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Biomedical subjects

J D Kemp

Publications and source records attributed to J D Kemp.

At least 91 records · Page 5Linked to original sources
PubMed1979

Purification and characterization of the crown gall specific enzyme nopaline synthase.

Nopaline synthase of sunflower (Helianthus annuus L.) crown gall tissue induced by Agrobacterium tumefaciens strain C58 or T37 (nopaline utilizers) was purified to homogeneity as judged by analytical disc gel electrophoresis. The native enzyme elutes from a column of Ultrogen AcA 34 as a single peak with an estimated molecular weight of 158,000. The dissociated enzyme migrates on NaDodSO4-polyacrylamide gels as a single band with a molecular weight of 40,000. Thus, the native enzyme appears to be composed of four equal-weight subunits. Nopaline synthesizing activity is found exclusively in crown gall tissues induced by strains of A. tumefaciens that utilize nopaline (e.g., C58 and T37). We found the same tissue specificity for the purified protein that we believe represents nopaline synthase. The results of kinetic studies of the purified enzyme are consistent with a ter-bi rapid-equilibrium random-order mechanism. Nopaline synthase is probably responsible for the in vivo synthesis of both N2-(1,3-dicarboxypropyl)arginine (nopaline) and N2-(1,3-dicarboxypropyl)ornithine (ornaline) in crown gall tissues since substrate specificities and Km values do not change during purification.

Amino Acid Oxidoreductases↗
PubMed1979

Transcription of Ti plasmid-derived sequences in three octopine-type crown gall tumor lines.

Total RNA isolated from three octopine-type crown gall lines contains sequences homologous to specific regions of the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens strain 15955. A comparison of transcripts in these three tumor lines suggests that tumor cells transcribe various sequences within a sector of plasmid DNA of 13 x 10(6) daltons and that transcription may not be uniform across the plasmid derived sequences (T-DNA). Transcription of T-DNA by octopine-type tumors occurs at four major sites. The levels of transcription occurring at three of these sites appear to vary considerably among the three tumor lines investigated. Part of this variability may reflect differences in the organization and copy number of T-DNA. One of the transcription sites maps within a region of DNA with common sequence homology with all Ti plasmids. Varying amounts of transcript homologous to this region of T-DNA are present in all three tumor lines. It is suggested that transcription of these conserved sequences in the plant may have significance regarding the mechanism of tumorigenesis.

Arginine↗
PubMed1979

Isolation of a non-tumor-inducing mutant of the Ti plasmid of Agrobacterium tumefaciens strain B6.

A nonpathogenic mutant of Agrobacterium tumefaciens strain B6 was isolated and its properties compared with the parental strain in an effort to localize the mutation. Both B6 and its mutant (B6-95) had similar colony color and morphology, were ketolactose positive, utilized octopine, and contained plasmid DNA. Kinetic analysis of DNA reannealing showed that total DNA homology and plasmid DNA homology between B6 and B6-95 was at least 90%. The length of both plasmids was found to be 58 micrometer. Plasmid DNA from both B6 and the mutant was digested with endonucleases and the fragments separated by agarose gel electrophoresis. In all cases the pattern for B6 was identical with that of B6(-95). The Ti plasmid from B6 and the mutant was transferred to an avirulent, plasmidless strain of A. tumefaciens by in vitro conjugation and transformation. All of the B6 transconjugants and transformants were virulent, whereas all of the mutant transconjugants and transformants were avirulent. Electrophoretic patterns of endonuclease-digested plasmid DNA from transformants were identical to those of plasmid DNA from B6. Therefore, we conclude that the virulence mutation lies on the Ti plasmid.

Conjugation, Genetic↗
PubMed1979

Induction of suppressor cells in rat spleen: influence of microbial stimulation.

The role of the products of prostaglandin synthetase in the suppression of in vitro secondary antibody responses by rat spleen cells was examined. By including cells from both germfree and conventional rats in these studies, the effect of in vivo microbial stimulation on this suppression was also determined. We found that 1) the suppression that is normally present in the spleen cells of rats can be eliminated by the use of glass wool or indomethacin; 2) germfree rats do not display this suppressor activity, and 3) this "deficit" in the germfree rat may be due to a lack of a microbial-induced signal from a nonadherent cell to an adherent prostaglandin-containing macrophage, implying that at least two cells are involved in the prostaglandin-synthetase dependent suppressor effect. It is possible that large amounts of some or all microbial products activate "suppressor" macrophages and that inbred rats behave like "chronically infected" mice because of an inability to regulate their "normal" microbial flora.

