induced 10-Kd protein (INP10), which has been previously localized to 4q21. Analysis of lambda genomic clones demonstrate that the beta TG1 and PF4 genes are separated by less than 7 kb, and the beta TG2 and PF4alt genes by approximately 5 kb. Within each beta TG/PF4 duplication, the beta TG-like gene is upstream of its linked PF4-like gene. Thus, the beta TG/PF4 genes appear to form a close-linked complex expressed in a megakaryocyte-specific fashion. Further genomic studies may provide additional insights into the regulation of the tissue-specific expression of the beta TG/PF4 gene complex, while further analysis of the linked CXC SIG cytokine family may provide further understanding of their evolutionary history.


Characterization of urokinase receptor expression by human placental trophoblasts. The processes of implantation and placentation are both dependent on the invasion and remodeling of the uterine endometrium and vasculature by trophoblasts. Because the secretion and autocrine binding of urokinase (uPA) appears to be a common mechanism used by cells to facilitate plasmin-dependent tissue invasion, we measured the production of uPA and expression of uPA receptors by trophoblasts. Prourokinase bound specifically, reversibly, and with high affinity to cultured trophoblasts, via the uPA epidermal growth factor-like domain. Trophoblasts derived from two first-trimester placentae bound more prourokinase than cells isolated from term placentae. Furthermore, in vitro differentiation of cultured cytotrophoblasts into syncytiotrophoblasts was associated with diminished expression of urokinase receptors and a parallel decrease in the cellular content of uPA receptor mRNA. Trophoblasts also secreted prourokinase and plasminogen activator inhibitors types 1 and 2 (PAI-1 and PAI-2). Although prourokinase was secreted in amounts sufficient to endogenously saturate trophoblast uPA receptors, trophoblasts secreted greater amounts of PAI-1 and PAI-2 than uPA, and no net plasminogen activator activity was detected in trophoblast conditioned medium. In contrast, plasminogen added directly to cultured trophoblasts was readily converted to plasmin. Although the invasion and remodeling of uterine tissues by trophoblasts is a complex process dependent on several proteases of varying specificity, our findings suggest that the expression and modulation of urokinase receptors on the trophoblast cell surface may play an important role in this process.


An atherogenic stimulus homocysteine inhibits cofactor activity of thrombomodulin and enhances thrombomodulin expression in human umbilical vein endothelial cells. Thrombomodulin plays a role as a cofactor for thrombin-catalyzed activation of protein C on endothelial cells. We examined the effect of homocysteine, a stimulant of atherosclerosis and thrombotic disease, on the cofactor activity and protein level of thrombomodulin and also on the expression of thrombomodulin in endothelial cells. Homocysteine inhibited the cofactor activity of thrombomodulin both on the surface of endothelial cells and in the whole cells dose- and time-dependently, and maximal inhibition of the cofactor activity occurred after a 3- to 6-hour incubation with 10 mmol/L homocysteine (10% of initial activity). Homocysteine also decreased the amount of intact (unreduced) thrombomodulin in endothelial cells. However, at the same condition the total protein level (reduced and unreduced form) of thrombomodulin, determined by dot immunoblot analysis using the monoclonal antibody that recognized both reduced and unreduced thrombomodulin, decreased slightly, and the mRNA level of thrombomodulin showed a twofold to three-fold increase. After 24 hours of incubation, the cofactor activity and total protein level of thrombomodulin were 60% and 165% of the initial values, respectively. When purified thrombomodulin fixed to a microwell plate was treated with homocysteine, both cofactor activity and thrombin-binding ability to the thrombomodulin were decreased in proportion to the concentration of homocysteine. These findings suggest that homocysteine directly inhibited the cofactor activity of thrombomodulin on endothelial cells by reducing the disulfide-bond rich epidermal growth factor-like structures of thrombomodulin. This would a result in the decrease of the antithrombotic property of endothelium and may also trigger off the synthesis of mRNA and protein of thrombomodulin to maintain the antithrombotic properties of the cells.


