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M Arens

Publications and source records attributed to M Arens.

52 records · Page 3Linked to original sources
PubMed1981

[A contribution to stepless antibody determination by the "enzyme linked immunosorbent assay" (ELISA) (author's transl)].

A simple method for the mathematical calculation of ELISA results is described. Using the formula (formula: see text) (Eu, En, Ep = extinction values of the unknown serum, the negative, and positive standard serum, respectively) an index value Iu is calculated for the testserum, whereby the optical density of the testserum is put in relation to the optical density of a negative and positive standardserum. Using three different ELISA test systems, the Iu values of a number of human, bovine and rabbit testsera were relatively uniform and reproducible in repeated investigations. A statistically calculated tolerance limit for the index values of negative sera can be introduced into the formula described, thus facilitating the decision "negative" or "positive" for unknown sera.

Animals↗
PubMed1980

Purification and properties of a DNA ligase from a soluble DNA replication complex.

A DNA ligase has been purified from a subnuclear soluble replication complex isolated from adenovirus type 2-infected human KB cells. DNA ligase activity could not be demonstrated using an exogenous template until the complex was dissociated, suggesting that the ligase activity may be a component of the complex. The purified enzyme was free of endonuclease, exonuclease, 5'-nucleotidase, and phosphatase activities, and had a molecular weight of 105 000, as estimated by sedimentation in a glycerol gradient. The ligase requires ATP and a divalent cation for activity. The optimum of the reaction is at pH 7.8 in 50--100 mM Tris-HCl buffer and 10--20 mM MgCl2. Monovalent salts greatly stimulate ligase activity and the optimum was found at 150 mM. The reaction is very sensitive to high temperature; maximum activity was observed at 25--30 degrees C. ATP is the sole required cofactor and NAD, dATP and GTP could not replace the requirement for ATP. The Km for ATP is 60 microM. The Km for DNA is 250 microgram/ml or 1.6 nmol of terminal phosphate/ml and thus the enzyme shows relatively weak affinity for exogenous DNA. The maximum conversion of 32P into a phosphatase-resistant form is approximately 1.3% of the total, whereas T4 ligase, under the same conditions, can convert more than 25% of phosphate into a resistant form.

Adenoviruses, Human↗
PubMed1979

Protein synthesized early after infection is linked to the termini of adenovirus type 2 DNA synthesized in vivo and in vitro.

The human adenovirus DNA genome contains a protein (CBP, or covalently bound protein) linked to each 5' terminus. To assess whether CBP is synthesized early, infected cells were incubated with hydroxyurea from 1 to 18 h postinfection, the hydroxyurea was removed, cycloheximide was added, and viral DNA was labeled with [3H]thymidine from 18 to 23 h postinfection. Removal of hydroxyurea at 18 h postinfection permits the synthesis of viral DNA, whereas cycloheximide maintains the block in late viral protein synthesis. Three lines of evidence are presented to show that viral 3H-labeled DNA prepared by this procedure was linked to CBP: (I) the DNA sedimented more rapidly than protein-free DNA (i.e., protinase treated) in neutral sucrose gradients containing guanidine hydrochloride; (ii) the DNA banded at a lower density than protein-free DNA in CsCl gradients containing guanidine hydrochloride; and (iii) neither the 3H-labeled DNA nor the end fragments produced by EcoRI digestion entered a 1.4% agarose gel during electrophoresis. These experiments are strong evidence that CBP is not a product of a late viral gene and is therefore the product of either an early viral gene or a cell gene. Experiments were performed to test whether CBP is attached to viral DNA synthesized in vitro by a soluble complex that synthesizes exclusively viral DNA as completed viral genomes in vitro. In vitro-labeled DNA was analyzed by velocity sedimentation, equilibrium sedimentation, and agarose gel electrophoresis as described above. Our results indicate that the majority of in vitro-synthesized DNA molecules were attached to CBP. These results, which indicate that CBP is synthesized early after infection and is attached to viral DNA labeled in vitro by a soluble replication complex, are consistent with the idea that CBP may play a role in viral DNA replication.

