Targeted degradation of the retinoblastoma protein by human papillomavirus E7-E6 fusion proteins. The E6 and the E7 proteins of the oncogenic human papillomavirus types 16 and 18 can stably associate with p53 and the retinoblastoma protein, respectively. The E6-p53 interaction results in the accelerated degradation of p53 in vitro via the ubiquitin-dependent proteolysis system. In this study we demonstrate that a fusion protein consisting of the N-terminal half of the HPV-16 E7 protein and the full length HPV-16 E6 protein promotes the in vitro degradation of the retinoblastoma protein. This indicates that the property of the HPV-16 E6 protein to stimulate the degradation of p53 can be targeted to other proteins. Unlike the HPV-16 or HPV-18 E6 protein, the E6 proteins of HPV-6 and 11 do not bind to p53 and consequently do not target p53 for degradation. Analogous E7-E6 fusion proteins using the E6 proteins of HPV-6 and HPV-11, however, also have the ability to promote the degradation of the retinoblastoma protein, indicating that the property to target associated proteins for degradation is shared by the anogenital specific HPV E6 proteins.


The 'second-codon rule' and autophosphorylation govern the stability and activity of Mos during the meiotic cell cycle in Xenopus oocytes. The c-mos proto-oncogene product, Mos, functions in both early (germinal vesicle breakdown) and late (metaphase II arrest) steps during meiotic maturation in Xenopus oocytes. In the early step, Mos is only partially phosphorylated and metabolically unstable, while in the late step it is fully phosphorylated and highly stable. Using a number of Mos mutants expressed in oocytes, we show here that the instability of Mos in the early step is determined primarily by its penultimate N-terminal residue, or by a rule referred to here as the 'second-codon rule'. We demonstrate that unstable Mos is degraded by the ubiquitin-dependent pathway. In the late step, on the other hand, Mos is stabilized by autophosphorylation at Ser3, which probably acts to prevent the N-terminus of Mos from being recognized by a ubiquitin-protein ligase. Moreover, we show that Ser3 phosphorylation is essential for Mos to exert its full cytostatic factor (CSF) activity in fully mature oocytes. Thus, a few N-terminal amino acids are primary determinants of both the metabolic stability and physiological activity of Mos during the meiotic cell cycle.


Identification of a putative membrane receptor for the bioactive phospholipid, lysophosphatidic acid. Lysophosphatidic acid (LPA) is a naturally occurring phospholipid with hormone- and growth factor-like activities. Exogenous LPA stimulates GTP-dependent phosphoinositide hydrolysis and inhibits adenylate cyclase in its target cells, but the site of action of LPA is unknown. We now report the identification by photoaffinity labeling of a putative LPA membrane receptor in various LPA-responsive cell types. A 32P-labeled LPA analogue containing a photoreactive fatty acid, [32P]diazirine-LPA, labels a membrane protein of apparent molecular mass of 38-40 kDa in various cell types, including neuronal cells, brain homogenates, carcinoma cells, leukemic cells and normal fibroblasts. Labeling of the 38-40 kDa protein is competitively inhibited by unlabeled 1-oleoyl-LPA (IC50 approximately 10 nM), but not by other phospholipids. Specific labeling is not detected in rat liver membranes or in human neutrophils, which are physiologically unresponsive to LPA. Suramin, an inhibitor of both early and late events in the action of LPA, completely inhibits the binding of photoreactive LPA. We suggest that the 38-40 kDa protein represents a specific LPA cell surface receptor mediating at least part of the multiple cellular responses to LPA.


Structure-function analysis of hepatocyte growth factor: identification of variants that lack mitogenic activity yet retain high affinity receptor binding. Hepatocyte growth factor (HGF) is a potent mitogen for parenchymal liver, epithelial and endothelial cells. Structurally, it has similarities to kringle-containing serine proteases, although it does not possess proteolytic activity. A structure-activity relationship study of human HGF was performed by functional analysis of HGF substitution and deletion variants. Analysis of HGF variants was accomplished by defining their ability to induce DNA synthesis on hepatocytes in primary culture and to compete with wild-type HGF for binding to a soluble form of the HGF receptor. Three groups of variants were made: (i) substitutions at the cleavage site, (ii) substitutions within the protease-like domain and (iii) deletions of the beta-chain and/or kringle domains. Our results show that: (i) single-chain HGF is a zymogen-like promitogen in that cleavage into a two-chain form is required for biological activity, however, the single chain form of HGF still retains substantial receptor binding capacity; (ii) certain mutations in the protease-like domain result in variants that are completely defective for mitogenic activity, yet exhibit apparent receptor binding affinities similar to wild-type HGF (Kd approximately 50-70 pM); and (iii) a variant containing the N-terminal 272 residues of mature HGF showed only a 4-fold increase in Kd when compared with wild-type HGF indicating that a primary receptor binding determinant is located within this sequence.


Yeast mitochondrial DNA mutators with deficient proofreading exonucleolytic activity. The MIP1 gene which encodes yeast mitochondrial DNA polymerase possesses in its N-terminal region the three motifs (Exo1, Exo2 and Exo3) which characterize the 3'-5' exonucleolytic domain of many DNA polymerases. By site directed mutagenesis we have substituted alanine or glycine residues for conserved aspartate residues in each consensus sequence. Yeast mutants were therefore generated that are capable of replicating mitochondrial DNA (mtDNA) and exhibit a mutator phenotype, as estimated by the several hundred-fold increase in the frequency of spontaneous mitochondrial erythromycin resistant mutants. By overexpressing the mtDNA polymerase from the GAL1 promoter as a major 140 kDa polypeptide, we showed that the wild-type enzyme possesses a mismatch-specific 3'-5' exonuclease activity. This activity was decreased by approximately 500-fold in the mutant D347A; in contrast, the extent of DNA synthesis was only slightly decreased. The wild-type mtDNA polymerase efficiently catalyses elongation of singly-primed M13 DNA to the full-length product. However, the mutant preferentially accumulates low molecular weight products. These data were extended to the two other mutators D171G and D230A. Glycine substitution for the Cys344 residue which is present in the Exo3 site of several polymerases generates a mutant with a slightly higher mtDNA mutation rate and a slightly lower 3'-5' exonucleolytic activity. We conclude that proofreading is an important determinant of accuracy in the replication of yeast mtDNA.


Role of associated gamma-chain in tyrosine kinase activation via murine Fc gamma RIII. Type III receptors for the Fc portion of IgG (Fc gamma RIII), initially characterized on macrophages and NK cells, are also expressed on several pre-B cell lines. Surface expression of Fc gamma RIII requires the association of the ligand binding alpha-chain with homodimeric gamma-chains. Type II Fc gamma R is homologous to Fc gamma RIII alpha-chain in the extracellular portion and differs in the transmembrane and cytoplasmic domains. The role of Fc gamma R in cell activation was investigated by expressing Fc gamma RIII and the lymphocyte-specific b1 isoform of Fc gamma RII (Fc gamma RIIb1) in an Fc gamma R-negative, sIgG-positive B-cell line. We found that, in contrast to Fc gamma RIIb1, Fc gamma RIII triggers the same events of cell activation as sIG i.e. Ca2+ mobilization, tyrosine phosphorylation and IL-2 secretion. By expressing cytoplasmic domain-lacking Fc gamma RIII alpha-chain in the absence or in the presence of gamma-chains, we demonstrated that cell activation via Fc gamma RIII requires the co-expression of gamma-chains, and is independent of the cytoplasmic portion of the alpha-chain. Furthermore, the cytoplasmic portion of the gamma-chain, fused to the extracellular and transmembrane domains of Fc gamma RII confers on the chimeric receptor the ability to trigger cell activation. Mutation of one tyrosine residue in the cytoplasmic domain of the gamma-chain prevented triggering of cytoplasmic signals. We therefore demonstrate that a tyrosine-containing motif, present in the cytoplasmic domain of the associated gamma-chain, is necessary and sufficient to trigger cell activation via Fc gamma RIII.


Effect of intravenous hypnotics on the actions of pipecuronium. Seventy-five ASA Grades I-III patients (18-85 years, 45-90 kg) were randomized into five groups. All patients received N2O/O2 (2/1) and alfentanil: loading dose (LD) 0.015 mg kg-1 and maintenance dose (MD) 0.045 mg kg-1 h-1 (groups 1-4). Group 1 received propofol (LD 2 mg kg-1 and MD 6 mg kg-1 h-1); Group 2 etomidate (LD 0.3 mg kg-1 and MD 0.6 mg kg-1 h-1); Group 3 midazolam (LD 0.2 mg kg-1 and MD 0.120 mg kg-1 h-1); Group 4 methohexitone (LD 1.5 mg kg-1 and MD 4 mg kg-1 h-1); Group 5 dehydrobenzperidol 0.05-0.23 mg kg-1 and alfentanil (LD 0.100 mg kg-1 and MD 0.060 mg kg-1 h-1). The neuromuscular block induced by pipecuronium (50 micrograms kg-1) was evaluated. No statistically significant differences were found between the five groups as concerned degree of block (expressed as % twitch amplitude in response to the first of the TOF stimuli (Ta1) at intubation, T1 minimum and recovery to Ta1 = 20%, 25% and 75%. Slightly faster intubation was possible when midazolam was used in comparison with propofol, methohexitone or NLA and when etomidate was used in comparison with propofol. A wide range of individual values of maximal neuromuscular blocking activity was found.


A comparison of the catalytic properties of cellobiose:quinone oxidoreductase and cellobiose oxidase from Phanerochaete chrysosporium. Several catalytic properties of the FAD enzyme cellobiose:quinone oxidoreductase (CBQ) and the heme/FAD enzyme, cellobiose oxidase (CBO) have been investigated and compared. Dichlorophenol-indophenol was found to be a very good electron acceptor for cellobiose oxidation by both enzymes. The optimal pH value for this oxidation with dichlorophenol-indophenol as a co-substrate was observed around pH 4 for both enzymes. The turnover numbers of this reaction were also very similar. The Km values for cellobiose oxidation were identical, whereas the Km for CBO with dichlorophenol-indophenol is lower than that of CBQ. Atmospheric oxygen is a very poor electron acceptor for both CBO and CBQ, however, CBO can utilize cytochrome c as an effective electron acceptor, while CBQ cannot. The specific activity of CBO for cytochrome c is thus about 200-times higher than for oxygen. Thus, one way to distinguish the two enzymes is by the cytochrome-c-reducing ability of CBO. Therefore, we propose that the nomenclature for CBO is tentatively changed to cellobiose:cytochrome c oxidoreductase until a rational name can be installed. Both enzymes have radical-reducing activities. The cation radical, derived from 1,2,4,5-tetramethoxybenzene, was reduced by both enzymes at almost the same reaction rate. The phenoxyradical produced by lignin peroxidase, catalyzing the oxidation of acetosyringon, was also reduced by both enzymes. The reduction of phenoxyradicals formed by phenoloxidases (lignin peroxidases, as well as laccases) may be important in preventing repolymerization reactions which we suggest would significantly facilitate lignin degradation.