Interleukin-5 selectively enhances the chemotactic response of eosinophils obtained from normal but not eosinophilic subjects. We have attempted to determine whether interleukin-5 (IL-5), a cytokine that selectively affects eosinophil (as opposed to neutrophil) differentiation and activation, also modulates eosinophil migrational responses. Using a modified Boyden chemotaxis assay, IL-5, IL-3, and granulocyte-macrophage colony-stimulating factor (GM-CSF) gave a weak locomotory response for eosinophils from normal nonatopic subjects (optimal at 10(-11), 10(-8), and 10(-9) mol/L, respectively), but not for eosinophils from subjects with an eosinophilia associated with asthma and/or allergic rhinitis. In contrast, IL-5 and IL-3 had no effect on neutrophils, while GM-CSF was chemotactic for neutrophils over a limited concentration range, optimal at 10(-8) mol/L. When eosinophils from normal subjects were incubated with IL-5 (10(-9) mol/L), the locomotory response to platelet-activating factor (PAF; 10(-8) mol/L, P less than .05), leukotriene B4 (LTB4; 10(-6) mol/L, P less than .01), and N-formyl-methionyl-leucyl-phenylalanine (FMLP; 10(-8) mol/L, P less than .01) was significantly enhanced. The percentage enhancement of eosinophil locomotion by IL-5 was greater for eosinophils from normal as compared with subjects with an eosinophilia associated with asthma (P less than .05 for PAF and LTB4; P less than .01 for FMLP). Preincubation of eosinophils from normal subjects with IL-5 (10(-9) mol/L) attenuated the subsequent locomotory response to IL-5 (10(-12) and 10(-11) mol/L, P less than .05). Therefore, the observed refractoriness of eosinophils from eosinophilic subjects to both directional migratory and priming effects of IL-5 in vitro, may reflect a deactivation process resulting from prior exposure in vivo. The selective priming of eosinophil but not neutrophil locomotion by IL-5 suggests that this cytokine may play a significant role in the preferential accumulation of eosinophils at sites of allergic inflammation.


Familial and congenital polycythemia in three unrelated families. Three families with polycythemia inherited through apparently different modes are described. Secondary causes of polycythemia were ruled out. Erythropoietin (EPO) levels were normal or low, even after phlebotomy. In vitro erythroid colony growth in standard assay cultures containing EPO was normal; however, in the absence of added EPO, a few progenitors from most of the affected individuals were able to generate recognizable colonies of mature erythroblasts, although these were smaller and proportionately less numerous than seen in polycythemia vera (PV). To search for EPO-receptor changes as a possible pathophysiologic mechanism, we examined, by Southern blot analysis, genomic DNA samples from affected and nonaffected family members, as well as three patients with PV. Two different probes, derived from the human EPO-receptor, were used. We found no evidence for chromosomal rearrangements or gene amplification in hereditary polycythemia or PV patients. Further, no nucleotide sequences were found that were homologous to the Friend spleen focus-forming virus glycoprotein gp55, which has been shown to bind to and activate the murine EPO-receptor. Functional studies examining number and binding affinity of the EPO-receptor on erythroid progenitors from three hereditary polycythemia patients demonstrated no abnormalities. We conclude that the mechanism(s) for the erythrocytosis in familial and congenital polycythemia and in PV may not involve the EPO-receptor and, therefore, may result from alterations of postreceptor responses.


