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2.  Adenovirus type 2 activates cell cycle-dependent genes that are a subset of those activated by serum. 
Molecular and Cellular Biology  1985;5(11):2936-2942.
We have studied a panel of 10 genes and cDNA sequences that are expressed in a cell cycle-dependent manner in different types of cells from different species and that are inducible by different mitogens. These include five sequences (c-myc, 4F1, 2F1, 2A9, and KC-1) that are preferentially expressed in the early part of the G1 phase, three genes (ornithine decarboxylase, p53, and c-rasHa) preferentially expressed in middle or late G1, and two genes (thymidine kinase and histone H3) preferentially expressed in the S phase of the cell cycle. We have studied the expression of these genes in nonpermissive (tsAF8) and semipermissive (Swiss 3T3) cells infected with adenovirus type 2. Under the conditions of these experiments, adenovirus type 2 infection stimulates cellular DNA synthesis in both tsAF8 and 3T3 cells. However, four of the five early G1 genes (c-myc, 4F1, KC-1, and 2A9) and one of the late G1 genes (c-ras) are not induced by adenovirus infection, although they are strongly induced by serum. The other sequences (2F1, ornithine decarboxylase, p53, thymidine kinase, and histone H3) are activated by both adenovirus and serum. We conclude that the cell cycle-dependent genes activated by adenovirus 2 are a subset of the cell cycle-dependent genes activated by serum. The data suggest that the mechanisms by which serum and adenovirus induce cellular DNA synthesis are not identical.
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PMCID: PMC369104  PMID: 2427924
3.  Chloride secretory mechanism induced by prostaglandin E1 in a colonic epithelial cell line. 
Journal of Clinical Investigation  1985;76(5):1828-1836.
Confluent T84 monolayers grown on permeable supports and mounted in a modified Ussing chamber secrete chloride (Cl-) in response to prostaglandin E1. The threshold stimulation was observed at 10(-9) M and a maximal effect at 10(-6) M. Unidirectional flux studies showed an increase in both serosal to mucosal and mucosal to serosal Cl- fluxes with 10(-6) M prostaglandin E1; the increase in serosal to mucosal Cl- flux exceeded the increase in mucosal to serosal flux, resulting in net Cl- secretion. Na+ transport was not affected in either direction and the changes in net Cl- flux correlated well with the changes in short circuit current. To identify the electrolyte transport pathways involved in the Cl- secretory process, the effect of prostaglandin E1 on ion fluxes was tested in the presence of putative inhibitors. Bumetanide was used as an inhibitor for the basolaterally localized Na+,K+,Cl- cotransport system whose existence and bumetanide sensitivity have been verified in earlier studies (Dharmsathaphorn et al. 1984. J. Clin. Invest. 75:462-471). Barium was used as an inhibitor for the K+ efflux pathway on the basolateral membrane whose existence and barium sensitivity were demonstrated in this study by preloading the monolayers with 86Rb+ (as a tracer for K+) and simultaneously measuring 86Rb+ efflux into both serosal and mucosal reservoirs. Both bumetanide and barium inhibited the net chloride secretion induced by prostaglandin E1 suggesting the involvement of the Na+,K+,Cl- cotransport and a K+ efflux pathways on the basolateral membrane in the Cl- secretory process. The activation of another Cl- transport pathway on the apical membrane by prostaglandin E1 was suggested by Cl- uptake studies. Our findings indicate that the prostaglandin E1-stimulated Cl- secretion, which is associated with an increase in cyclic AMP level, intimately involves (a) a bumetanide-sensitive Na+,K+,Cl- cotransport pathway that serves as a Cl- uptake step across the basolateral membrane, (b) the stimulation of a barium-sensitive K+ efflux mechanism on the basolateral membrane that most likely acts to recycle K+, and (c) the activation of a Cl- transport pathway on the apical membrane that serves as a Cl- exit pathway.