A hybrid protein of urokinase growth-factor domain and plasminogen-activator inhibitor type 2 inhibits urokinase activity and binds to the urokinase receptor. The binding of urokinase-type plasminogen activator (uPA) to its specific cell-surface receptor (uPAR) localises the proteolytic cascade initiated by uPA to the pericellular environment. Inhibition of uPA activity or prevention of uPA binding to uPAR might have a beneficial effect on disease states wherein this activity is deregulated, e.g. cancer and some inflammatory diseases. To this end, a bifunctional hybrid molecule consisting of the uPAR-binding growth-factor domain of uPA (amino acids 1-47; GFuPA) at the N-terminus of plasminogen-activator inhibitor type 2 (PAI-2) was produced in Saccharomyces cerevisiae. The purified protein inhibited uPA with kinetics similar to placental or recombinant PAI-2 and was also found to bind to U937 cells and to FL amnion cells. GFuPA-PAI-2 competed with uPA, the N-terminal fragment of uPA and a proteolytic fragment of uPA (amino acids 4-43) in cell binding experiments, indicating that the molecule bound to the cells via uPAR. Hence, both the uPA-inhibitory and uPAR-binding domains of the hybrid molecule were functional, demonstrating the feasibility of the novel concept of introducing an unrelated, functional domain onto a member of the serine-protease-inhibitor superfamily.


Polymyxin B inhibits insulin-induced glucose transporter and IGF II receptor translocation in isolated adipocytes. In isolated adipocytes, polymyxin B inhibited insulin-induced glucose incorporation into lipids in a dose-dependent manner, while polymyxin E, a structurally related antibiotic, was ineffective. To approach the mechanism of this effect, the subcellular distribution of the glucose transporter Glut 4 was investigated. Adipocytes were pretreated without or with polymyxin B before insulin stimulation, subcellular fractionation was performed and Glut 4 was detected by immunodetection. Incubation of adipocytes with polymyxin B prevented the insulin-induced appearance of Glut 4 in the plasma membranes, but did not prevent their decrease from the low-density microsomal fraction. A lower purity of the plasma membrane fractions, a detergent effect of polymyxin B on the membranes or an interference of the substance with the immunodetection of the Glut 4 molecules were excluded. These results suggest that polymyxin B was interfering with the Glut 4 translocation process stimulated by insulin in adipocytes. In a similar fashion, polymyxin B inhibited the insulin-induced increase in IGF II binding to adipocytes. This resulted from a blockade of the appearance of IGF II receptors in the plasma membranes. Since low-molecular-mass GTP-binding proteins have been implicated in the regulation of vesicular trafficking, we have used [alpha-32P]GTP binding to analyze such proteins in adipocyte fractions, after SDS/PAGE and transfer to nitrocellulose. Specific and distinct subsets of GTP-binding proteins were revealed in plasma membrane and low-density microsomal fractions of control adipocytes, whether they were stimulated or not with insulin. Polymyxin B treatment of adipocytes markedly modified the profile of the low-molecular-mass GTP-binding proteins in plasma membranes, but not in low-density microsomal fractions. Our results suggest that polymyxin B was interfering with the exocytotic process of the Glut 4 and IGF II receptor-containing vesicles, perhaps at the fusion step between vesicles and plasma membranes.


Enhanced cell-free transcription of the ribosomal protein L32 gene by the polyoma virus enhancer PEA3 DNA-binding protein. The mouse-ribosomal-protein-L32-gene promoter contains a 12-bp sequence motif within the 5'-upstream region termed the beta element which shows significant similarity with the consensus sequence of the polyoma-virus-enhancer PEA3. A cloned PEA3 DNA-binding protein, expressed in Escherichia coli and purified, activates the expression of the ribosomal-protein-L32 gene in a cell-free system. Moreover, the PEA3 protein participates in the formation of the ribosomal-protein-L32-promoter-preinitiation-transcription complex. The preinitiation complex formed with PEA3 is resistant to competition by oligonucleotides containing the beta element. In addition anti-PEA3 serum interacts with a factor in mouse L1210 nuclear extract that binds to the beta element, causing a supershift in a mobility-shift assay. Our study demonstrates for the first time that the PEA3 protein can transactivate a cellular gene in a cell-free transcription system.


Purification and partial characterization of a beta-1,3-glucan-binding-protein membrane receptor from blood cells of the crayfish Pacifastacus leniusculus. A receptor for the 100 kDa beta-1,3-glucan-binding protein [Duvic, B. and Söderhäll, K. (1990) J. Biol. Chem. 265, 9327-9332] has been purified from hemocyte membranes of the crayfish Pacifastacus leniusculus. The purification was achieved by DEAE-cellulose chromatography of detergent-solubilized membranes. The receptor had an apparent molecular mass of 350 kDa when subjected to native polyacrylamide-gel electrophoresis and was composed of two non-covalently associated subunits of about 230 kDa and 90 kDa, as judged by SDS/polyacrylamide-gel electrophoresis or two-dimensional electrophoresis. The receptor could only bind the beta-1,3-glucan-binding protein if this protein had previously reacted with a beta-1,3-glucan, laminarin, and the binding site was located on the 230 kDa subunit. The binding of laminarin-treated beta-1,3-glucan-binding protein to its receptor was a saturable process and binding data indicated a single high-affinity-binding site with a Kd of 0.35 +/- 0.15 microM as determined by Scatchard analysis. The receptor had a requirement for divalent cations and a pH optimum of 6.5 for binding the laminarin-treated beta-1,3-glucan-binding protein. Laminarin, as well as oligosaccharides such as D-glucose, sialic acid, N-acetyl glucosamine or methyl-alpha-D-mannoside, could not affect the binding of the beta-1,3-glucan-binding protein to its receptor.


Structural requirements for the occupancy of pituitary adenylate-cyclase-activating-peptide (PACAP) receptors and adenylate cyclase activation in human neuroblastoma NB-OK-1 cell membranes. Discovery of PACAP(6-38) as a potent antagonist. In these structure activity studies, the 46 analogs of the 27-amino-acid form of the pituitary-adenylate-cyclase-activating peptide, PACAP(1-27), and the 38-amino-acid form, PACAP(1-38), were either monosubstituted or bisubstituted at positions 1-3, 20 and 21 or N-terminally shortened. All analogs were compared on human neuroblastoma NB-OK-1 cell membranes for their ability to occupy 125I-[AcHis1]PACAP(1-27)-labelled receptors (AcHis, N alpha-acetylhistidine) and to activate adenylate cyclase (in terms of potency and intrinsic activity). The monophasic slope of dose/effect curves on both parameters suggested interaction with one class of PACAP receptor. Residues 28-38 in the C-terminally extended peptide, PACAP(1-38), played a favorable role in recognition, in that receptors coupled to adenylate cyclase were, in general, more sensitive to PACAP(1-38) analogs than to the corresponding PACAP(1-27) analogs. At variance with PACAP(6-27), PACAP(6-38) was well recognized and acted as a potent competitive antagonist (Ki 1.5 nM). Residues 1-3 were all important in enzyme activation: modification of the beta-turn potential gave full agonists (the LAla2 and DAla2 derivatives) or partial agonists (LPhe2 and DPhe2; LArg2 and DArg2; Glu3 and Asn3). Finally, a proper alpha-helix was also important: the combined substitution of Lys21/Lys22 by Gly21/Gly22 decreased the binding affinity sharply.


Transcript levels for nuclear-encoded mammalian mitochondrial respiratory-chain components are regulated by thyroid hormone in an uncoordinated fashion. Thyroid hormone is one of the few known physiological regulators of mammalian mitochondrial biogenesis. Although it exerts a global effect on biogenesis, it does so by regulating the expression of a limited number of unidentified mitochondrial proteins. We have investigated these hormone-regulated proteins in rat liver. Hormone injection induced a 30-fold increase in the levels of cytochrome-c1 mRNA after 3 d. In addition, the mRNA for the growth-activated adenine-nucleotide translocator, ANT2, was increased 13-fold and that for the ATPase N,N'-dicyclohexylcarbodiimide-binding protein increased 4-5-fold. Mitochondrial transcripts of cytochrome-oxidase subunit I also increased. No changes were found in the mRNA levels for the F1-ATPase beta-subunit or cytochrome oxidase IV. A single low dose of triiodothyronine induces rapid increases in cytochrome-c1 and ANT2 mRNA species which parallel changes in the activity of the hormone-responsive malic enzyme, but are earlier than other mitochondrial biogenetic events. These data strengthen the view that thyroid hormone regulates synthesis of specific components within each respiratory-chain complex and that these products apparently play key roles in inner-membrane biogenesis and assembly. The significance of ANT2 induction is also discussed with respect to the rapid respiratory response induced by thyroid hormone.


ADP receptor-induced activation of guanine-nucleotide-binding proteins in human platelet membranes. ADP receptor-regulated binding of the labeled GTP analog, guanosine 5'-O-(3-[35S]thiotriphosphate) ([35S]GTP[gamma S]), to guanine-nucleotide-binding proteins (G proteins) was studied in human platelet membranes. The potent ADP receptor agonist, 2-methyl-thio-adenosine 5'-diphosphate (2MeSADP), a non-hydrolyzable analog of ADP, increased the binding of [35S]GTP[gamma S] without apparent lag phase. Under optimal conditions, i.e. in the presence of GDP (1-10 microM), 2MeSADP increased the binding up to about threefold, with half-maximal and maximal increase observed at 10 nM and 1 microM 2MeSADP, respectively. ADP itself increased the binding of [35S]GTP[gamma S] by maximally about twofold, with half-maximal increase occurring at 0.1 microM ADP. The agonist-induced stimulation was competitively antagonized by the ADP receptor(s) antagonist, (1S)-adenosine 5'-O-(1-thiotriphosphate) [(Sp)-ATP[alpha S]]. Other platelet receptor agonists known to act through receptors coupled to G proteins also increased binding of [35S]GTP[gamma S] in human platelet membranes, but without being inhibited by (Sp)-ATP[alpha S]. The data presented indicate that the platelet ADP receptor(s) can interact with and efficiently activate G proteins, the nature of which remains to be identified.


Large-scale purification and characterisation of a recombinant epidermal growth-factor receptor protein-tyrosine kinase. Modulation of activity by multiple factors. The human epidermal-growth-factor receptor (EGF-R) is a 170-kDa transmembrane glycoprotein that mediates the mitogenic response of cells to EGF and transforming growth factor alpha. Culture conditions have been developed for the large-scale expression of the cytoplasmic domain of the EGF-R in insect cells using a recombinant baculovirus. From 61 Sf9 cells, grown to high density using a bioreactor, 20 mg of the EGF-R kinase was purified to greater than 95% purity. Purification, which was carried out in the absence of detergents using classical purification methods, yielded an EGF-R protein that was not phosphorylated on tyrosine. This procedure has enabled us to produce high quality enzyme for both structural and biochemical studies on the EGF-R kinase. The in vitro activity of the cytoplasmic domain of the EGF-R kinase was modulated by multiple assay factors which include substrates, divalent cations and conformational modulators. Kinetic analysis in the presence of Mn2+ gave an apparent Vmax value of 20 nmol min-1 mg-1 and Km values of 4.5 microM for ATP and 1.43 mM for angiotensin II. This corresponds to a turnover number of 1.4 mol min-1 mol-1. Ammonium sulfate (1 M) resulted in an eightfold stimulation of kinase activity when assayed using angiotensin II as substrate. The specific activity of the intracellular domain of the EGF-R, when assayed at 20 degrees C in the presence of 1M ammonium sulfate, was 160 nmol min-1 mg-1. Activation of the EGF-R kinase by ammonium sulfate was found to be substrate-specific. No activation was found when assayed using polymeric substrates. Addition of Me(2+)-ATP to the purified enzyme resulted in autophosphorylation and was accompanied by retardation of SDS/PAGE migration. Kinetic constants and metal ion preferences of a number of co-polymers and peptide substrates have been compared. Dramatic differences in kinetic constants were found which were dependent on both the substrate and metal ion used. Activation of EGF-R autophosphorylation was found to be influenced by the use of charged polymers. The random polymer of Glu, Lys, Ala, Tyr (2:5:6:1), which was not phosphorylated by the EGF-R kinase, dramatically activates autophosphorylation of the EGF-R. Thus the intracellular domain of the EGF-R appears to be in a low-activity conformation which, under appropriate assay conditions, can be activated to a similar specific activity to that reported for the purified EGF-R holoenzyme.