Bone marrow transplant recipients have defective MHC-unrestricted cytotoxic responses against cytomegalovirus in comparison with Epstein-Barr virus: the importance of target cell expression of lymphocyte function-associated antigen 1 (LFA1). Cytomegalovirus (CMV) remains the most common single infective cause of death following allogeneic bone marrow transplantation (BMT) from major histocompatibility complex (MHC)-identical siblings, whereas Epstein-Barr virus (EBV)-related disease is infrequent. We show here that MHC-unrestricted cytotoxic effector cells in the peripheral blood of BMT recipients are highly effective at killing EBV-infected target cells, but are inactive against CMV-infected target cells. Differential cytotoxicity is associated with disparate target structure expression. Although both EBV- and CMV-infected target cells express viral antigens, it is only those infected with EBV that express the adhesion molecule lymphocyte function-associated antigen 1 (LFA1; CD11a/18). Thus, EBV-infected target cells are able to interact with the principal LFA1 ligand, intercellular adhesion molecule 1 (ICAM1; CD54), which is expressed on posttransplant peripheral blood mononuclear (PBM) effector cells. CMV-infected target cells cannot utilize this ligand. Posttransplant cytotoxicity against EBV-infected target cells is abolished by target and effector cell blockade with monoclonal antibodies (MoAbs) to LFA1 and ICAM1, respectively, demonstrating the functional relevance of this additional ligand interaction. These results provide an illustration both of the importance and of the limitations of MHC-unrestricted cytotoxicity in vivo and may explain the frequency of CMV disease and the relative rarity of EBV-related disease following allogeneic transplantation from MHC-matched siblings. The increased immunosuppression used following MHC-mismatched/matched unrelated-donor BMT may cause this MHC-unrestricted defense mechanism to fail and may contribute to the greatly increased incidence of EBV lymphoproliferative syndrome in these patients.


Ileal pouch-anal anastomosis: state of the art. IPAA surgery has evolved to assume a major role in the operative management of CUC and FAP. In experienced centres, the safety of performing this somewhat complex procedure, often in gravely ill patients, has been confirmed. A significant decrease in morbidity has accompanied increased experience and simplification of the operative techniques. Two major issues await resolution. The first has to do with the less than totally predictable functional results of IPAA surgery. While many patients do well, others, for no apparent reason, do poorly with excess frequency, urgency and incontinence. Whether operative modifications or preoperative testing can alter this outcome is at this time unclear. The second issue has to do with the potential long-term sequelae of IPAA surgery. Pouchitis and nutritional and metabolic consequences, including the potential for malignant transformation of ileal mucosa or of retained rectal mucosa, cannot be ignored. At present, these risks seem remote but only long-term follow-up will determine whether IPAA surgery deserves its current enthusiasm.


Medical management of severe inflammatory disease of the rectum: nutritional aspects. It is clear that the nutritional state of patients with inflammatory bowel disease is often impaired and can be improved by the provision of nutritional support. Improvement in nutritional status can be achieved as effectively with enteral as with parenteral nutrition. Nutritional support appears to have no primary therapeutic effect in patients with ulcerative colitis. With regard to nutritional support in Crohn's disease, parenteral nutrition should be restricted to use as supportive rather than primary therapy. Available information now seems to suggest that most of the benefits of parenteral nutrition in Crohn's disease are related to an improvement in nutritional state rather than as primary therapy, and its use should be restricted to the treatment of specific complications of Crohn's disease, such as intestinal obstruction related to stricture formation or short bowel syndrome following repeated resection. Although some doubt exists over the efficacy of oligopeptide-containing elemental and polymeric enteral diets, the present evidence indicates that chemically defined free amino acid-containing elemental diets have primary therapeutic efficacy in the management of acute exacerbations of Crohn's disease. As such, these diets are worthy of therapeutic trial in patients with severe Crohn's disease involving the distal colon and rectum, particularly in those patients who are malnourished and who prove to be resistant to treatment with a combination of topical corticosteroids and 5-aminosalicylic acid-containing compounds. Clinicians should be aware, though, that the beneficial effects are likely to be restricted to the short term, with high relapse rates by 1 year, this being particularly so in patients with distal Crohn's proctocolitis (Teahon et al, 1988). Volatile fatty acid enemas clearly have potential in the management of patients with severe steroid-resistant proctitis. Finally, one of the most important observations made in recent years is the one concerning the large losses of nitrogen that will occur in patients with inflammatory bowel disease treated with corticosteroids in the absence of adequate protein intake (O'Keefe et al, 1989). Hopefully the days of treating patients with severe inflammatory bowel disease with high dose corticosteroids and a peripheral dextrose or dextrose-saline drip have passed into history.