PMCID: PMC424218  PMID: 2997290
4.  Indirect enzyme-linked immunosorbent assay for the mycotoxin zearalenone. 
A competitive indirect enzyme-linked immunosorbent assay (ELISA) was developed for the detection of zearalenone, an estrogenic mycotoxin. Zearalenone was converted to zearalenone-6'-carboxymethyloxime and conjugated to bovine serum albumin and poly-L-lysine for use as immunogen and solid-phase marker, respectively. Immunization of rabbits with the bovine serum albumin conjugate resulted in zearalenone antibody titers of 20,480 in 11 weeks. A competitive indirect ELISA was conducted by simultaneously incubating zearalenone with zearalenone antiserum over zearalenone-6'-carboxymethyloxime poly-L-lysine solid phase and then determining the bound rabbit immunoglobulin with goat anti-rabbit peroxidase conjugate. Response range for zearalenone in the resulting competition curve was between 1 and 50 ng/ml. Reactivities of this antiserum for alpha-zearalenol, beta-zearalenol, alpha-zearalanol, and beta-zearalanol were, respectively, 50, 12, 6, and 3% of that found for zearalenone. By using the competitive indirect ELISA, zearalenone was detectable in methanol-water extracts of corn, wheat, and pig feed samples.
PMCID: PMC238624  PMID: 2932054
5.  Isolation and Some Properties of the Enzyme That Transforms Eremofortin C to PR Toxin 
PR toxin and eremofortin C are secondary metabolites of Penicillium roqueforti. The chemical structures of these two compounds are closely related to each other and differ only by an aldehyde and an alcohol group at the C-12 position. In an effort to better understand the biosynthesis of PR toxin, we discovered the enzyme of P. roqueforti that is responsible for the transformation of eremofortin C to PR toxin. The maximum activity of the enzyme in the culture medium was found to occur on day 13, which corresponded to the maximal production of PR toxin in the medium. The enzyme was isolated and purified from the culture medium and the mycelium of the fungus, respectively, through a procedure involving ammonium sulfate fractionation and DEAE-cellulose chromatography. The specific activity increased 20- and 8-fold, respectively, and the yield was 33.3 and 21.6%, respectively, for the enzyme from the medium and mycelium. The optimal pH for the enzyme reaction was ca. pH 5.6. The enzyme reaction was temperature dependent. The rates followed a linear time course when it catalyzed the transformation at 30°C and decayed with time when reacted at higher temperatures. At 100°C, the enzyme activity was completely lost. The Km and Vmax of the enzyme as determined at 30°C were 0.02 mM and 4.0 μmol/min per mg, respectively. The molecular weight of the enzyme was estimated by gel filtration on a high-pressure liquid chromatography I-250 protein column to be ca. 40,000.
PMCID: PMC241746  PMID: 16346812
6.  Transformation of Halogen-, Alkyl-, and Alkoxy-Substituted Anilines by a Laccase of Trametes versicolor† 
The laccase of the fungus Trametes versicolor was able to polymerize various halogen-, alkyl-, and alkoxy-substituted anilines, showing substrate specificity similar to that of horseradish peroxidase, whereas the laccase of Rhizoctonia praticola was active only with p-methoxyaniline. The substrate specificities of the enzymes were determined by using gas chromatography to measure the decrease in substrate concentration during incubation. With p-chloroaniline as the substrate, the peroxidase and the Trametes laccase showed maximum activity near pH 4.2. The transformation of this substrate gave rise to a number of oligomers, ranging from dimers to pentamers, as determined by mass spectrometry. The product profiles obtained by high-pressure liquid chromatography were similar for the two enzymes. A chemical reaction was observed between p-chloroaniline and an enzymatically formed dimer, resulting in the formation of a trimer. All three enzymes oxidized p-methoxyaniline to 2-amino-5-p-anisidinobenzoquinone di-p-methoxyphenylimine, but only the T. versicolor laccase and the peroxidase caused the formation of a pentamer (2,5-di-p-anisidinobenzoquinone di-p-methoxyphenylimine). Our results demonstrate that in addition to horseradish peroxidase, a T. versicolor laccase can also polymerize aniline derivatives.
PMCID: PMC238501  PMID: 16346778
7.  Magnetic resonance imaging: present and future applications 
Magnetic resonance (MR) imaging has created considerable excitement in the medical community, largely because of its great potential to diagnose and characterize many different disease processes. However, it is becoming increasingly evident that, because MR imaging is similar to computed tomography (CT) scanning in identifying structural disorders and because it is more costly and difficult to use, this highly useful technique must be judged against CT before it can become an accepted investigative tool. At present MR imaging has demonstrated diagnostic superiority over CT in a limited number of important, mostly neurologic, disorders and is complementary to CT in the diagnosis of certain other disorders. For most of the remaining organ systems its usefulness is not clear, but the lack of ionizing radiation and MR's ability to produce images in any tomographic plane may eventually prove to be advantageous. The potential of MR imaging to display in-vivo spectra, multinuclear images and blood-flow data makes it an exciting investigative technique. At present, however, MR imaging units should be installed only in medical centres equipped with the clinical and basic research facilities that are essential to evaluate the ultimate role of this technique in the care of patients.