Phorbol esters, bombesin and insulin elicit differential responses on the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase system in primary cultures of foetal and adult rat hepatocytes. The effects of 4 beta-phorbol 12-myristate 13-acetate (PMA), bombesin and insulin on 6-phosphofructo-2-kinase (PFK-2) activity, on fructose 2,6-bisphosphate concentration and on the phosphorylation state of PFK-2 were investigated in primary cultures of hepatocytes from foetal and adult rats. Bombesin stimulated PFK-2 activity and increased hexose phosphate (glucose 6-phosphate and fructose 6-phosphate) and fructose 2,6-bisphosphate content in hepatocytes both in the foetal and adult state. However, PMA-treated foetal cells exhibited a marked stimulation in fructose 2,6-bisphosphate concentration and in PFK-2 activity as well as in the content of hexose phosphates, while no response was found in the case of adult hepatocytes. Moreover, the effect of PMA on foetal hepatocytes was suppressed when cells were incubated with cycloheximide, but not when this effect was elicited by bombesin or insulin. These results, and those obtained on the phosphorylation state of PFK-2, suggest that there are different pathways that modulate fructose 2,6-bisphosphate content and, therefore, the control mechanisms of glycolysis and gluconeogenesis at this regulatory step, both in adult and foetal rat liver.


Biochemical characterization of Epstein-Barr virus nuclear antigen 2A and an associated ATPase activity. A partial purification of the Epstein-Barr-virus nuclear antigen 2A (EBNA 2A) protein from the Epstein-Barr-virus-infected lymphoblastoid cell line, Cherry, has been designed. The main purification step was immunoaffinity chromatography, based on the mAb, 115E, directed towards the carboxy terminus of EBNA 2A. This was followed by chromatography over a Blue Sepharose column. According to silver-stained SDS/PAGE, EBNA 2A was estimated to be 20% pure. The purified fractions contained an ATPase activity that was inhibited by the mAb 115E. Immunopurification of six EBNA-2A-positive cell lines and their negative counterpart showed that only fractions from EBNA-2A-positive lines contained ATPase activity. In gel-filtration experiments EBNA 2A eluted as a 75-kDa protein in conjunction with an ATPase activity. The EBNA 2A protein was covalently labeled by the ATP analog [14C]5'-[p-(fluorosulfonyl)benzoyl]adenosine. The ATPase activity was found to be optimal in the presence of 0.25 mM MgCl2 or CaCl2, whereas, in the presence of MnCl2 and ZnCl2, the activity was only about 50% of the control. High concentrations of Na2VO3 and heparin do not interfere with the activity, while 2.5 mM NaF or 0.5 M NaCl give a 50% reduction of the activity. The Km for ATP and for GTP was 13 microM and 11 microM, respectively, and the Vmax for ATP was about six-times higher than with GTP as substrate. Other low-molecular-mass non-protein phosphate esters, such as phosphoserine or phosphothreonine inhibited the ATPase activity with a Ki of 18 and 32 microM, respectively. Phosphotyrosine had a Ki of 480 microM. Serine, threonine and tyrosine had no inhibitory effect on the ATPase activity.


EPR and redox characterization of iron-sulfur centers in nitrate reductases A and Z from Escherichia coli. Evidence for a high-potential and a low-potential class and their relevance in the electron-transfer mechanism. The redox properties of the iron-sulfur centers of the two nitrate reductases from Escherichia coli have been investigated by EPR spectroscopy. A detailed study of nitrate reductase A performed in the range +200 mV to -500 mV shows that the four iron-sulfur centers of the enzyme belong to two classes with markedly different redox potentials. The high-potential group comprises a [3Fe-4S] and a [4Fe-4S] cluster whose midpoint potentials are +60 mV and +80 mV, respectively. Although these centers are magnetically isolated, they are coupled by a significant anticooperative redox interaction of about 50 mV. The [4Fe-4S]1+ center occurs in two different conformations as shown by its composite EPR spectrum. The low-potential group contains two [4Fe-4S] clusters with more typical redox potentials (-200 mV and -400 mV). In the fully reduced state, the three [4Fe-4S]1+ centers are magnetically coupled, leading to a broad featureless spectrum. The redox behaviour of the high-pH EPR signal given by the molybdenum cofactor was also studied. The iron-sulfur centers of the second nitrate reductase of E. coli, nitrate reductase Z, exhibit essentially the same characteristics than those of nitrate reductase A, except that the midpoint potentials of the high-potential centers appear negatively shifted by about 100 mV. From the comparison between the redox centers of nitrate reductase and of dimethylsulfoxide reductase, a correspondence between the high-potential iron-sulfur clusters of the two enzymes can be proposed.


The pattern of growth and growth retardation of patients with hypophosphataemic vitamin D-resistant rickets: a longitudinal study. Growth in height of 16 patients (5 boys and 11 girls) with hypophosphataemic rickets (HR) was studied in a longitudinal survey. The data shortly before and during puberty were analysed on the basis of Preece Baines curves, fitted to the original data; for the analysis at the age of 5 years, the original data were used. It appeared that the overall shape of the individual and average growth pattern could be adequately described by the Preece Baines method. The results further showed that from the age of 5 years onwards, average height was approximately two standard deviations below the normal mean for Dutch children. The patients showed a normal pubertal growth spurt which was, in general, insufficient to restore the growth retardation already established before adolescence. The four children who did show catch-up growth between the age of 5 years and adulthood had minimal rachitic lesions. The greater impact of the disease on growth in early childhood than on adolescent growth could be explained by the fact that HR mainly affects the growth of the legs, the major contributor to body size in early childhood. Finally, it was found that the difference between bone age, as determined by the Tanner Whitehouse (TW2)-method, and chronological age was not significant and the adult height in all patients except two could be adequately predicted from bone age and height.


Effects of novel uracil analogs on proliferation and differentiation of human myeloid leukemia cells. Twenty-seven novel nucleobases and nucleosides were synthesized by structural modification of uracil, and their effects on growth and differentiation of human myeloid leukemia HL-60 cells were examined. Some of the compounds inhibited the growth of HL-60 effectively. The nitroblue tetrazolium (NBT)-reducing activities of cells treated with the concentrations of these compounds for 50% inhibition of growth were compared. TI-66 (2,4-dibenzyl-6-fluoro-7,7,8,8-tetramethyl-cis-2,4-diazabicyclo-[4.2.0] octane-3,5-dione) was the most effective inducer of NBT-reducing activity and morphological differentiation of HL-60 cells into cells of the myelomonocytic lineage. TI-66 was also effective for induction of differentiation of another human myelogenous leukemia cell line, ML-1 cells, but not for differentiation of human erythroid leukemia K562 or HEL cells, or monocytic U937 cells. The effect of TI-66 in inducing differentiation of HL-60 cells was additive or more than additive in combination with retinoic acid or vitamin D3. Adenine or hypoxanthine alone induced NBT-reducing activity of the cells, and at suboptimal concentrations these compounds enhanced the effect of TI-66, but the enhanced NBT-reducing activities did not exceed the maximal activity induced by TI-66 alone. Simultaneous treatment of HL-60 cells with hypoxanthine reduced the growth inhibition by TI-66 alone. TI-66 was about 150 times more potent on a molar basis than adenine in inducing differentiation of HL-60 cells. These results suggest that nucleobase analogs such as TI-66 should be useful for differentiation therapy of some types of myelogenous leukemia.


Effects of tumor necrosis factor-alpha on normal feline hematopoietic progenitor cells. The effects of tumor necrosis factor-alpha (TNF-alpha) on feline bone marrow hematopoietic progenitors were evaluated by exposing bone marrow mononuclear cells from specific pathogen-free cats to different concentrations of TNF-alpha (ranging from 50 to 800 pg/ml) for 2 h before plating for clonal assays of colony-forming units. TNF-alpha caused a dose-dependent suppression of feline erythroid colony-forming units (CFU-E) and erythroid burst-forming units (BFU-E), whereas granulocyte-macrophage colony-forming units (CFU-GM) were minimally affected. TNF-alpha concentrations as low as 200 pg/ml significantly inhibited growth of erythroid progenitors. Addition of polyclonal rabbit anti-TNF-alpha antibodies completely neutralized the suppressive effect of TNF-alpha on erythroid progenitors. At higher concentrations of TNF-alpha (800 pg/ml), 35% of CFU-E and 21% of BFU-E still survived, indicating that some erythroid progenitors are not sensitive to a single exposure of TNF-alpha in vitro. These results suggest that TNF-alpha may play a role in regulating hematopoiesis in cats and may be involved in the pathogenesis of erythroid aplasia in cats infected with feline leukemia virus.


Ultrastructural, immunochemical, and cytochemical study of myeloperoxidase in myeloid leukemia HL-60 cells following treatment with succinylacetone, an inhibitor of heme biosynthesis. Myeloperoxidase (MPO) is a heme-containing glycoprotein found in the primary granules (or azurophilic granules) of human polymorphonuclear leukocytes. In the present study, cultured myeloid leukemia HL-60 cells were exposed for 0-72 h to 250 microM 4,6-dioxoheptanoic acid (succinylacetone, SA), a specific inhibitor of heme biosynthesis, and the effects were evaluated using ultrastructural, immunochemical, and cytochemical methods. En bloc peroxidase staining of glutaraldehyde-fixed cells was accomplished with a 30-min exposure to 3,3'-diaminobenzidine (DAB) tetrahydrochloride. Ultrastructural examination revealed that peroxidase reactivity in the endoplasmic reticulum (ER) was relatively unchanged for 8 h and decreased between 12 and 24 h; however, ER lacked DAB-reactive peroxidase at 48-72 h. Peroxidase reactivity in the ER reappeared within 4 h after removal of SA. Seventy-two hours after exposure to SA the number of condensed cytoplasmic granules stained with DAB was significantly decreased, and many of the granules had a "target" appearance with a central DAB-reactive dense core. Staining of mitochondria was observed with overnight exposure to DAB and persisted in HL-60 cells treated 72 h with SA. Mitochondrial and nuclear morphology appeared unaltered. Immunostaining of MPO in thin sections of paraformaldehyde/glutaraldehyde-fixed unosmicated HL-60 cells, embedded in Lowicryl K4M, was accomplished with sequential exposure to an affinity-purified monospecific rabbit antibody to HL-60-MPO and protein A conjugated to 5- or 10-nm colloidal gold. Compared to untreated control HL-60 cells, cells exposed to SA for 48 h exhibited comparable to increased immunoreactive MPO in the ER, despite the absence of heme-dependent peroxidase reactivity. The data indicate that SA inhibits formation of enzymatically active MPO and that in the presence of SA, the ER contains a form(s) of MPO that lacks enzymatic reactivity.