Adenoviruses, Human↗
PubMed1979

[A method for production of Coxiella burnetii antigen and cell walls by guanidiniumchloride extraction (author's transl)].

By use of 6-molar guanidinium chloride a potent Coxiella burnetii antigen could be produced for diagnostic purposes from infected yolk sacs, with little technical expense. This treatment did not only a cause remarkably purifying effect (Tab. 1) but also the extraction of soluble cytoplasmic substance. From guanidinium chloride-treated suspensions a highly purified and uniform suspension of cell walls could be separated by Saccharose Density Gradient centrifugation (Fig. 2). Guanidine extracted organisms retained their full antigenic potential with respect to Phase I and Phase II and lacked anticomplementary activity. Such preparations can be used for serological tests like complement fixation reaction or Enzyme Linked Immunosorbent Assay and are particularly suitable for biochemical studies of Phase antigens of Coxiella burnetii.

Antigens, Bacterial↗
PubMed1978

An endodeoxyribonuclease of human KB cells. Purification and properties of the enzyme.

An endodeoxyribonuclease has been purified 750-fold from human KB cells. The purified endonuclease requires Mg2+ for maximum activity: Mn2+ was less than half as active and Ca2+ inhibited the reaction. The optimum pH is 8.8 in Tris-HCl and the optimum buffer concentration is 10 mM. KCl (and NaCl), --SH-reacting reagents, and tRNA strongly inhibit the reaction. An apparent molecular weight of 54,000 was determined by sedimentation in a glycerol gradient. The purified endonuclease cleaved native, double-stranded adenovirus 2 DNA, and the reaction proceeded stepwise during the initial stage of degradation by cleavage of the DNA substrate in half, then in half again, etc. At longer digestion times, single strand scissions were detected. RNA was not a substrate for the enzyme. Poly(dG) . poly(dC) was susceptible but poly(dA) . poly(dT) was resistant to degradation. Hydrolysis of adenovirus 2 DNA yielded double-stranded polynucleotides containing 5'-phosphoryl and 3'-hydroxyl termini with short, single-stranded regions presumably at the ends. More than 50% of the product of a limit digest had a chain length greater than 35 to 40 nucleotides. Analysis of the 5' and 3' end groups of the digestion products indicated a preference for the site of the enzymatic cleavage; thymidylic acid residues were present at the 5' end and deoxyguanosine residues at the 3' end, each with a frequency of 40 to 50%.

Cell Line↗
PubMed1978

In vitro termination of adenovirus DNA synthesis by a soluble replication complex.

The double-stranded DNA from a soluble DNA replication complex that was labeled with deoxyribonucleoside triphosphates and completed in vitro was digested with EcoRI, Sma I, and Hpa I restriction endonucleases. All regions of the adenovirus type 2 genome were labeled in vitro, but restriction fragments derived from the ends of the DNA molecules were relatively more highly labeled than those derived from internal regions. The in vitro endogenous DNA polymerase reaction also exhibited strand-specific labeling near the molecular ends, in that restriciton fragments from the left end were labeled predominantly in the r strand and fragments from the right end were labeled predominantly in the l strand.

Adenoviruses, Human↗
PubMed1976

Characterization of DNA polymerase associated with nuclear membrane fractions from uninfected and adenovirus 2-infected KB cells.