Management of familial adenomatous polyposis. The management of FAP involves treatment of affected individuals and their families. Such an approach is best coordinated by registrars working in dedicated registries, in close collaboration with nurses, physicians, surgeons, clinical geneticists and others who become involved in the care of these patients. The large bowel of patients with FAP should be removed (totally or subtotally) by the third decade of life. Screening of other areas at risk is recommended to document the natural history of extracolonic manifestations and to allow study of the effects of intervention. Despite these other, sometimes life-threatening manifestations, a near to normal life span is possible in the majority of patients with FAP. The aims of management of the individual and of the family are to ensure that their quality of life is optimal, that support is provided in times of emotional need, that anxiety is minimized and that relatives are adequately screened and treated.


Effects of recombinant granulocyte-macrophage colony-stimulating factor on bovine peripheral blood and mammary gland neutrophil function in vitro. Modulation of peripheral blood and mammary gland neutrophil function following in vitro exposure to recombinant bovine granulocyte-macrophage colony-stimulating factor (rBoGM-CSF) was studied. Bovine blood and mammary gland neutrophils were cultured for 9 h in media containing 0.005, 0.05 or 0.5 microgram/mL rBoGM-CSF. Neutrophils treated with rBoGM-CSF exhibited significantly more chemotactic and bactericidal activities and tended to produce more superoxide anion than control cells. The effects of rBoGM-CSF on bovine neutrophil populations appeared to be dose-dependent. The production of superoxide anion and the bactericidal activity of mammary gland neutrophils were consistently higher than blood neutrophils. Only moderate increases in lipopolysaccharide-induced mammary gland neutrophil functions were observed following incubation with rBoGM-CSF which suggests that there may be a threshold of immunomodulation for these prestimulated cells. It may be possible to augment the functional capacity of bovine neutrophil populations in vivo through the therapeutic application of rBoGM-CSF and consequently enhance resistance of dairy cattle to bacterial infections.


Detection of infectious laryngotracheitis virus infected cells with cloned DNA probes. A genomic library of infectious laryngotracheitis virus (ILTV) DNA BamH1 fragments was prepared and two cloned fragments were evaluated for their potential as probes for the detection of ILTV infected cells. The virus was purified by a modified sucrose density gradient procedure for the isolation of pure ILTV DNA. A genomic library was constructed using BamH1-digested ILTV DNA and pGEM7 as a vector. A 1.1 kb cloned BamH1 fragment of ILTV DNA was tested in a slot or dot blot assay for the detection of ILTV infected cells. The limit of detection for this probe was at least 0.12 ng of pure ILTV DNA. The probe was able to identify both chicken embryo liver (CELi) cells and choriallantoic membranes infected with ILTV. Chicken embryo liver cells infected with several field isolates and a vaccine strain of ILTV were positive by dot blot analysis using this probe. Some qualitative differences in the degree of hybridization to cells infected by different ILTV isolates were observed. Uninfected cells and cells infected with fowlpox virus, turkey herpesvirus, Marek's disease virus or Newcastle disease virus were negative by the same assay. Compared with the 1.1 kb fragment, a larger 6 kb cloned BamH1 fragment of ILTV DNA showed a stronger hybridization signal to DNA from ILTV infected cells.


Study of the potential involvement of pseudorabies virus in swine respiratory disease. In order to investigate the potential involvement of pseudorabies virus (PRV) in swine respiratory disease, nine week old pigs were intranasally inoculated with the PRV strain 4892. Two doses of infection were used: 10(4.5) median tissue culture infectious doses (TCID50)/pig and 10(3.5) TCID50/pig, with ten pigs per group. In the group of pigs inoculated with 10(4.5) TCID50, seven out of ten pigs died within six days after inoculation. The mortality rate in the group of pigs inoculated with the lower dose was only two out of ten and, there were several pigs in this group that showed signs of respiratory distress besides some mild nervous signs. Pseudorabies virus was isolated from various tissues collected postmortem, including alveolar macrophages. Virus localization in tissues was also detected by in situ hybridization. The histopathological examination of the respiratory tract tissues revealed a pathological process that was progressing from mild pneumonia to severe suppurative bronchopneumonia. The isolation of virus from alveolar macrophages provides support to the hypothesis that replication of PRV during the course of infection produces an impairment of the defense mechanisms in the respiratory tract.