Combined chemotherapy including rifabutin for rifampicin and isoniazid resistant pulmonary tuberculosis. G.E.T.I.M. (Group for the Study and Treatment of Resistant Mycobacterial Infection). A prospective multicentre open study has been conducted in France in order to assess the efficacy and tolerability of an antimycobacterial regimen including rifabutin in the treatment of patients with pulmonary tuberculosis due to rifampicin and isoniazid resistant bacilli. Patients were treated with daily rifabutin (450-600 mg), associated with companion drugs to which the organisms remained susceptible; in most cases the regimen included a fluoroquinolone. The duration of treatment was initially scheduled for a minimum period of 12 months after sputum culture conversion. Thirty nine patients were enrolled, 23 of whom were treated for at least 12 months. Culture conversion was obtained at the end of the twelfth month in 14 out of 23 patients. Twenty one out of 39 patients experienced adverse events. These were, however, serious enough to discontinue treatment in only four patients. These results suggest that an antimycobacterial combination including rifabutin might contribute to the treatment of multi-resistant pulmonary tuberculosis.


Urinary leukotriene E4 in bronchial asthma. Leukotriene E4 (LTE4) is excreted into the urine in a relatively constant proportion of 4-7% when either leukotriene C4 (LTC4) or LTE4 is intravenously infused, regardless of the magnitude of the infused dose. Measurement of LTE4 in urine is, therefore, a convenient and non-invasive method for assessing changes in the rate of total body sulphidopeptide leukotriene production. We assayed urinary LTE4 in 17 normal subjects, 31 subjects with asthma without aspirin sensitivity, and 10 aspirin-sensitive subjects. The relationship between urinary LTE4 and nonspecific bronchial hyperresponsiveness, as assessed by the provocative dose producing a 20% fall in forced expiratory volume in one second (PD20) to inhaled histamine, was examined in 19 non-aspirin-sensitive asthmatic subjects. The urinary LTE4 values were log-normally distributed. Urinary LTE4 was detected in 28 of the 31 non-aspirin-sensitive asthmatic subjects, and the geometric mean (95% confidence interval (CI) of 43 (32-57) pg.mg-1 creatinine was no different to that of 34 (25-48) pg.mg-1 creatinine measured in the normal subjects. The geometric mean of 101 (55-186) pg.mg-1 creatinine measured in the aspirin-sensitive asthmatics was significantly higher than that measured in the normal subjects (p less than 0.005) and in the asthmatic subjects who were non-aspirin-sensitive (p less than 0.002), but there was considerable overlap between the three groups. There was no relationship between urinary LTE4 and PD20, or between urinary LTE4 and baseline forced expiratory volume in one second (FEV1) (% predicted). Thus, measurement of LTE4 in a single sample of urine will not predict the extent of bronchial hyperresponsiveness or degree of airflow obstruction.


In vivo and in vitro effects of glucocorticosteroids on arachidonic acid metabolism and monocyte function in nonasthmatic humans. Glucocorticosteroids are used as anti-inflammatory agents in a range of diseases, however, their mechanism of action is unknown. Recently, inhibition of arachidonic acid metabolism has been suggested as one possible mechanism of action. A series of experiments were undertaken in nonasthmatic humans to examine the effects of oral prednisolone and dexamethasone and inhaled budesonide on the excretion of the urinary leukotriene E4 (LTE4), an established marker of total body leukotriene generation in vivo. In addition, the effect of the drugs on the in vitro and ex-vivo function of monocytes was examined. In vitro dexamethasone greater than 10(-8) M inhibited the thromboxane A2 (TxA2) release from human monocytes, an effect which recovered within 24 h. In vivo, neither inhaled budesonide (1.6 mg.day-1 for 7 days), nor a standard therapeutic dose of oral prednisolone (30 mg.day-1 for 3 days), nor high doses of oral dexamethasone (8 mg.day-1 for 2 days) altered the excretion of urinary LTE4, despite the latter completely suppressing endogenous cortisol production. The ex-vivo zymosan stimulated release of TxA2 release from monocytes was not altered by the standard dose prednisolone, but was reduced by high dose dexamethasone and inhaled budesonide. This study shows that high doses of systemic steroids have little effect on arachidonic acid metabolism in normal nonasthmatic humans. Inhaled budesonide, however, does reduce arachidonic acid metabolism in circulating monocytes, presumably by affecting these cells during their passage through the lung.


Oxidation of methylamine by a Paracoccus denitrificans mutant impaired in the synthesis of the bc1 complex and the aa3-type oxidase. Evidence for the existence of an alternative cytochrome c oxidase in this bacterium. A Paracoccus denitrificans fbcC-ctaDII double mutant strain impaired in the synthesis of both the bc1 complex and the aa3-type oxidase has been constructed. This mutant strain, which is still able to grow on methylamine as sole carbon and energy source, exhibits unimpaired oxygen consumption with succinate, methylamine and endogenous substrates as electron donors. From kinetic studies of the oxidation and reduction rates of cytochromes c, it can be concluded that P. denitrificans contains a second cytochrome c oxidase, different from the aa3-type.


Polymerase chain reactions using Saccharomyces, Drosophila and human DNA predict a large family of protein serine/threonine phosphatases. Using the polymerase chain reaction (PCR) to examine the protein serine/threonine phosphatase (PP) family which includes PP1, PP2A and PP2B, we have identified two, seven, and four novel protein phosphatase genes in Saccharomyces cerevisiae, Drosophila melanogaster and Homo sapiens, respectively. Consequently, the genes in the PP1/PP2A/PP2B family now number 11, 15 and 12 in these species respectively, and the data predicts still more unidentified phosphatases in higher eukaryotes. The PCR analyses also point to the presence in Drosophila and mammals of three or more different genes encoding PP2B, the enzyme recently identified as the target of certain immunosuppressant drugs.


The lysine-rich H1 histones from the slime moulds, Physarum polycephalum and Dictyostelium discoideum lack phosphorylation sites recognised by cyclic AMP-dependent protein kinase in vitro. Calcium chloride-extracted histones were prepared from nuclei of the slime moulds, Physarum polycephalum and Dictyostelium discoideum, and phosphorylation by purified preparations of cyclic AMP-dependent protein kinase (cAMP-d PK) and growth-associated H1 histone kinase (HKG) examined and compared. Among the major histone fractions and other proteins in the two preparations, the H1 histones from both organisms were found to be effective and exclusive substrates for HKG. cAMP-d PK, which phosphorylates mammalian H1 histone and certain, in particular H2B, of the mammalian core histones, phosphorylated several of the core histones from both slime moulds but did not phosphorylate H1 histone from either. The slime mould H1s remained ineffective substrates for cAMP-d PK even after extensive alkaline phosphatase treatment of the histone preparations. Additional studies demonstrated that the lack of slime mould H1 phosphorylation by cAMP-d PK was not due to competition of the H1 molecules with the core histones for the kinase. Our studies suggest that H1 histones from these organisms, whilst clearly containing sites for phosphorylation by HKG, apparently lack phosphorylation sites recognised by cAMP-d PK. Thus, the mediation of specific nuclear functions by cAMP-dependent phosphorylation of H1 in higher organisms may not occur or be required in these lower eukaryotes.


Cyclin A-Cys41 does not undergo cell cycle-dependent degradation in Xenopus extracts. Truncated cyclin A and cyclin B lacking the N-terminal domain comprising the 'destruction box' escape from proteolysis and arrest cells at metaphase. Mutation of a conserved arginine residue of the destruction domain makes cyclin B resistant to proteolysis. Here we show that mutation of the same residue also makes cyclin A resistant to proteolysis, in either of two situations in which the cyclin degradation pathway is turned on: (i) in Xenopus extracts of activated eggs where the degradation pathway has been permanently turned on by adding a recombinant undegradable cyclin B in which the arginine residue of the destruction box has been substituted by alanine; (ii) in extracts of metaphase II-arrested oocytes after Ca(2+)-dependent inactivation of the cytostatic factor (CSF).


A G-rich sequence element common to Dictyostelium genes which differ radically in their patterns of expression. Removal of a G-rich element from the DIF-inducible, prestalk-, and stalk-specific ecmB gene reduces expression but cell-type specificity is retained. The ecmB element will functionally substitute for a homologous sequence upstream of CP2, a cAMP-inducible gene and is bound by GBF, the factor which interacts with the CP2 G box. These results suggest that the G box may play a similar stimulatory role in these two independently regulated genes where it presumably interacts with different ancillary promoter elements.


Expression of the fibroblast growth factor receptor FGFR-1/flg during gastrulation and segmentation in the mouse embryo. Recent evidence from studies in both amphibians and mammals suggest that the fibroblast growth factor (FGF) family of signaling molecules and their receptors may play regulatory roles during early embryogenesis. We have used both standard and whole-mount in situ hybridization techniques to analyze the temporal and spatial expression patterns of the murine fibroblast growth factor receptor-1 (FGFR-1) in order to help define the role of FGFs in the processes of gastrulation and segmentation. FGFR-1 transcripts were detected in the primitive ectoderm of the egg cylinder embryo but not in the primitive endoderm or ectoplacental cone. During gastrulation, FGFR-1 mRNA were expressed at high levels in the migrating embryonic mesoderm of the mid-streak-stage embryo. Late-streak-stage embryos displayed strong expression in both the embryonic ectoderm and mesoderm. Within the ectodermal lineage, FGFR-1 mRNA later became localized to the neural ectoderm during its formation and continued to be expressed at high levels throughout neural development. In the mesodermal lineage, FGFR-1 transcripts became concentrated in the posterior medial mesoderm of the embryo as it condensed to form paraxial mesoderm. The most striking expression patterns were observed before and during segmentation where FGFR-1 was strongly expressed in the presomitic mesoderm and the rostral half of the newly formed somites. The patterns of expression are consistent with a role for FGFR-1 in posterior mesoderm formation. FGFR-1 may also play significant roles in the formation of neural ectoderm and the early events that establish compartments within the developing somites.


Phosphoinositide metabolism in the developing conceptus. Effects of hyperglycemia and scyllo-inositol in rat embryo culture. Culture of the postimplantation rat conceptus in hyperglycemic medium causes developmental abnormalities and is associated with a diminished water-soluble myo-inositol content. We investigated the effect myo-inositol depletion has on lipid-soluble phosphoinositides, precursors, and water-soluble inositol phosphates. Rat conceptuses were cultured from gestational day 9.5 (presomite, early head fold) to day 10.5 (7-15 somites) in 6.7-73.3 mM D-glucose. Significant decreases in the phosphoinositides of the embryo were observed with increased culture D-glucose concentrations. PI was reduced 15-34%, PIP 18-46%, and PIP2 26-46%. Yolk sac phosphoinositides also were reduced but to a lesser degree. Culture in hyperglycemic media also mediated significant reductions of conceptus inositol phosphates. To investigate whether effects similar to those induced by D-glucose could be mediated by another agent capable of decreasing myo-inositol content, we used scyllo-inositol, a transported but nonmetabolized isomer of myo-inositol. Conceptuses cultured in medium containing scyllo-inositol (0.06-16.7 mM) had dose-dependent decreases of myo-[3H]inositol in water-soluble and lipid-soluble fractions. Incorporation of myo-[3H]inositol into phosphoinositides and inositol phosphates was decreased concomitantly. Developmental effects of D-glucose and scyllo-inositol were assessed in rat conceptuses cultured from day 9.5 (presomite, early head fold) to day 11.5 (22-28 somites). Culture in 40.0-73.3 mM glucose and 0.06-33.3 mM scyllo-inositol impaired growth while increasing dysmorphogenesis in a dose-dependent manner. The results suggest that decreases in conceptus myo-inositol and associated diminution of phosphoinositides, which are the inositol/lipid cycle precursors, are dysmorphogenic and may contribute to the etiology of diabetic embryopathy.


