Studies on the bacteriophage PS8 of Agrobacterium tumefaciens (Smith and Townsend) Conn: physico-chemical properties of its DNA.

DNA from the bacteriophage PS8 was extracted and purified. The buoyant density was determined was 1.716 cm3/g. The guanine-cytosine content was calculated to be 57%. DNA molecules which looked like circles were found among linear strands in an electron-microscopic study. With an endonuclease from Streptomyces albus G the DNA was digested to 19 fragments, with molecular weights ranging from 600 to 7,400 daltons. The molecular weight of the DNA was determined to be 38.8 X 10(6) daltons +/- 8.7%.     

Activation of the tumor-inducing ability of Agrobacterium tumefaciens (Smith and Townsend) Conn. by means of capsicin

Summary Treating cultures of Agrobacterium tumefaciens in liquid medium with capsicin in doses of 0.0025, 0.005 and 0.01 g, especially in the highest dose, greatly increases its tumor-inducing ability, causing intense tumor growth; no such growth is produced by an untreated culture. Under the same influence the bacterium also increases greatly its multiplication ability, and to a certain degree — its virulence, too.According to Thresh, capsicin means the raw oily alkaloidal substance extracted from the fruit of red peppers (Capsicum annuum L.), containing also the alkaloid with the same taste-capsicin; the latter term means the chemically pure alkaloid.     

Agrobacterium tumefaciens Conn IV. Bacteriophage PB21 and Its Inhibitory Effect on Tumor Induction

Strain B2 of Agrobacterium tumefaciens Conn produces plaques when seeded against strain B6-806 of the same organism. From such a plaque, a highly virulent bacteriophage was obtained by use of D'Herelle's technique of selecting for virulent phage. On nutrient agar, this phage, PB2₁, produced large clear plaques which did not overgrow. Plaques produced on a glutamate medium and on White's plant tissue culture medium were even larger and in White's medium had a three-dimensional appearance. PB2₁ does not appear to be an oncogenic virus. To the contrary, the addition of phage under circumstances which insure mass lysis completely inhibited tumor initiation. Fewer than 10 phage particles present at the beginning of a 21-hr induction period were able, at times, to inhibit completely tumor induction by highly virulent bacteria (strain B6). The data lend further support to the concept that anything which interferes with the metabolic activity associated with the growth of the bacteria interferes with the tumor-inducing process. Attempts to use the phage to rid crown gall tissue of bacteria were unsuccessful.     

Agrobacterium tumefaciens Conn. 3. Effect of thermal shock on bacteria in relation to tumorinducing ability

Stonier, Tom (Manhattan College, Bronx, N.Y.), Robert E. Beardsley, Lowell Parsons, and James McSharry. Agrobacterium tumefaciens Conn. III. Effect of thermal shock on bacteria in relation to tumor-inducing ability. J. Bacteriol. 91:266-269. 1966.-Bacteria heated to 42 C for 30 min exhibit a decrease in tumor-initiating ability without a detectable loss in viability. The thermal shock inhibits subsequent bacterial growth for up to 1.5 hr. As bacterial growth recovers, so does tumor-initiating ability. Respiration of the culture is somewhat increased by the heat treatment. The data suggest that living, actively respiring bacteria do not induce tumors unless they are also growing. The results also point to the necessity for excluding bacterial growth inhibition when interpreting data on the effect of various agents on tumor initiation.     

Enfuvirtide effects on human erythrocytes and lymphocytes functional properties.

Enfuvirtide (T-20) is the first inhibitor of human Immunodeficiency Virus type-1 (HIV-1) entrance on a target cell approved for clinical use. Recent studies indicated that its action mechanism involves the interaction with the membrane surface, increasing the concentration in the site of action. In the present study, the in vitro interaction between enfuvirtide and blood cells of healthy human donors, namely erythrocytes and lymphocytes, and the peptide effect on plasma and lymphocyte suspensions supernatant ions were evaluated, in order to better characterize the action of this peptide. Enfuvirtide causes a decrease in the concentration of hemoglobin and in the percentages of methemoglobin and carboxyhemoglobin, together with increased values of P50, pCO2, and [HCO3-], and significant decreases of pO2 and pH, in blood plasma. The supernatants of lymphocyte suspensions derived from blood incubated with enfuvirtide presented a decrease in pH and [HCO3-]. Fluorescence anisotropy measurements of 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-(trimethylamino)-phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), used to assess erythrocyte and lymphocyte membrane fluidity, did not yield enfuvirtide-induced changes (an effect could be expected due to peptide partition to lipid bilayers). Erythrocytes incubated with high enfuvirtide concentrations showed a significant decrease in osmotic fragility. As for erythrocyte deformability, enfuvirtide leads to increased elongation indexes for low shear stress values, whereas for high shear stress values it has the opposite effect. Despite the observed statistically significant variations in several parameters, these enfuvirtide-induced changes are not expected to lead to any detectable biomedical outcome for enfuvirtide-treated patients.     