Seroconversion of pigs in contact with dogs exposed to canine coronavirus. In order to determine if canine coronavirus (CCV) could be transmitted to pigs, two dogs were inoculated orally with virulent CCV. After 24 h, the dogs were moved to an isolation room that contained three three-day-old pigs. A wire mesh fence, allowing close contact between the animals, separated the dogs from the pigs. The dogs and pigs were observed for 14 days for clinical signs of disease. Samples of blood were obtained from dogs and pigs immediately before the dogs were inoculated with virus and 14 and 28 days later. The dogs developed mild clinical signs of an infection, but the pigs remained normal throughout the observation period. The dogs shed CCV for eight days after exposure. All three pigs developed neutralizing antibodies against CCV and transmissible gastroenteritis virus by 14 days after they were exposed to the dogs.


Penicillins, cephalosporins, quinolones. This article details the mechanisms of action, antimicrobial spectrum, pharmacokinetics, and adverse reactions of these three antibiotics.


Thrombin stimulates L-type calcium channels of guinea pig cardiomyocytes in cell-attached patches but not after intracellular dialysis. The action of the blood clotting enzyme thrombin on single channel and whole cell Ca(2+)-currents was studied in isolated mammalian cardiac myocytes. Thrombin, at a concentration of 10(-8) mol/l, increased the Ca(2+)-channel activity in cell-attached patches. The mean open probability of the channel was enhanced, while the number of sweeps without openings, which reflects the availability of the channel, was significantly reduced. Neither the single channel conductance nor the activation curve were affected by thrombin. Thrombin was added to the bath solution, and its effect is therefore indirect and probably mediated via a second messenger. However, thrombin did not affect whole-cell Ca(2+)-currents, whereas a beta-adrenergic stimulation in the same cell increased the Ca(2+)-current. It is concluded that thrombin affects an intracellular mechanism for Ca2+ channel current regulation, which is still unknown and which is rapidly lost during conventional whole-cell Ca2+ current measurements.


Relationship between cytosolic calcium and oxygen consumption in isolated rat hearts. Maximum oxygen consumption was attained in isolated perfused rat hearts using high perfusate calcium and/or isoproterenol, or phenylephrine. The amplitude of calcium transients was directly related to oxygen consumption until oxygen consumed per beat reached maximum. At saturating oxygen consumption the amplitude of [Ca2+]i transients continued to increase, indicative of a calcium overload. In all cases +dP/dt correlated proportionately with +dCa2+/dt. Augmented developed pressure, related to isoproterenol-induced increase in cytosolic cAMP, cannot be attributed totally to elevated levels of [Ca2+]i transients. Adenosine (10(-5) M) added to the medium containing isoproterenol (10(-6) M) negated the isoproterenol-induced increase in cAMP and returned cardiac performance, oxygen consumption, and amplitude of [Ca2+]i transients to control state.


A brefeldin A-like phenotype is induced by the overexpression of a human ERD-2-like protein, ELP-1. Brefeldin A (BFA) is a unique drug affecting the molecular mechanisms that regulate membrane traffic and organelle structure. BFA's ability to alter retrograde traffic from the Golgi to the endoplasmic reticulum (ER) led us to ask whether the ERD-2 retrieval receptor, proposed to return escaped ER resident proteins from the Golgi, might either interfere with or mimic the effects of the drug. When either human ERD-2 or a novel human homolog (referred to as ELP-1) is overexpressed in a variety of cell types, the effects are phenotypically indistinguishable from the addition of BFA. These include the redistribution of the Golgi coat protein, beta-COP, to the cytosol, the loss of the Golgi apparatus as a distinct organelle, the mixing of this organelle with the ER, the addition of complex oligosaccharides to resident ER glycoproteins, and the block of anterograde traffic. Thus, these receptors may provide signals that regulate retrograde traffic between the Golgi and the ER.