Disruption of a silencer domain by a retrotransposon. A galactose-inducible Ty element carrying the HIS3 gene has been used as an insertional mutagen to generate alpha-factor resistant mutants. This collection of Ty-induced mutations includes insertions into the gene for the alpha-factor receptor (STE2), several nonspecific STE genes, and mutations that lead to the expression of the normally silent HML alpha locus. The hml alpha "on" mutations fall into two classes, those that disrupt trans-acting regulators involved in silencing HML alpha and a novel class of mutations that activate HML alpha by insertion at that locus. The hml alpha::Ty "on" mutations illustrate the unusual ability of these retrotransposons to activate genes by overcoming gene silencing mechanisms. The hml alpha::Ty "on" mutations include examples of multimeric Ty arrays. Single Ty and solo delta insertion derivatives of these Ty multimers restore the ability of the silencing mechanism to repress HML alpha.


A genetic mapping system in Caenorhabditis elegans based on polymorphic sequence-tagged sites. We devised an efficient genetic mapping system in the nematode Caenorhabditis elegans which is based upon the differences in number and location of the transposable element Tc1 between the Bristol and Bergerac strains. Using the nearly completed physical map of the C. elegans genome, we selected 40 widely distributed sites which contain a Tc1 element in the Bergerac strain, but not in the Bristol strain. For each site a polymerase chain reaction assay was designed that can distinguish between the Bergerac Tc1-containing site and the Bristol "empty" site. By combining appropriate assays in a single reaction, one can score multiple sites within single worms. This permits a mutation to be rapidly mapped, first to a linkage group and then to a chromosomal subregion, through analysis of only a small number of progeny from a single interstrain cross.


latheo, a new gene involved in associative learning and memory in Drosophila melanogaster, identified from P element mutagenesis. Genetic dissection of learning and memory in Drosophila has been limited by the existence of ethyl methanesulfonate (EMS)-induced mutations in only a small number of X-linked genes. To remedy this shortcoming, we have begun a P element mutagenesis to screen for autosomal mutations that disrupt associative learning and/or memory. The generation of "P-tagged" mutant alleles will expedite molecular cloning of these new genes. Here, we describe a behavior-genetic characterization of latheoP1, a recessive, hypomorphic mutation of an essential gene. latheoP1 flies perform poorly in olfactory avoidance conditioning experiments. This performance deficit could not be attributed to abnormal olfactory acuity or shock reactivity-two task-relevant "peripheral" behaviors which are used during classical conditioning. Thus, the latheoP1 mutation appears to affect learning/memory specifically. Consistent with chromosomal in situ localization of the P element insertion, deficiencies of the 49F region of the second chromosome failed to complement the behavioral effect of the latheoP1 mutation. Further complementation analyses between latheoP1 and lethal alleles, produced by excision of the latheoP1 insert or by EMS or gamma-rays, in the 49F region mapped the latheo mutation to one vital complementation group. Flies heterozygous for latheoP1 and one of two EMS lethal alleles or one lethal excision allele also show the behavioral deficits, thereby demonstrating that the behavioral and lethal phenotypes co-map to the same locus.


The Ac and Uq transposable element systems in maize: interactions among components. Components of the Uq and Ac transposable element systems interact. A large sample of Ds-containing and ruq-containing alleles were tested against Uq and Ac. The Uq elements elicit a mutable response from only one of the classes of Ds elements (Ds1) in the Ac family. This response is similar to the response from ruq to Uq. In contrast, Ac elicits mutable responses from all Ds and ruq elements tested. This represents a lack of reciprocity of interaction for the components of the two elements, Ac and Uq. Further, two atypical Ac and Uq elements (Ac2 and Uq-Mn) were examined. All ruq and Ds elements tested respond to four doses of Ac2. (Responses to lower doses were not compared.) Only the ruq (Ds1) containing alleles respond to Uq-Mn. The other Ds containing alleles were nonresponsive. The finding of nonreciprocating interaction between components suggests a heterogeneous nature for transposable element systems in maize.


Production of human papillomavirus and modulation of the infectious program in epithelial raft cultures. OFF. Human papillomaviruses trophic for anogenital epithelia cause benign warts, and certain genotypes are closely associated with cervical neoplasia. By using our modifications of the epithelial raft culture system, we were able to recapitulate and modulate the infectious program of a papillomavirus in vitro for the first time. Small pieces of a condyloma containing human papillomavirus type 11 were explanted onto a dermal equivalent consisting of a collagen matrix with fibroblasts and were cultured at the medium-air interface. The infected stem cells proliferated rapidly across the matrix, stratified, and differentiated, as judged by histology. The results correlated with the state of epithelial differentiation, which, in turn, was dependent on the type of fibroblast in the matrix. Under conditions where the epithelial outgrowth underwent terminal differentiation, the entire productive program took place, leading to virion assembly. In contrast, using an alternative condition where the outgrowth failed to achieve terminal differentiation, only the E-region RNAs from the E1 promoter accumulated to any appreciable extent. The proliferating cell nuclear antigen was induced in the differentiated suprabasal cells in the productive cyst growth, which also exhibited high copy viral DNA and abundant E6-E7 RNAs. Comparable cells in the nonproductive cyst outgrowth were negative for all three. These results suggest that the E6 and E7 proteins may play a role in establishing a cellular environment conducive to vegetative viral replication. The culture conditions described should be useful for genetic analysis of this family of important human pathogens and for testing potential pharmacological agents.


Trans and cis requirements for intron mobility in a prokaryotic system. Intron mobility requires cleavage of an intronless allele by an intron-encoded endonuclease, followed by transfer of the intron into the cleaved recipient. The mobile phage introns provide an opportunity to identify accessory functions involved in the intron inheritance process. To test for trans and cis requirements of mobility in Escherichia coli, we have exploited the td intron of phage T4 in both phage T4 and lambda genetic backgrounds. Mobility depends on host or phage recombinase functions, RecA or UvsX, respectively. The process also requires a phage-encoded 5'----3' exonuclease activity and associated annealing function that can be provided by phage lambda. Finally, host-encoded 3'----5' exonuclease activities are also implicated in intron inheritance. We demonstrated further that restriction enzymes could substitute for the intron-encoded endonuclease, indicating that the endonuclease does not have an essential role in recombination. Neither the precise position nor the nature of the double-strand break was critical to intron transfer. These features provide insight into the recombination pathway and are factors impacting on the spread of introns throughout natural populations.


Topoisomerases and yeast rRNA transcription: negative supercoiling stimulates initiation and topoisomerase activity is required for elongation. Previous work has shown that rRNA synthesis is strongly inhibited in yeast top1-top2 double mutants. Here, we show that inactivation of yeast topoisomerases can have paradoxical effects on transcription by RNA polymerase I. For example, transcription of ribosomal minigenes on extrachromosomal plasmids is greatly stimulated in top1-top2 cells while accumulation of full-length endogenous rRNA is strongly inhibited. We present evidence for a mechanism that can partly account for these opposing effects on transcription. On the one hand, transcription initiation can be stimulated owing to an accumulation of negative superhelicity because polymerase I prefers to initiate on negatively supercoiled templates. Conversely, synthesis of full-length rRNA is inhibited owing to the fact that chain elongation requires a DNA relaxing activity.


New IS10 transposition vectors based on a gram-positive replication origin. We describe below a set of plasmid-based vehicles which can be used for delivery of IS10-derived transposons into Gram- bacteria. These vehicles replicate via a Gram+ plasmid origin that is inactive in Escherichia coli; they are easily maintained in Bacillus subtilis. Transposons are introduced by electroporation or transformation with the plasmid, and as in previous delivery systems, transpositions are selected with the appropriate antibiotic. This system should be particularly useful in situations where the standard delivery vehicles, based on bacteriophage lambda, are inappropriate. The system described incorporates a number of useful features: a variety of antibiotic markers (Er, Cm, Km or Tc), a polylinker containing restriction sites for rare-cutting endonucleases to facilitate physical mapping of chromosomal insertions, a mutant transposase that confers a relaxation in insertion specificity and positioning of the transposase-encoding gene outside of the transposing segment to ensure the stability of insertions once isolated.


Oxidation of 3-hydroxykynurenine catalyzed by methemoglobin with hydrogen peroxide. Methemoglobin (metHb) with H2O2 catalyzed the oxidation of 3-hydroxykynurenine (3-HKY) in the reaction mixture of metHb, 3-HKY, and H2O2. The spectrophotometric experiments suggest the following mechanism for the 3-HKY oxidation by metHb with H2O2. MetHb first reacts with H2O2 to form the ferryl complex of Hb. This species then oxidizes 3-HKY, while it returns to metHb. 3-HKY was more reactive with the ferryl complex than glutathione but less reactive than ascorbic acid. Scavengers of the hydroxyl radical, dimethyl sulfoxide and ethanol, scarcely inhibited the 3-HKY oxidation by metHb with H2O2. Desferrioxamine, a metal chelator, hardly suppressed the 3-HKY oxidation. These results indicate that the hydroxyl radical is not involved in the 3-HKY oxidation by metHb with H2O2.


Aluminum(III) facilitates the oxidation of NADH by the superoxide anion. Al(III) augments the oxidation of reduced nicotinamide adenine dinucleotide (NADH) by enzymatic or photochemical sources of O2-. Superoxide dismutase, but not catalase, inhibited this action of Al(III). It thus appears that Al(III) forms a complex with O2-, which is a stronger oxidant than is O2- itself and which may contribute to the adverse biological effects of Al(III).


Intracellular free iron in liver tissue and liver homogenate: studies with electron paramagnetic resonance on the formation of paramagnetic complexes with desferal and nitric oxide. Treatment of intact liver and liver homogenate with sodium nitrite, or desferal, brings about the appearance of g = 2.03 and g = 4.3 electron paramagnetic resonance spectroscopy (EPR) signals, respectively. The g = 2.03 signal is conditioned by the formation of dinitrosyl complexes of Fe(II); the g = 4.3 signal is related to the appearance of paramagnetic desferal-Fe(III) complexes. Desferal and sodium nitrite were administered successively into liver homogenate, resulting in only a g = 4.3 EPR signal. And, vice versa, if desferal was administered after sodium nitrite, there appeared only the signal with g = 2.03. These data testify to the fact that one and the same endogenous free iron is included in both paramagnetic centers. The concentration of iron ions was measured in intact tissue according to the formation of dinitrosyl-iron complexes and desferal-iron complexes. It was 33.2 +/- 4.6 and 20.3 +/- 4.0 nmol/g of tissue weight, respectively. The data obtained testify to the fact that free endogenous iron is present in intact tissue. Possibilities of the EPR method for estimation of the content of intracellular free iron are discussed.