We have isolated a nuclear membrane fraction from KB cells infected with human adenovirus 2 that synthesizes exclusively small viral DNA chains (approx. 9 S) in vitro (Yamashita, T., Arens, M. and Green, M. (1975) J. Biol. Chem. 250, 3273-3279). The DNA polymerase activity present in the adenovirus 2 DNA-nuclear membrane complex was purified through chromatography on phosphocellulose and DEAE-cellulose, glycerol gradient centrifuation and DNA-cellulose chromatography. A single peak of enzymatic activity sedimented in glycerol gradients at about 6.7 S which corresponds to a molecular weight of 125000. The enzyme preparation in the step of glycerol gradient centrifugation utilized activated calf thymus, KB cell and adenovirus 2 DNA as template-primer in the presence of Mg2+; Km values for these DNAs were 5.5, 4.0, and 0.8 mug/ml, respectively. The partially purified enzyme preparation was characterized by several criteria which were compared to the properties of the three major mammalian DNA polymerases, alpha, beta, and psi. On the basis of template-primer preference, effect of salt, inhibition by N-ethylmaleimide and Km for dTTP, the DNA polymerase activity from the membrane complex can be distinguished from the alpha and beta DNA polymerases. The elution profile from DNA cellulose revealed a minor peak (I) and a major peak (II) of DNA polymerase activity utilizing poly(A) -(dT)10 as template-primer in the presence of Mn2+ - Peak II from DNA cellulose, which contained about 90% of the total DNA polymerase activity eluted from the column, was 2-3 times as active with poly(A) - (dT)10 as template-primer in the presence of Mn2+ than with activated calf thymus DNA in the presence of Mg2+. On the other hand, peak I had a low ratio of poly(A) - (dT)10 to activated calf thymus DNA activity. DNA polymerase was also purified from the nuclear membrane fraction of uninfected KB cells by the same procedures as those used in enzyme purification from the adenovirus 2 DNA-nuclear membrane complex. A minor peak and a major peak of DNA polymerase activity utilizing poly(A) - (dT)10 as template primer in the presence of Mn2+ were again observed that eluted from DNA cellulose at the same KCl concentrations as peak I and II from adenovirus 2-infected cells. The enzymes of the nuclear membrane fraction of uninfected KB cells could not be differentiated from the enzymes of the adenovirus 2 DNA-nuclear membrane complex through any of the purification steps nor by their template specificities. These results indicate that the predominant enzyme in the adenovirus 2 DNA-nuclear membrane complex and in the KB cell nuclear membrane complex belongs to the class of DNA polymerase psi.

Adenoviruses, Human↗
PubMed1975

Adenovirus deoxyribonucleic acid replication. II. Synthesis of viral deoxyribonucleic acid in vitro by a nuclear membrane fraction from infected KB cells.

A cell-free system for the study of viral DNA replications was developed by the isolation of a nuclear membrane fraction "DNA replication complex" from adenovirus 2-infected human KB cells late after infection. This complex which possesses both DNA polymerase activity and a virus-specific endonuclease synthesizes exclusively virus-specific DNA sequences in vitro by a semiconservative mechanism. Analysis by rate zonal sedimentation in alkaline sucrose gradients showed that the products of the reaction are small DNA chains approximately 6 to 9 S, presumably "Okazaki intermediates," that are not sealed under our in vitro conditions. Analysis by rate zonal sedimentation in neutral sucrose gradients showed that labeled viral DNA fragments are hydrogen bonded to viral 18 S DNA segments, 0.25 the size of the linear, viral 31 S DNA genome. The 18 S DNA is probably a specific cleavage product of the viral endonuclease found in the replication complex and could represent intermediates in viral DNA replication or degradation products.

Adenosine Triphosphate↗
PubMed1975

DNA binding proteins in the cytoplasm and in a nuclear membrane complex isolated from uninfected and adenovirus 2 infected cells.