Animals↗
PubMed1979

Immunity to Plasmodium yoelii and Babesia microti: modulation by the CBA/N X-chromosome.

CBA/N mice carry an X-linked recessive defect expressed in cells of the B cell lineage. The major deficiency in these mice is an almost complete inability to respond to certain thymus-independent antigens, such as pneumococcal polysaccharide type III (S III). We have examined the responses of mice carrying the CBA/N X-chromosome to the malaria parasite Plasmodium yoelii and the piroplasm Babesia microti. We have found that the duration and severity of these infections is increased in mice carrying the CBA/N X-chromosome and that this is associated with a markedly defective IgM antibody response to the parasitized red cell and a failure to produce autoantibodies to bromelain-treated mouse RBCs. An autoimmune response directed at modified determinants on the red cell membrane may be one of the factors involved in the control of these infections.

Babesia↗
PubMed1979

Experimental malaria in the CBA/N mouse.

CBA/N mice carry an X-linked, recessive gene, which results in the absence of a B cell subset, and is expressed primarily as an inability to respond to a certain class of thymus-independent antigens. We have examined the responses of these mice to the malaria parasite Plasmodium yoelii and found that primary infections induced by this parasite are more severe and last longer in mice with X-linked defect than in normal controls. The decreased resistance of the defective mice is associated with a striking deficiency in their IgM antibody response. After recovery from a primary infection, defective mice resist reinfection with the homologous parasite as well as normal mice. Although as resistant as normal controls, B cells from defective mice transfer considerably less immunity to naive recipients than B cells from normal animals. Hence, two modes of thymus-dependent protective immunity may contribute to the host response to P. yoelii. Control of an acute primary infection appears to involve a thymus-dependent antibody response that CBA/N mice are deficient in. Resistance to reinfection may be mediated primarily by a different mechanism.

Animals↗
PubMed1978

Metastasizing atypical fibroxanthoma. Coexistence with chronic lymphocytic leukemia.

Atypical fibroxanthoma (AFX) is one of a group of cutaneous lesions with a malignant histological appearance but a generally benign clinical course. A 79-year-old white man had AFX of the cheek that recurred and metastasized to buccal and cervical lymph nodes three months after initial diagnosis. When careful physical and and laboratory examinations were done, the patient was found to have concomitant chronic lymphatic leukemia, "null cell" type. In view of the low incidence of metastasizing AFX and the increased occurrence of tumors in patients with lymphomatous disorders, an important association is suggested. Before establishing the prognosis for patients with pseudomalignancies of the skin, an evaluation of their general health and immunological status should be made.

Aged↗
PubMed1978

In Vivo Synthesis of Crown Gall-specific Agrobacterium tumefaciens-directed Derivatives of Basic Amino Acids.

Several kinds of primary sunflower (Helianthus annuus) crown gall tissues were established in tissue culture and then labeled in vivo with either [(14)C]arginine, [(14)C]histidine, [(3)H]lysine, or [(3)H]ornithine. Crown gall tissues incited by Agrobacterium tumefaciens strains that utilize octopine as a sole source of carbon or nitrogen for growth synthesized the four members of the N(2)-(1-carboxyethyl)-amino acid family: octopine, histopine, lysopine, and octopinic acid. Those tissues incited by A. tumefaciens strains that utilize nopaline synthesized nopaline and two new compounds, a lysine and an ornithine derivative (ornaline). A normal tissue culture, a habituated tissue culture, and a crown gall culture from a strain of the bacteria unable to utilize either octopine or nopaline did not synthesize any of the amino acid derivatives. We could not detect any other crown gall-specific derivatives of the four basic amino acids.

Journal Article↗
PubMed1978

Characterization of the enzyme responsible for nopaline and ornaline synthesis in sunflower crown gall tissues.

Extracts prepared from sunflower (Helianthus annuus L.) crown gall tissues induced by Agrobacterium tumefaciens strains C58 and T37 (nopaline utilizers) catalyze the synthesis of nopaline and ornaline. These compounds are not synthesized in extracts of crown gall tissues induced by strains B6, 15955 (octopine utilizers), and AT1 (utilizes neither octopine nor nopaline) or in extracts of habituated sunflower callus. Both synthetic activities require NADPH, alpha-ketoglutarate, and either arginine or ornithine; histidine and lysine will not substitute. Incorporation of arginine or ornithine into product is inhibited by the other substrate but not by histidine or lysine. On the basis of inhibition and K(m) data, both activities appear to be catalyzed by one enzyme and the same enzyme is apparently present in crown gall tissues induced by strains C58 and T37.