Inactivation of rabbit muscle creatine kinase by hydrogen peroxide. The effects of xanthine + xanthine oxidase-generated reactive oxygen species (ROS) on rabbit muscle creatine kinase (CK) were studied. Xanthine (0.1 mM) + xanthine oxidase (30 mU/ml) inhibited activity of rabbit muscle CK (1.2 mU/ml). Catalase (100 U/ml), but not SOD (100 Uml), deferoxamine (100 microM) or mannitol (20 mM), protected CK from inactivation; suggesting that H2O2 was responsible for inactivation. These results were different from previously reported findings on bovine heart CK that superoxide radicals inactivate the enzyme. Thus, enzymes with homologous structures may have different reactivities to different ROS. H2O2-induced inactivation of rabbit muscle CK was accompanied by a decrease in its thiol group content, whereas no significant changes in the protein structure were detected by SDS-PAGE or carbonyl content. These results suggest that oxidation of -SH groups by H2O2 seems to be a major mechanism of activation of rabbit muscle CK by xanthine + xanthine oxidase. Such inactivation of CK by H2O2 may be important in ROS-induced pathology.


The measurement of oxidative damage to DNA by HPLC and GC/MS techniques. Oxidative damage to DNA has been measured by quantitating 8-hydroxy-2'-deoxyguanosine (8-OHdGuo) after enzymic digestion of DNA, followed by HPLC separation and electrochemical detection. Alternatively, 8-hydroxyguanine (and a wide range of other base-derived products of free radical attack) may be measured after acidic hydrolysis of DNA or chromatin, followed by derivatization and gas-chromatography/mass spectrometry. Both techniques have comparable sensitivity, but GC/MS enables determination of a wide variety of chemical changes to all four DNA bases and it can be applied to DNA-protein complexes. However, the two techniques do not always give similar results. Potential reasons for this are discussed. Greater attention to methodological questions is required before using measurement of 8-OHdGuo as a "routine" marker of oxidative DNA damage in vivo.


Oxidative stress in lungs of mice infected with influenza A virus. As oxidative stress has been implicated in the pathogenesis of certain viral diseases we determined antioxidant and prooxidant parameters in lungs and bronchoalveolar lavage fluid (BALF) of mice infected with a lethal dose of influenza A/PR8/34 virus. Viral infection was characterized by massive infiltration of leukocytes, mainly polymorphonuclear leukocytes, into the alveolar space. The total number of BALF cells increased up to 8-fold (day 3 post-infection) and these cells appeared activated as judged by their increased rates of superoxide anion radical (O2-.) generation upon stimulation. Maximal rates of radical generation by BALF cells during the early stages of infection were 15- or 70-fold higher than those of cells from control animals when expressed per cell or total BALF cells, respectively. At the terminal stages of infection the total capacity of BALF cells to release O2-. declined to approximately 35-fold the control values. Infection also resulted in increased in vivo formation of hydrogen peroxide (H2O2) within the lungs at a time that coincided with the maximal capacity of BALF cells to release O2-.. Whereas pulmonary activities of glutathione peroxidase and reductase remained unaltered, levels of ascorbate in the cell-free BALF decreased significantly during the early stages of the infection and then returned to normal levels and above, late in infection. The oxidation state of the dehydroascorbic acid/ascorbate couple increased concomitantly with the decrease in ascorbate concentrations early in infection and remained elevated throughout the infection. As assessed by the prevention of peroxyl radical-induced loss of phycoerythrin fluorescence, the total antioxidant capacity present in lung tissue homogenate from terminally ill animals was not diminished when compared to that prepared from lungs of control mice. We conclude that although early stages of influenza infection are associated with the presence of oxidative stress in the lung tissue and alveolar fluid lining the epithelial cells, this stress does not appear to overwhelm local antioxidant defenses. The results therefore do not support a direct causative role of oxidative tissue damage in the pathogenesis of influenza virus infection.


Sodium lactate reversal of electrophysiological effects of imipramine in guinea-pig ventricular myocardium. Overdose cardiac effects of imipramine are due to fast Na channel blockade and are clinically reversed by administration of sodium lactate which induces alkalosis (about pH 7.50) and hypernatremia (about 8 mM). The mechanisms of this beneficial effect of Na lactate were explored in vitro on guinea-pig ventricular myocardium using the microelectrode technique. The time-course effects of the clinically relevant concentration of 10 microM imipramine on action potential characteristics were examined at pH 7.20 and pH 7.50. To test whether alkalinisation per se is important or whether an increase in Na concentration plays a major role in the reversal effect, preparations were exposed to increasing concentrations (1, 3, 10, 30, 100 mM) of either Na lactate, bicarbonate or chloride in the absence or in the presence of 10 microM imipramine at pH 7.50. The influence of elevating osmolality was evaluated with equivalent concentrations of sucrose. Imipramine alone significantly depressed Vmax and shortened action potential duration at all phases of repolarisation. All three high sodium solutions reversed imipramine effects. However the reversal effect was already obvious with 10 mM Na lactate and 10 mM NaHCO3 but not 10 mM NaCl. Osmolality did not reverse the imipramine-induced Vmax depression. The results suggest that at the clinically relevant 10 mM concentration, sodium lactate and bicarbonate may displace imipramine from its receptor site on the Na channel by causing alkalosis at the membrane level without profoundly affecting the driving force of the Na current, whereas at the upper concentrations, the increase in Na ion concentrations is predominantly involved in the reversal of imipramine effects.


Contractile effect of alpha,beta-methylene ATP on the guinea-pig isolated trachea. The effects of alpha,beta-methylene adenosine 5'-triphosphate (alpha,beta-methylene ATP), 2-chloroadenosine and R-PIA (N6-(L-2-phenylisopropyl)-adenosine) and their interaction with dipyridamole, indomethacin, 8-phenyl-theophylline, diazepam and other agonists of central or peripheral benzodiazepine receptors were studied on the guinea-pig isolated trachea. alpha,beta-methylene ATP exerted contractile effects on the guinea-pig isolated trachea; - log EC50 and E(max) values were 8.86 +/- 0.19 and 31.3 +/- 2.3 (n = 31) (% vs acetylcholine 10(-3) M) respectively. In comparison with other purinergic receptor agonists, the rank order of potency was: alpha,beta-methylene ATP greater than 2-chloroadenosine greater than R-PIA. alpha,beta-methylene ATP and 2-chloroadenosine had significantly (P less than 0.05) greater efficacy (E(max)) than R-PIA. Indomethacin (3 x 10(-6) M) and 8-phenyltheophylline (10(-7) to 10(-5) M) significantly reduced the contractile effect of 2-chloroadenosine and R-PIA but did not affect alpha,beta-methylene ATP-induced contraction. Conversely, dipyridamole significantly reduced (10(-7) M) or suppressed (10(-6) M) the contractile effects of alpha,beta-methylene ATP whereas it only partially reduced (10(-6) M) the contractile effects of high concentrations of 2-chloroadenosine or R-PIA. Diazepam potentiated the efficacy of alpha,beta-methylene ATP. The E(max) (% vs acetylcholine 10(-3) M) values were 26.1 +/- 2.0 (n = 10) in control conditions and 45.9 +/- 4.6 (n = 5; P less than 0.05) in the presence of diazepam 10(-5) M. Diazepam did not modify the contractile effects of 2-chloroadenosine or R-PIA. Ro5-4864 (10(-7) to 10(-5) M), an agonist of peripheral benzodiazepine receptors, potentiated the contractile effects of alpha,beta-methylene ATP to the same extent as diazepam. Clonazepam, an agonist of central benzodiazepine receptors, did not modify these effects. Antagonists of central (flumazenil) or peripheral (RP 52028) benzodiazepine receptors had no influence on the interaction between diazepam or Ro-4864 and alpha,beta-methylene ATP. In conclusion, alpha,beta-methylene ATP exerts on guinea-pig isolated trachea a contractile effect which is not modified by indomethacin and 8-phenyltheophylline, but is reduced by dipyridamole. It is suggested that this effect might involve P2 chi receptor stimulation.


Imbalance between tumour necrosis factor-alpha and soluble TNF receptor concentrations in severe meningococcaemia. The J5 Study Group. The extracellular domain of tumour necrosis factor-alpha (TNF-alpha) receptors have inhibitory properties against TNF-alpha. The relative ratio between ligand and ligand inhibitors may influence the outcome of meningococcaemia. To test this hypothesis, levels of TNF-alpha and of each of the soluble inhibitory fragments originating from two distinct TNF-alpha receptors (sTNF-RI and sTNF-RII) were measured in sera of children with severe meningococcaemia. On admission to the hospital the levels of sTNF-RI, -RII and TNF-alpha were markedly increased and all three correlated with the outcome of the disease. A correlation was found between TNF-alpha and sTNF-RI (P less than 0.001 by Pearson rank correlation coefficient) or sTNF-RII (P = 0.012). For TNF-alpha concentrations below 500 pg/ml, the increase of TNF-alpha was proportional to that of sTNF-RI and RII; however, when TNF-alpha levels exceeded 500 pg/ml, sTNF-RI and RII concentrations did not increase proportionally. At admission, in patients with fatal outcome, the ratios TNF-alpha/sTNF-RI and -RII were higher than in survivors. During the first 6 hr, the kinetics of TNF-alpha, sTNF-RI and -RII were different. Naturally occurring TNF-alpha inhibitors may play an important role in modulating the biological activity of TNF-alpha in severe meningococcaemia.


C-reactive protein (CRP) peptides inactivate enolase in human neutrophils leading to depletion of intracellular ATP and inhibition of superoxide generation. The nature and the biochemical mechanism of inhibition of neutrophil membrane-associated oxidative metabolism by two synthetic peptides p77-82 and p201-206 (amino acid sequences Val-Gly-Gly-Ser-Glu-Ile and Lys-Pro-Gln-Leu-Trp-Pro respectively, from the primary amino acid sequence of C-reactive protein) have been ascertained. Preincubating neutrophils for 15 min with 50 microM of p77-82 or p201-206 resulted in superoxide generation by opsonized zymosan stimulated neutrophils being inhibited by 34 +/- 2% (P less than 0.005) and 29 +/- 2% (P less than 0.005) respectively. With a 60-min preincubation period 6.25 microM of p77-82 or p201-206 was effective in inhibiting this superoxide generation by 12 +/- 2% (P less than 0.01) and 10 +/- 1% (P less than 0.01) respectively. Neither peptide inhibited neutrophil arachidonic acid release, transmembrane potential or transductional events preceding superoxide generation. Inhibition of neutrophil functions was found to be due to the ability of each peptide (50 microM) following a 15-min preincubation period to inhibit both neutrophil glycolysis and ATP generation by approximately 30%. The inhibition of ATP generation and glycolysis in neutrophils is attributable to the ability of these peptides to inhibit uncompetitively the glycolytic enzyme enolase. Using purified enolase the relative Ki values for p77-82 and p201-206 were 27 and 19 microM respectively. Inhibition of neutrophil function by the peptides is concluded to be due to effective interference of neutrophil energy metabolism.