Studies on Agrobacterium tumefaciens. IV. Nonreplication of the bacterial DNA in mung beam (Phaseolus aureus).

DNA·DNA filter hybridization and DNA solution enrichment reassociation experiments showed that no $\text{Agrobacterium tumefaciens}$ DNA was replicated in mung bean seedlings under the conditions specified in published reports for the uptake, integration, and replication of bacterial DNA in higher plants. Residual presumptive DNA hybrids that formed in a few instances were characterized by thermal chromatography on hydroxylapatite. The Tm and melting profiles of these hybrids from DNA-treated plants were the same as those from untreated control plants. The sensitivities of these procedures were sufficient to detect $\text{A. tumefaciens}$ DNA in the order of 0.005% to 0.01% of the plant genome. These results do not concur with previous reports that large pieces of DNA (at least 30%) of the plant genome of bacterial-DNA-treated-plants is made up of bacterial donor DNA.     

DNA topoisomerase from Agrobacterium tumefaciens: purification and catalytic properties.

The DNA topoisomerase from Agrobacterium tumefaciens has been purified to apparent homogeneity. The enzyme is a single polypeptide of about 100,000 in molecular weight. No apparent separation of the nicking and sealing activities could be obtained in attempts to separate the two activities by a variety of methods, including limited protease digestion, thermal denaturation, and differential inhibition. Monoclonal antibodies obtained from hybridomas likewise did not preferentially inhibit one of the two activities. These results suggest that the two catalytic functions are carried by the same essential residues of the active enzyme site.     

Distinctive functional properties of human blood L lymphocytes: a comparison with T lymphocytes, B lymphocytes, and monocytes.

Human blood lymphocytes with high affinity Fc receptors have been operationally named L lymphocytes because of membrane-labile IgG markers. L lymphocytes lack membrane-incorporated immunoglobulin and do not form rosettes with sheep red blood cells coated with IgM antibody and mouse complement. These lymphocytes are capable of binding IgG in normal human serum at 4 degrees C and will form rosettes with human lymphocytes coated with Ripley IgG. In this study, functional in vitro properties of isolated L lymphocytes were compared with T lymphocytes, B lymphocytes, and monocytes. To obtain these mononuclear populations, first, plastic adherent monocytes were harvested. T lymphocytes were then isolated by centrifugation of E rosette-forming cells, and other rosetting techniques were employed to isolate L and B lymphocytes by negative selection. The functional properties of L lumphocytes were completely unlike those of T cells, B cells, or monocytes. L lymphocytes did not proliferate in response to mitogens, soluble antigens, or cell surface antigens. Moreover, this population could not replace monocytes in helping T lymphocytes respond to concanavalin A and pokeweed mitogen. Once T cells were supplemented with monocytes, however, the addition of L lymphocytes to the culture greatly enhanced the T lymphocytes proliferative response to phytohemagglutinin, concanavalinA, purified protein derivative (PPD), and streptokinase/streptodornase. L lymphocytes were not a subset of B cells. They did not spontaneously develop surface Ig in culture, and pokeweek mitogen could not induce them to transform and generate cytoplasmic Ig detectable by immunofluorescence. Mixtures of B cells and T cells responded to pokeweed mitogen better than do T cells alone. In contrast, enhanced reactivity with L and T cell combinations was not observed. Another sharp difference between these two populations was the stimulator capacity of each in mixed lymphocyte culture. When B and L lymphocytes were carefully monocyte-depleted, only B cells were effective stimulators of autologous and allogeneic lymphocytes. In comparison with T cells, B cells, and monocytes, L lymphocytes were the only effective killers of human blood lymphocytes sensitized with IgG. L lymphocytes, then, have cytotoxic potential, but cannot proliferate in response to various stimulants or become antibody-producing cells. These findings suggest that L lymphocytes comprise a third lymphocyte population.     

Physical and functional map of supervirulent Agrobacterium tumefaciens tumor-inducing plasmid pTiBo542.

Agrobacterium tumefaciens strains carrying pTiBo542 induce large, fast-appearing tumors and have an unusually wide host range. A clone bank was made from this 250-kilobase plasmid in a wide-host-range vector, and restriction maps were determined for BamHI and SalI. The virulence genes, transferred DNA genes, plasmid incompatibility region, and a region that inhibits growth of certain A. tumefaciens strains were localized. The six virulence genes and two tms genes were highly homologous to the genes of pTiA6, but the tmr gene was not. Mutations in each of the six vir loci of pTiA6 were complemented by clones from the vir region of pTiBo542.     