Transgenic mouse mammary tumor virus superantigen expression prevents viral infection. Endogenous mouse mammary tumor virus (MMTV) proviruses have recently been shown to cosegregate genetically with the minor lymphocyte-stimulating loci, also termed self-superantigens. The antigenic activity has been localized to the open reading frame (ORF) protein encoded in the long terminal repeat of MMTV. We show here that unlike their nontransgenic littermates, transgenic mice expressing high levels of an ORF protein derived from the C3H exogenous MMTV specifically delete their V beta 14+ T cells and do not become infected with this virus when it is present in their mother's milk. Thus, it appears that MMTV utilizes cells of the immune system in its infection pathway, and mice that retain endogenous MMTVs should be immune to infection by exogenous virus. These results offer possible new approaches to anti-viral therapy or immunization.


Drosophila retinal degeneration C (rdgC) encodes a novel serine/threonine protein phosphatase. The Drosophila retinal degeneration C (rdgC) gene is required to prevent light-induced retinal degeneration. Molecular analysis shows that the rdgC transcription unit encodes a novel serine/threonine protein phosphatase. Amino acids 153-393 define a domain that has 30% identity with the catalytic domains of types 1, 2A, and 2B serine/threonine protein phosphatases. A putative regulatory domain is appended that contains multiple potential Ca(2+)-binding sites or "EF hand motifs." Thus, the analysis suggests that the rdgC protein is a novel type of serine/threonine protein phosphatase that is directly regulated by Ca2+. rdgC is expressed in the visual systems of the fly, as well as in the mushroom bodies of the central brain.


[Effectiveness of phosphonoformic acid in herpes infections]. The antiviral efficacy of phosphonoformic acid (PFA) was examined on tissue cultures of the human embryonal lungs against the viruses herpes simplex type 1 and 2, herpes zoster, and the human cytomegalovirus using the method of the inhibition of the cytopathic effect and against herpes simplex type 1 and 2 on the tissue cultures of Vero cells using the methods of the inhibition of plaque formation. PFA was demonstrated to inhibit the reproduction of the viruses under study in both tests on tissue cultures. In persistence studies, the cytomegalovirus was not isolated back from the cells of the human embryonal lungs, which gave evidence of its greater sensitivity to PFA. In in vivo experiments, PFA in the form of a 3% ointment suppressed herpetic dermatitis on the guinea-pig skin, when treatment started 6, 24 and 48 hours after infection. In a preliminary experiment on rabbit corneas, an ointment with 3% PFA inhibited herpetic keratoconjunctivitis in time intervals of 1 and 3 hours after infection.


A prognostic-factor risk index in advanced non-small-cell lung cancer treated with cisplatin-containing combination chemotherapy. Prognostic factors for response and survival were retrospectively evaluated in 192 previously untreated patients with advanced non-small-cell lung cancer (NSCLC) who had received either vindesine plus cisplatin or mitomycin plus vindesine plus cisplatin as initial treatment. Univariate analysis demonstrated that squamous-cell histology, early stage, and a small number of metastatic sites were favorable prognostic factors for response to chemotherapy. Multivariate analysis using Cox's proportional hazard model indicated that the number of metastatic sites was the only significant pretreatment factor for response (P = 0.0005). Multivariate regression analysis revealed that the number of metastatic sites (P = 0.0002), sex (P = 0.0009), serum albumen levels (P = 0.0018), performance status (P = 0.0026) and lactic dehydrogenase values (P = 0.0026) contributed independently to survival. On the basis of these five prognostic factors, a prognostic index for survival was used to define three prognostic groupings (good, intermediate, and poor) for survival (median survival, 16.5 vs 9.4 vs 4.6 months; P = 0.0001). This particular regression model should aid in the design and analysis of new treatment strategies and may be useful for indirect comparisons of different studies carried out in similar patient populations.