Effects of mucoid and non-mucoid Pseudomonas aeruginosa isolates from cystic fibrosis patients on inflammatory mediator release from human polymorphonuclear granulocytes and rat mast cells. Mucoid Pseudomonas aeruginosa causing chronic bronchopulmonary infection in cystic fibrosis (CF) patients may interfere with host defence mechanisms. We investigated 13 P. aeruginosa strains isolated from sputa of CF patients with regard to the induction or modulation of inflammatory mediator release from human neutrophils (PMN) and rat mast cells. The effects of mucoid as compared to non-mucoid bacteria were studied using a mucoid strain and its non-mucoid revertant. The release of leukotrienes (LT) and histamine in response to the majority of the CF strains was insignificant. However, preincubation of PMN with P. aeruginosa caused a dose-dependent decrease (50-95%) of LTB4 and LTC4 generation and LTB4 metabolism induced by the Ca(2+)-ionophore A23187 or opsonized zymosan (ZX) (P less than 0.001). The mucoid strains caused a three- to 10-fold higher impairment of LTB4 release (P less than 0.05) and a concomitant down-regulation of LTB4 receptors on neutrophils. Inhibitory effects were also obtained for mucoid and non-mucoid bacteria when the phorbol-ester or the Ca(2+)-ionophore induced luminol enhanced chemiluminescence response (P less than 0.001) or the histamine release from rat peritoneal mast cells (P less than 0.01) was studied. The bacteria-cell contact with non-mucoid strains was associated with an increased Ca2+ influx into PMN, whereas mucoid bacteria had no effect. In addition, a protein kinase C-dependent decrease of the C3bi receptor was suppressed by the mucoid--and less effectively--by the non-mucoid strain. The results suggest that the impairment of the phagocytic and inflammatory system may contribute to the pathogenesis and persistence of mucoid P. aeruginosa infection in CF.


The effect of potassium on the uptake of 137Cs in food crops grown on coral soils: coconut at Bikini Atoll. The soils of Bikini Atoll (11 degrees 35'N, 165 degrees 25'E) were contaminated by fallout from a thermonuclear explosion in 1954. Today, in the absence of any treatment, intake of 137Cs via the terrestrial food chain could account for 70% of the radiological dose received by a returning population. Therefore, we examined the effectiveness of potassium applications, alone and combined with nitrogen and phosphorus, in reducing 137Cs uptake by coconut (Cocos nucifera L.), a major food crop. Mean pretreatment concentrations of 137Cs in "drinking-nut" meat of approximately 17-y palms ranged from approximately 0.5 to 7.0 kBq kg-1 (approximately 14 to 190 pCi g-1) (wet mass) in the seven primary experiments reported. These values were reduced to approximately 0.06 to approximately 1.0 kBq kg-1 (approximately 1.6 to approximately 27 pCi g-1) following soil additions of potassium-chloride at rates from 670 to 6270 kg potassium ha-1. Major reductions were complete within 9-12 mo after single large applications and persisted for at least 3 y. Proportional reductions in associated drinking-nut fluid and in mature "copra nut" meat from the same palm also occurred. Comparable but shorter-lived reductions occurred in grass and herbaceous species beneath the palms. A combined nitrogen-phosphorus treatment had no additive effect in the presence of potassium but, by itself, reduced plant uptake by about 50%. The persistence of this effect suggested that phosphorus alone was the major factor. Periodic addictions of potassium at rates of approximately 1000 kg ha-1 would provide a feasible and highly effective means of reducing 137Cs in coconut food products.


Detection and quantitation of 17F formation during the cyclotron production of 13N. The positron emitter 17F has been identified for the first time as a radioactive contaminant formed during 13N production. It was determined that 17F made up 18% of the short-lived radioactivity previously thought to be exclusively 15O. Mathematical deconvolution of decay curve data was employed as a complement to radioanalytical techniques. The nuclidic composition of the effluents from the proton bombardment of (a) water, (b) dilute ethanol and (c) 13C powder/water slurry was determined and compared with previous literature values.


Design and performance of a mass spectrometric facility for measuring helium isotopes in natural waters and for low-level tritium determination by the 3He ingrowth method. The design and performance of a mass spectrometric system for the measurement of helium isotopes and very low tritium concentrations in natural waters are described and discussed in the light of analytical precision and accuracy. The system consists of a VG 3000 mass spectrometer with a fully automated inlet system for preparation and purification of the samples. Along with this mass spectrometric system, different custom-fabricated units are described, especially designed for taking samples, extracting helium or degassing tritium samples prior to the mass spectrometric analysis. The 3He detection limit of the system is close to 10(-16) cm3 STP corresponding to a tritium level of 0.003 TU for a 500 g water sample stored six months for 3He regrowth. A vertical oceanic tritium profile from the south hemisphere is presented as an illustration of the system's capability to detect very low tritium concentrations in the environment.


Preparation of the renal function and imaging agent 99mTc-MAG3 starting from S-unprotected mercaptoacetyltriglycine. A new approach that obviates inconvenient heating of the labelling solution when preparing the renal function and imaging agent 99mTc-MAG3 is described. The labelling procedure involves reduction of 99mTc-generator eluate with stannous chloride in alkaline solution in the presence of S-unprotected mercaptoacetyltriglycine (MAG3) and the co-ligand sodium tartrate, followed by neutralization with a phosphate buffer solution. After optimization of the kit preparation the radiochemical purity of the radiopharmaceutical amounts to greater than 98%. During the labelling process intermediate Tc-species occur.


Production of fluorine-18 labeled (3-N-methyl)benperidol for PET investigation of cerebral dopaminergic receptor binding. The multi-millicurie synthesis of the D-2 receptor ligand [18F](3-N-methyl)benperidol (NMB; 1-[3-(4'- [18F]fluorobenzoyl)propyl]-4-(2-keto-3-methyl-1- benzimidazolinyl)piperidine) is described. [18F]NMB was produced via a 3-step reaction sequence with an overall radiochemical yield of 5-10% and a specific activity greater than 3000 Ci/mmol within 100 min. In vitro binding assays indicated that NMB has high affinity for D-2 receptors in primate brain (K1 = 3.6 nM), with a receptor specificity exceeding that of spiperone. The technique described here permits the routine production of 10-20 mCi of this promising radiopharmaceutical for PET study of D-2 receptor binding in vivo.


Cytomegalovirus-induced flare of systemic lupus erythematosus. Exacerbations of systemic lupus erythematosus (SLE) may be brought about by a number of factors, including stress, surgery, or infection. By interacting with immunologic mediators or by producing immune complexes, infections may exacerbate underlying autoimmune diseases such as SLE. This illustrative case report describes a woman who had SLE for 20 years who contracted cytomegalovirus mononucleosis that induced an exacerbation of her SLE.


Site-specific incorporation of [125I]iododeoxyuridine into DNA. A procedure for the incorporation of [125I]IdU into specific sites in DNA is described. The approach depends upon attachment of radioiododeoxyuridine to a controlled pore glass support which is then used for automated synthesis of an oligomer. The resulting oligomer, containing a terminal 3'[125I]iododeoxyuridine, is used as a primer during DNA synthesis catalyzed by the Taq polymerase employing thermal cycling. The product formed includes the radioiodonucleotide at a single internal site determined by the length of the oligomer.


Differential elimination of radioiodine from single- and double-stranded 125I-DNA. [125I]Iododeoxyuridyl residues in heat-denatured I-DNA underwent deiodination in solutions of sodium bisulfite. In 0.5 M bisulfite containing 1 M trimethylamine, 92% of the iodine was released from denatured DNA in 2.5 min at 60 degrees C. By contrast iodine in native I-DNA remained stably bound under the same condition. The deiodination of [125I]I-deoxyuridine residues incorporated by enzymatic copying of oligomers 60 nucleotides in length was found to be pairing dependent. While 125I in iododeoxyuridine paired to deoxyadenosine in a complementary oligomer remained approximately equal to 90% covalently bound in the presence of 1 M bisulfite at 60 C, when the iodouridylate residue was mismatched greater than 96% was eliminated in 4 min. The presence of mismatches in the two nucleotides adjacent to a [125I]iododeoxyuridine/deoxyadenosine pair increased the elimination of iodine. The results indicate that the deiodination reaction might be applied for the rapid detection of variations in natural DNA sequences.


Measurement of radon/thoron and its daughter nuclides in room air. Pumping air through a soft tissue which acts as a membrane is a relatively easy and quick method to collect and measure radon/thoron and its daughter nuclides in air. Analysis of the activity of the radionuclides can be calculated using an alpha counter which has been calibrated. In this method the activity of radon/thoron cannot be separated from the activity of radionuclides already present in the aerosol or dust particles.


A simplified method of 85Kr measurement for dating young groundwaters. The technique of gas extraction from water and gas sample preparation for 85Kr low-level measurements was further developed to improve its applicability to isotope-hydrological "age" determinations of young groundwaters. A water sample of about 200 L is flushed with helium to extract the dissolved gases. Krypton is separated from the extracted gas mixture by repeated adsorption on charcoal, fractionated desorption and subsequent gas chromatography. The detection limit for a 10,000 min counting time in a 10 mL counter containing the krypton (15 microL) and methane as counting gas corresponds to 25 mBq 85Kr per mL (STP) krypton or 2.5% of the mean atmospheric 85Kr activity concentration in the year 1990.


Synthesis, characterization and biodistribution of a new hexadentate aminethiol ligand labeled with Tc-99m. A new hexadentate aminethiol ligand (TACNS) derived from triazacyclononane was synthesized and characterized for the development of technetium radiopharmaceuticals. The ligand formed a neutral, lipophilic and stable complex with [99mTc]pertechnetate in the presence of tin(II)tartarate as a reducing agent. The biodistribution of [99mTc]TACNS indicates slight uptake in brain (0.23% ID/organ at 5 min) with a washout at 30 min to 0.14% ID/organ. A small uptake in heart (0.48% ID at 5 min) was also observed. The characterization of [99mTc]TACNS complex using single crystal x-ray analysis and mass spectroscopy has shown that an Sn-N3S3 complex was formed in which tin is oxidized from Sn(II) to Sn(IV). Pertechnetate was incorporated into the complex as counter anion. The nature of the species formed with Tc-99 and "no-carrier-added" [99mTc]pertechnetate is different as confirmed by ratio TLC. From these results, it is demonstrated that sometimes it may be difficult to predict the structure of new technetium radiopharmaceuticals, especially when stannous ion is used as a reducing agent. Moreover, the nature of the chemical species may not be the same at millimolar and at nanomolar levels.


Effect of reinnervation on collagen synthesis in rat skeletal muscle. The effect of reinnervation on the activities of prolyl 4-hydroxylase (PH) and galactosylhydroxylysyl glucosyltransferase (GGT), both enzymes of collagen biosynthesis, and on the concentration of hydroxyproline (Hyp) was studied in gastrocnemius, soleus, and tibialis anterior muscles of rat 19, 26, 40, and 61 days after crush denervation of the sciatic nerve. The GGT activity was elevated in denervated gastrocnemius and soleus muscles and the PH activity in gastrocnemius. Muscular Hyp concentration was increased in denervated tibialis anterior muscle. Both the PH and GGT activities and the Hyp concentration returned to the control level during the reinnervation period (19-61 days from the start of denervation). It seems that denervation atrophy of skeletal muscle is associated with an increased rate of muscular collagen biosynthesis and that during reinnervation collagen synthesis rate decreases despite accelerated muscular growth. The results thus suggest that innervation is a powerful suppressive regulator of muscular collagen biosynthesis.