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PMCID: PMC1345865  PMID: 3884120
8.  Methanosarcina mazei LYC, a New Methanogenic Isolate Which Produces a Disaggregating Enzyme 
A methanogenic coccoid organism, Methanosarcina mazei LYC, was isolated from alkaline sediment obtained from an oil exploration drilling site. The isolate resembled M. mazei S-6 by exhibiting different morphophases during its normal growth cycle. It differed from M. mazei S-6 by undergoint a spontaneous shift from large, irregular aggregates of cells to small, individual, irregular, coccoid units. In batch cultures at pH 7.0, M. mazei LYC grew as aggregates during the early growth stage. As the batch culture began exponential growth, the cell aggregates spontaneously dispersed: the culture liquid became turbid, and myriads of tiny (diameter, 1 to 3 μm) coccoid units were observed under phase-contrast microscopy. Disaggregation apparently was accomplished by the production of an enzyme which hydrolyzed the heteropolysaccharide component of the cell wall; the enzyme was active on other Methanosarcina strains as well. Although the enzyme was active when tested at pH 6.0, it apparently was not produced at that pH: when strain LYC was grown at pH 6.0, only cell aggregates were present throughout batch growth. Individual coccoid cells of M. mazei LYC were sensitive to sodium dodecyl sulfate, but the large aggregates of cells were not. Strain LYC rapidly used H2-CO2, in addition to methanol, and mono-, di-, and trimethylamine as methanogenic substrates; acetate was used very slowly. Its optimum growth temperature was 40°C, and its optimum pH was 7.2.
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PMCID: PMC373557  PMID: 16346753
9.  A thermolabile mutant of adenovirus 5 resulting from a substitution mutation in the protein VIII gene. 
Journal of Virology  1985;53(3):920-925.
The mutant adenoviruses H5sub304 and H5RIr were isolated sequentially from adenovirus 5 wild type by selection for the loss of EcoRI restriction endonuclease sites by Jones and Shenk (Cell 13:181-188, 1978). sub304 lacks the site at 84.0 map units (m.u.), and RIr lacks both that and the site at 75.9 m.u. A set of derivatives of RIr that lack the site at 75.9 m.u. accumulated virus more slowly at 38.8 or 39.5 degrees C than those with the site present, as measured by low-multiplicity passage or single-step replication cycles, respectively. Since the EcoRI site at 75.9 m.u. is predicted to lie in the gene encoding the precursor to virion polypeptide VIII (pVIII), the failure to accumulate virus rapidly could lie either in some step in processing and assembly of virions or in an increased virion thermolability. The latter possibility was shown to be the case, as all strains mutated at the EcoRI 75.9 m.u. site were extremely thermolabile in vitro, even at 37 degrees C. CsCl equilibrium density centrifugation of heated crude stocks of RIr and sub304 demonstrated that loss of infectivity in RIr was accompanied by physical disruption of virions. Polyacrylamide gel electrophoresis of infected cell extracts or of purified virions showed that pVIII of RIr had an apparent molecular weight that was slightly greater than that of sub304, and mature RIr and sub304 virions displayed polypeptide VIIIs which appeared to be of identical molecular weights. Nucleotide sequence analysis of RIr demonstrated that it contained a 9-base-pair (bp) substitution for 6 bp found in sub304, leading to a loss of the EcoRI site and a predicted insertion of a single amino acid. Comparison of the sequence of sub304 with the published sequence of adenovirus 2 revealed two changes, a single transversion at bp 1,722 and a bp deletion at 1,749, leading to the loss of a TaqI site. The predicted reading frame change would lead to a stop codon at bp 1,885. This raises the question of whether adenovirus 2 and adenovirus 5 use the same reading fame for pVIII.
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PMCID: PMC254727  PMID: 3973969

Results 1-9 (9)