Transfection and transformation of Agrobacterium tumefaciens.

Summary The freeze thaw transfection procedure of Dityatkin et al. (1972) was adapted for the transfection and transformation of A. tumefaciens. Transfection of the strains B6S3 and B6-6 with DNA of the temperate phage PS8cc186 yielded a maximum frequency of 2 10^-7 transfectants per total recipient population. In transformation of the strain GV3100 with the P type plasmid RP4 a maximum frequency of 3.5 10^-7 transformants per total recipient population was obtained. Agrobacterium Ti-plasmids were introduced in the strain GV3100 with a maximal efficiency of 4.5 10^-8. These experiments provide further evidence that the Ti-plasmid is responsible for the oncogenic properties of A tumefaciens and for its capacity to induce opine synthesis in Crown-gall plant cells.     

Effect of blood storage on radiation-induced micronuclei in human lymphocytes.

To evaluate the effect of blood storage on the yield of micronuclei (MN) in both irradiated (in vivo and ex vivo) and unirradiated peripheral blood lymphocytes (PBL), we applied the MN assay in cytokinesis-blocked (CB) PBL obtained from healthy subjects (n=11), and from cancer patients (n=10) who were undergoing fractionated partial-body radiotherapy (xRT). The heparinized blood samples were exposed to 137Cs-irradiation (0 Gy or 2 Gy) immediately after blood collection and were stored upright in test tubes either at room temperature (22 degrees C) or in the refrigerator (5 degrees C). Duplicate whole blood cultures from each sample were set up at 0 h, 96 h, and 120 h after ex vivo irradiation. Giemsa (10%) stained slides were prepared from each culture. MN yield was determined per 1000 binucleated cells. As compared to that obtained from the corresponding fresh blood samples, we found that (1) the 22 degrees C blood storage temperature did not affect MN yields in PBL of either healthy subjects or cancer patients up to 96 h, either with or without ex vivo irradiation; and (2) while blood samples were stored at 5 degrees C, the MN yield increased significantly in PBL of healthy subjects (with or without ex vivo irradiation) at 120 h, and in cancer patients (with ex vivo irradiation) at 96 h and 120 h. Since handling of the blood sample is important for CBMN assay during shipment or in the laboratory, our findings showed that blood storage at 22 degrees C or at 5 degrees C up to 96 h appeared to provide insignificant variations of the MN results as compared to fresh blood samples. However, the 96 h of blood storage at 5 degrees C elevated the MN frequency in ex vivo irradiated PBL of cancer patients who were undergoing xRT.     

Purification and properties of cytochrome c-556 from Agrobacterium tumefaciens B2a.

Cytochrome c-556 from Agrobacterium mefaciens B2a was isolated in a pure, homoneous state. The best purification procedure volved ammonium sulphate fractionation, delting on Sephadex G-25, column chromatographic fractionation on DEAE- and CM-cellulose, and gel filtration on Sephadex G-75 superfine. Substitution of the CM-cellulose step by isoelectric focusing was successful. The purity of the final preparation is warranted by the purity index value, the electrophoretic patterns in the absence and presence of sodium dodecylsulphate, the sedimentation profile and the N-terminal amino acid analysis (alanine). The absorption spectrum of reduced cytochrome c-556 has maxima at 318, 419, 526 and 555.5 nm. The molar extinction coefficient for the alpha-band is 20 200M-1cm-1. The isoelectric point, determined both by preparative and analytical isoelectric focusing, is 5.55 +/- 0.10. The molecular weight of cytochrome c-556 was determined by gel filtration as 12000 and by dodecylsulphate gel electrophoresis as 11 500.     

On the isolation of TI-plasmid from Agrobacterium tumefaciens.

An efficient lysis method for Agrobacterium cells was developed, which allows a reproducible isolation of the tumor inducing (TI)-plasmid. The lysis method is based on the sensitivity of this bacterium to incubation with lysozyme, n-dodecylamine,EDTA, followed by Sarkosyl, after growth in the presence of carbenicillin. We also present a procedure for the isolation of the TI-plasmid on a large scale, that might be used for the mass isolation of other large plasmids which like the TI-plasmid, can not be cleared with earlier described procedures. The purity of the plasmid preparations was determined with DNA renaturation kinetics, which method has the advantage that the plasmid need not to be in the supercoiled or open circular form.