Chromosomal, mitochondrial and metabolic alterations in SV40-transformed rabbit chondrocytes. Karyotype, mitochondrial ultrastructure and several enzymatic activities were studied in two clones, D22 and D27, from SV40-transformed rabbit chondrocytes. Similar chromosome alterations, with recurrent losses and gains were observed at the various passages. Mitochondria were rare, with increase in size and crest alterations. By comparison to non-transformed rabbit chondrocytes, activities of superoxide dismutase 1 and 2 (SOD) and glutathione peroxidase were increased, those of glutathione reductase, glucose-6-phosphate dehydrogenase and glutathione-S-transferase fluctuated according to passages, thymidylate synthase decreased, thymidine kinase and hypoxanthine-phosphoribosyl-transferase increased and the ratio lactate dehydrogenase B/A increased. In most cases, these variations were correlated with the number of chromosomes carrying the genes encoding for corresponding enzymes. These results, compared to those obtained in SV40-transformed human fibroblasts, demonstrate that the two cell types behave differently for detoxication systems against oxygen radicals, in particular for SOD2 activity, and have opposite imbalances of chromosomes carrying the corresponding genes.


In vitro and in vivo evidence for the formation of methyl radical from procarbazine: a spin-trapping study. Electron spin resonance (ESR) analysis combined with the use of 4-pyridyl-1-oxide-t-butyl nitrone (4-POBN) and dibromonitroso benzenesulfonic acid (DBNBS) as spin-trapping agents was used to characterize free radical generation during the metabolism of the anticancer agent procarbazine [N-isopropyl-a-(2-methylhydrazino)-p-toluamide hydrochloride]. The formation of free radical species, identified as methyl radicals, was observed during oxidation of procarbazine in rat liver microsomes and isolated hepatocytes in vitro, as well as in several organs following administration of the drug in vivo. A cytochrome P450-mediated reaction, involving P450IA and IIB isoenzymes, was responsible for the activation process. The metabolic pathway leading to free radical formation was characterized using various procarbazine metabolites and revealed strict analogies with previously published data on methane production from procarbazine. These results supported the identification of the trapped species as methyl free radical and suggested that C-oxidation of azoprocarbazine is the main source of radical intermediates derived from this anticancer drug.


The level of aryl hydrocarbon (Ah) receptor and of 4S polycyclic aromatic hydrocarbon (PAH) binding protein in diploid and polyploid hepatocytes of 2-acetylaminofluorene-treated rats. Sequential treatment of partially hepatectomized male Wistar rats with diethylnitrosamine (DEN) and 2-acetylaminofluorene (AAF) induces the emergence of diploid hepatocyte populations. These carcinogen-induced hepatocytes are thought to include the precursor cells of liver carcinomas that arise later in this treatment protocol. The growth of the diploid hepatocytes is promoted by AAF and it has been suggested that the action of the arylamine may be receptor-mediated. AAF has been shown to bind specifically to the aryl hydrocarbon (Ah) receptor and the so-called 4S polycyclic aromatic hydrocarbon (PAH) binding protein. The present study addresses the question of whether the concentrations of the two binding proteins differ in diploid and polyploid hepatocytes from DEN/AAF-treated rats. Hepatocytes from carcinogen-treated rats were isolated and diploid, and tetraploid hepatocytes separated by means of centrifugal elutriation. Whereas Ah receptor concentrations in diploid hepatocytes were insignificantly lower (21.8 +/- 5.9 versus 29.2 +/- 6.6 fmol/mg cytosolic protein; n = 4; P = 0.1), levels of the 4S PAH binding protein in diploid hepatocytes were twice as high as in tetraploid hepatocytes (252.3 +/- 93.6 versus 124.0 +/- 18.5 fmol/mg cytosolic protein; n = 4; P = 0.04). We conclude from our results that the differences in growth control in polyploid and carcinogen-induced diploid hepatocytes are not associated with changes in the levels of the Ah receptor. The role of the 4S PAH binding protein in the process of hepatocarcinogenesis remains to be established.