Influence of proton availability on intracapillary CO2-HCO3(-)-H+ reactions in isolated rat lungs. Transcapillary CO2 exchange entails a transient perfusate CO2-HCO3(-)-H+ disequilibrium, leading to net loading or unloading of blood HCO3-. Perfusate reequilibration may or may not reach completion during the time of capillary transit, depending on the rate of intracapillary CO2-HCO3(-)-H+ reactions. Failure to reestablish equilibrium within the "open" capillary system leads to continued reequilibration in the "closed" postcapillary vasculature with resultant shifts in postcapillary perfusate PCO2, pH, and [HCO3-]. In the present study, we determined the effects of perfusate nonbicarbonate buffer capacity (beta) on intracapillary CO2-HCO3(-)-H+ reactions in isolated saline-perfused rat lungs. Effects of beta on the rate of transcapillary CO2 excretion (VCO2) and the magnitude of the postcapillary perfusate pH disequilibrium were measured as a function of luminal vascular carbonic anhydrase (CA) activity. The data indicate that beta markedly influenced the kinetics and dynamics of intravascular CO2-HCO3(-)-H+ reactions. beta affected VCO2 and the relative enhancement of VCO2 by luminal vascular CA. The data emphasize the inadequacies of using traditional "equilibrium" models of the CO2-HCO3(-)-H+ system to investigate capillary CO2 transport and exchange, even in organs (e.g., lungs) that contain significant luminal vascular CA activity.


Gas exchange in dogs in the prone and supine positions. To determine the cause of the difference in gas exchange between the prone and supine postures in dogs, gas exchange was assessed by the multiple inert gas elimination technique (MIGET) and distribution of pulmonary blood flow was determined using radioactively labeled microspheres in seven anesthetized paralyzed dogs. Each animal was studied in the prone and supine positions in random order while tidal volume and respiratory frequency were kept constant with mechanical ventilation. Mean arterial PO2 was significantly lower (P less than 0.01) in the supine [96 +/- 10 (SD) Torr] than in the prone (107 +/- 6 Torr) position, whereas arterial PCO2 was constant (38 Torr). The distribution of blood flow (Q) vs. ventilation-to-perfusion ratio obtained from MIGET was significantly wider (P less than 0.01) in the supine [ln SD(Q) = 0.75 +/- 0.26] than in the prone position [ln SD (Q) = 0.34 +/- 0.05]. Right-to-left pulmonary shunting was not significantly altered. The distribution of microspheres was more heterogeneous in the supine than in the prone position. The larger heterogeneity was due in part to dorsal-to-ventral gradients in Q in the supine position that were not present in the prone position (P less than 0.01). The decreased efficiency of oxygenation in the supine posture is caused by an increased ventilation-to-perfusion mismatch that accompanies an increase in the heterogeneity of Q distribution.


Contributions of ventilation and perfusion inhomogeneities to the VA/Q distribution. The anatomic distributions of ventilation (VA) and perfusion (Q) in prone and supine dogs have been described in the literature. These data also provide frequency distributions, i.e., the distribution of lung units as a function of VA or Q. A comprehensive distribution that encompasses these two distributions is described, and the properties of the comprehensive distribution that determine the width of the VA/Q distribution are identified. Using data on the VA and Q distributions taken from various sources in the literature, we estimated the widths of the VA/Q distributions. The widths estimated from the independent data on the VA and Q distributions agree well with the widths obtained from gas exchange data. The analysis provides information about the relative contributions of the VA and Q distributions to the width of the VA/Q distribution. In the prone dog, the VA and Q distributions, as described by the available data, have different length scales, and we argue that these distributions are therefore not highly correlated. As a result, the variance of the VA/Q distributions is approximately the sum of the variances of the VA and Q distributions. Two-thirds of the variance in VA/Q is a result of nonuniform Q, and one-third is a result of nonuniform VA. In the supine dog, the variance of VA is larger than in the prone dog because of a vertical gradient and the variance of Q is larger, in part, because of a vertical gradient. Because the magnitudes of the vertical gradients of VA and Q are about equal, the vertical gradient of VA/Q is small, and these components of the VA and Q inhomogeneities contribute little to the width of the VA/Q distribution. The other components of Q inhomogeneity cause the additional variance of VA/Q in the supine dog.


Metabolic and febrile responses to typhoid vaccine in humans: effect of beta-adrenergic blockade. Fever and activation of acute phase responses were induced in human volunteers by intramuscular injection of typhoid vaccine. Vaccine injection caused a rapid (within 1 h) and sustained rise in metabolic rate (peak response 16%, 6-8 h), followed by later increases in white blood cell count (3-4 h), skin temperature (4-5 h), oral temperature (5-6 h), heart rate (6-8 h), and plasma cortisol (5-8 h). A peak fever [1.2 +/- 0.2 degree C (SE) rise] was recorded 12 h after vaccine injection. The involvement of the sympathetic nervous system in the development of these responses was investigated by the oral administration of propranolol before (80 mg) and 3 h after (40 mg) vaccine injection. Propranolol prevented the increases in metabolic rate, heart rate, and skin temperature but did not inhibit the rise in oral temperature or white cell count after vaccine administration. These data indicate that the sympathetic nervous system is responsible for the rise in energy expenditure associated with fever in humans. However, the rise in body temperature can develop in the absence of this increase in metabolic rate possibly by changes in heat loss.


Spontaneous injury in isolated sheep lungs: role of resident polymorphonuclear leukocytes. Perfusion of isolated sheep lungs with homologous blood caused pulmonary hypertension and edema that was not altered by depletion of perfusate polymorphonuclear (PMN) leukocytes (D. B. Pearse et al., J. Appl. Physiol. 66: 1287-1296, 1989). The purpose of this study was to evaluate the role of resident PMN leukocytes in this injury. First, we quantified the content and activation of lung PMN leukocytes before and during perfusion of eight isolated sheep lungs with a constant flow (100 ml.kg-1.min-1) of homologous blood. From measurements of myeloperoxidase (MPO) activity, we estimated that the lungs contained 1.2 x 10(10) PMN leukocytes, which explained why the lung PMN leukocyte content, measured by MPO activity and histological techniques, did not increase significantly with perfusion, despite complete sequestration of 2.0 x 10(9) PMN leukocytes from the perfusate. MPO activities in perfusate and lymph supernatants did not increase during perfusion, suggesting that lung PMN leukocytes were not activated. Second, we perfused lungs from 6 mechlorethamine-treated and 6 hydroxyurea-treated sheep with homologous leukopenic blood and compared them with 11 normal lungs perfused similarly. Despite marked reductions in lung PMN leukocyte concentration, there were no differences in pulmonary arterial pressure, lymph flow, or reservoir weight between groups. Extravascular lung water was greater in both groups of leukopenic lungs. These results suggest that resident PMN leukocytes did not contribute to lung injury in this model.


Regulation of skeletal muscle dihydropyridine receptor gene expression by biomechanical unloading. Biomechanical unloading of the rat soleus by hindlimb unweighting is known to induce atrophy and a slow- to fast-twitch transition of skeletal muscle contractile properties, particularly in slow-twitch muscles such as the soleus. The purpose of this study was to determine whether the expression of the dihydropyridine (DHP) receptor gene is upregulated in unloaded slow-twitch soleus muscles. A rat DHP receptor cDNA was isolated by screening a random-primed cDNA lambda gt10 library from denervated rat skeletal muscle with oligonucleotide probes complementary to the coding region of the rabbit DHP receptor cDNA. Muscle mass and DHP receptor mRNA expression were assessed 1, 4, 7, 14, and 28 days after hindlimb unweighting in rats by tail suspension. Isometric twitch contraction times of soleus muscles were measured at 28 days of unweighting. Northern blot analysis showed that tissue distribution of DHP receptor mRNA was specific for skeletal muscle and expression was 200% greater in control fast-twitch extensor digitorum longus (EDL) than in control soleus muscles. A significant stimulation (80%) in receptor message of the soleus was induced as early as 24 h of unloading without changes in muscle mass. Unloading for 28 days induced marked atrophy (control = 133 +/- 3 vs. unweighted = 62.4 +/- 1.8 mg), and expression of the DHP receptor mRNA in the soleus was indistinguishable from levels normally expressed in EDL muscles. These changes in mRNA expression are in the same direction as the 37% reduction in time to peak tension and 28% decrease in half-relaxation time 28 days after unweighting. Our results suggest that muscle loading necessary for weight support modulates the expression of the DHP receptor gene in the soleus muscle.


The cadC gene product of alkaliphilic Bacillus firmus OF4 partially restores Na+ resistance to an Escherichia coli strain lacking an Na+/H+ antiporter (NhaA). A 5.6-kb fragment of alkaliphilic Bacillus firmus OF4 DNA was isolated by screening a library of total genomic DNA constructed in pGEM3Zf(+) for clones that reversed the Na+ sensitivity of Escherichia coli NM81, in which the gene encoding an Na+/H+ antiporter (NhaA) is deleted (E. Padan, N. Maisler, D. Taglicht, R. Karpel, and S. Schuldiner, J. Biol. Chem. 264:20297-20302, 1989). The plasmid, designated pJB22, contained two genes that apparently encode transposition functions and two genes that are apparent homologs of the cadA and cadC genes of cadmium resistance-conferring plasmid pI258 of Staphylococcus aureus. E. coli NM81 transformed with pJB22 had enhanced membrane Na+/H+ antiporter activity that was cold labile and that decreased very rapidly following isolation of everted vesicles. Subclones of pJB22 containing cadC as the only intact gene showed identical complementation patterns in vivo and in vitro. The cadC gene product of S. aureus has been proposed to act as an accessory protein for the Cd2+ efflux ATPase (CadA) (K. P. Yoon and S. Silver, J. Bacteriol. 173:7636-7642, 1991); perhaps the alkaliphile CadC also binds Na+ and enhances antiporter activity by delivering a substrate to an integral membrane antiporter. A 6.0-kb fragment overlapping the pJB22 insert was isolated to complete the sequence of the cadA homolog. A partial sequence of a region approximately 2 kb downstream of the cadA locus shares sequence similarity with plasmids from several gram-positive bacteria. These results suggest that the region of alkaliphile DNA containing the cadCA locus is present on a transposon that could reside on a heretofore-undetected endogenous plasmid.


Construction of a Neisseria gonorrhoeae MS11 derivative deficient in NgoMI restriction and modification. We have cloned from Neisseria gonorrhoeae MS11 the gene encoding a methylase that modifies the sequence GCCGGC. The corresponding restriction enzyme was also encoded by this clone. Sequence analysis demonstrated that the methylase shares sequence similarities with other cytosine methylases, but the sequence organization of M.NgoMI is different from that seen for other cytosine methylases. A deletion was introduced into the chromosome of N. gonorrhoeae MS11 to produce strain MUG701, a strain that is inactivated in both the methylase and the restriction genes. Although this strain no longer methylated its DNA at the NgoMI recognition sequence, cells were viable and had no other significant phenotypic changes. Transformation data indicated that MS11 does not produce enough restriction activity to block plasmid transformation in the gonococcus, even though restriction activity could be demonstrated in E. coli containing the cloned gene.