The DNA binding proteins in a nuclear membrane fraction that can synthesize DNA in vitro (referred to as "nuclear membrane complex") and in the cytoplasm of adenovirus infected and uninfected cells were isolated and characterized. Suspension cultures of human KB cells infected with human adenovirus 2 were treated with 25 mu-g/ml of arabinosylcytosine starting at 2 hr to block the synthesis of viral structural proteins, and then labeled with (3H)leucine from 6 to 24 hr after infection. Uninfected cells were treated similarly and labeled with (14C)leucine. The 3H-labeled proteins (infected cells) and 14C-labeled proteins (uninfected cells) isolated from the cytoplasm were mixed, as were the corresponding proteins isolated from the membrane complex, and each mixture was fractionated by stepwise elution from single-stranded DNA-cellulose columns. From 50 to 60% of the labeled protein in the membrane complex from infected cells and 40 to 50% of that from uninfected cells bound to DNA-cellulose in 0.05 M NaCl. Much less protein from the cytoplasm was bound to DNA cellulose, 20% from infected cells and 11% from uninfected cells. Gel electrophoresis of the mixture of 3H- and 14C-labeled proteins eluted from DNA-cellulose by different concentrations of NaCl revealed the following. (1) The 0.15 and 0.40 M NaCl eluates from the membrane complex of infected and uninfected cells contained a heterogenous mixture of similar polypeptides. (2) The 0.6 M NaCl eluate from the membrane complex derived from infected cells contained two major DNA binding proteins with molecular weights of 75,000 and 45,000 that were absent from uninfected cells. Large quantities of these two proteins were present in highly purified form in the 0.6 M NaCl eluate from the cytoplasm of infected cells. The DNA binding proteins of molecular weight 75,000 and 45,000 that are present in the cytoplasm are identical with those present in the membrane complex, as established by coelectrophoresis. (3) Two major cell-specific proteins of molecular weight 40,000 and 15,000-17,000 were present in the 2 M NaCl eluate of the membrane complex from uninfected and infected cells. A major cell-specific protein of molecular weight 33,000 was present in the 0.15 and 0.4 M NaCl eluates of the uninfected and infected cell cytoplasmic fractions. Aanalysis of cells labeled at 2-6 hr after infection in the absence of arabinosyl cytosine indicated that the synthesis of the DNA binding proteins of molecular weight 75,000 and 45,000 begins early after infection prior to the onset of viral DNA replication.

Adenoviridae↗
PubMed1975

Isolation of DNA polymerase gamma from an adenovirus 2 DNA replication complex.

The major DNA polymerase in a nuclear membrane complex that is capable of synthesizing viral DNA sequences in vitro has been purified about 900-fold from adenovirus 2-infected KB cells. The enzyme was characterized as belonging to the class of mammalian DNA polymerases (DNA polymerase gamma) that can utilize poly(A) with oligo(dT) as template primer.

Adenoviridae↗
PubMedDate not supplied

Optimizing recovery of cytomegalovirus in the shell vial culture procedure.

We have investigated three factors that may be related to the recovery of cytomegalovirus (CMV) using the shell vial culture procedure. First, we compared fluorescent-antibody staining of shell vial cultures using a monoclonal antibody to a CMV immediate early antigen at 16 vs 40 hr after inoculation. Of 332 routinely submitted specimens cultured in duplicate and stained at the different times, 25 (7.5%) were positive at 16 hr and 32 (9.6%) were positive at 40 hr. The increased yield was 28%. Second, we analyzed the effect of using duplicate shell vials (both stained at 40 hr) for all routinely submitted CMV cultures. During a 6-month period, 272 (12.5%) of the 2157 cultures processed with duplicate shell vials were positive, including 222 positive in both vials and 50 positive in only one. Assuming that a single-vial setup would have detected 50% of those positive in only one of the two vials, the increased yield attributable to the duplicate vial was estimated at 10% (25/(222 + 25)). Third, we investigated the effects of seeding density and culture age on the shell vial assay. Cell age of greater than 1 day was associated with a decrease in sensitivity both in cultures that were confluent and in those that were subconfluent at the time of inoculation. Incorporating these findings in the routine shell vial culture procedure used in our Clinical Virology Laboratory has resulted in a greater overall detection of CMV in shell vial cultures than in conventional 6-week tube cultures.

Cell Line↗