Journal Article↗
PubMed1976

A chemical and physical method for determining the complete base composition of plant DNA.

Two physical methods are routinely used to determine the base composition of DNA. One measures the temperature corresponding to the midpoint of the absorbance rise (TM) and relates it to base composition with the equation, TM = 41 (dG + dC) + 69, the other measures buoyant density (rho) and relates it to base composition rho = 0.098(dG + dC) + 1.6535. The base composition of DNA from various sources was first determined by a chemical method and these values compared to those determined by the physical methods. Higher plants contained up to 7 mol% 5-methyldeoxycytidine in their DNA and in all cases tested deoxyguanosine = deoxycytidine + 5-methyldeoxycytidine. After determining that TM was unaffected by the amount of 5-methyldeoxycytidine in DNA, the mol% of dA, dT, dG, and the total of dC plus 5-methyldeoxycytidine for any DNA could be calculated. Buoyant density on the other hand, was lowered 0.004 g . cm-3 for every 6.3 mol% 5-methyldeoxycytidine. Therefore, both physical parameters were related to the mole fraction of 5-methyldeoxycytidine by the following equation: (see article). With a value of r 5-methyldeoxycytidine an estimation of deoxycytidine was made. The resultant values agreed with the chromatographic determinations.

Deoxyribonucleosides↗
PubMed1976

Attempts to Detect Agrobacterium tumefaciens DNA in Crown-Gall Tumor Tissue.

Primary and secondary crown gall tissue cultures were established from sunflower plants (Helianthus annuus, variety Mammoth Russian) wound-inoculated with Agrobacterium tumefaciens (Smith and Townsend) Conn strain B(6). Growth rates of tumor tissues and habituated healthy sunflower stem section tissues on basal medium lacking auxin and cytokinin were compared to those of healthy sunflower stem section tissue grown on the same medium with added phytohormones. No difference was detected in the thermal denaturation midpoints (74.8 C) and melting profiles in 25 mm sodium phosphate (pH 6.8), or the buoyant densities in cesium chloride equilibrium centrifugation (1.687 g cm(-3)), between deoxyribonucleic acids (DNAs) isolated from crude nuclear preparations of the four tissue types. No satellite DNA was observed in equilibrium centrifugation of unsheared plant DNAs.Heterologous DNA renaturation kinetic analyses were performed in 0.14 m sodium phosphate (pH 6.8) at 70 C. Thermal stability measurements of reassociated DNA revealed less than 1% of mismatched base pairs. Reannealing of sheared, denatured, radioactive A. tumefaciens B(6) DNA (molecular weight, 325,000 daltons) in the presence of a 5400-fold excess of sheared calf thymus, healthy tissue, or secondary sunflower crown gall DNA obeyed second order kinetics, with a Cot((1/2)) of 2.8, identical to that observed when B(6) DNA was reannealed in the absence of foreign DNA.Reannealing rates of B(6) DNA in the presence of 5400-fold excesses of DNA from two lines of primary sunflower crown gall were increased 2.24- or 1.47-fold. Digestion of the tumor DNA preparations with pancreatic deoxyribonuclease I until no detectable DNA remained, followed by restoration of solution viscosity by added calf thymus DNA, failed to remove the acceleration effect of the tumor DNA preparations. Reisolation of the reannealed nucleic acid formed in this experiment, and digestion with ribonuclease A or deoxyribonuclease I revealed that the double-stranded fraction was composed entirely of DNA-DNA duplexes, with no detectable DNA-RNA hybrids.The data indicate that tumor, but not healthy tissue DNA preparations contain some factor or factors (not DNA) which accelerate the reannealing of bacterial DNA. Sunflower tumor tissue DNAs, therefore, do not contain integrated A. tumefaciens DNA sequences in amounts greater than a random (1/5) of the bacterial genome per diploid amount of plant DNA, or a complete bacterial genome per five diploid plant cell DNA equivalents. Further, the possibility of the presence of many copies of a specific portion greater than 5% of the bacterial genome is excluded.

Journal Article↗