James J. Lightbody

Isolation and properties of plastocyanin from Anabaena variabilis

Abstract Plastocyanin is a copper protein found in photosynethetic tissue and it exhibits the properties of a physiological redox reagent. This protein has been purified from the blue-green alga Anabaena variabilis . Plastocyanin is required for a number of partial reactions of the photosynthetic electron transfer chain. These reactions include the transfer of electrons from reduced 2,3′,6-trichlorophenolindophenol, N , N , N ′, N ′- tetramethyl- p -phenylenediamine and 2,3,5,6-tetramethyl- p -phenylenediamine to low potential oxidants. Reduced cytochrome c photooxidation does not appear to be dependent on plastocyanin. Cytochrome f , isolated from this alga, will partially replace plastocyanin in many of these reations. Inhibition of photosynthetic reactions by copper chelators appears to occur at some site other than the site of plastocyanin function.

Spontaneous cytotoxicity of human lymphoblast cell lines mediated by normal peripheral blood lymphocytes. I. Differential susceptibility of T-versus B-cell lines.

Abstract Human lymphoblast cell lines of B- and T-cell origin have been tested for their ability to serve as targets in a 4-hr 51Cr release microcytotoxicity assay using normal human peripheral blood lymphocytes as effector cells. Cell lines of T-cell origin were susceptible to lysis in this assay by effector lymphocytes from all normal donors tested. Cell lines of B-cell origin were repeatably lysed by normal lymphocytes from some, but not all donors. Spontaneous cytotoxicity of B-cell lines, when observed, was also quantitatively less than was obtained using T-cell lines as targets. One cell line (RPMI-7666), of B-cell origin, was not susceptible to spontaneous cytotoxicity by almost all of the normal lymphocyte effectors tested. Lymphocytes from patients with acute lymphoblastic leukemia in remission were less capable of effecting lysis in this assay.

A disulfide-bonded trimer of myoglobin-like chains is the principal subunit of the extracellular hemoglobin of Lumbricus terrestris

The extracellular hemoglobin of Lumbricus terrestris (3900 kDa) consists of at least six different polypeptide chains: I through IV (16-19 kDa), V (31 kDa) and IV (37 kDa) (Vinogradov, S.N., Shlom, J.M., Hall, B.C., Kapp, O.H. and Mizukami, H. (1977) Biochim. Biophys. Acta 492, 136-155). SDS-polyacrylamide gel electrophoresis of the unreduced hemoglobin shows that chains II, III and IV form a disulfide-bonded 50 kDa subunit. This subunit was isolated by gel filtration of the hemoglobin on Sephacryl S-200 (a) at neutral pH in 0.1% SDS and (b) in 0.1 M sodium acetate buffer (pH 4.0); in the latter case it retains heme. The 50 kDa subunit obtained by method (b) was reduced and subjected to chromatofocusing on PBE 94 column: the elution pattern obtained with Polybuffer 74 (pH 4.5) and monitored at 280 nm, consisted of three peaks A, B and C; peaks A and B but not C, had absorbance at 410 nm. SDS-polyacrylamide gel electrophoresis showed that peaks A, B and C corresponded to chains II, IV and III, respectively. Amino acid analyses and N-terminal sequence determinations identified chain II as the whose primary structure had been determined (Garlick, R. and Riggs, A. (1982) J. Biol. Chem. 257, 9005-9015). Carbohydrate analysis of the native hemoglobin shows it to contain 2.0 +/- 0.5% carbohydrate consisting of mannose and N-acetylglucosamine in a mole ratio of about 9:1. The carbohydrate content of the 50 kDa subunit is 1.8 +/- 0.5%; it consists of mannose and N-acetylglucosamine in the same ratio and it appears to be associated with chain IV. Rabbit polyclonal antisera to 50 kDa subunit, prepared by method (a), and to the native hemoglobin were shown to cross-react with the hemoglobin and the 50 kDa subunit, respectively, by immunodiffusion. One of eight mouse monoclonal antibodies directed against the native hemoglobin reacted strongly with the 50 kDa subunit prepared by methods (a) and (b) in an enzyme-linked immunosorbent assay (ELISA). Another monoclonal antibody reacted strongly with the 50 kDa subunit obtained by method (b). Neither of the two hybridomas exhibited a strong reaction with any of the three constituent chains of the 50 kDa subunit. These results suggest that the unusual disulfide-bonded 50 kDa subunit, consisting of three myoglobin-like polypeptide chains of which only two have heme, is an integral part of the native Lumbricus hemoglobin molecule.

Determination of adenosine deaminase activity using high-pressure liquid chromatography.

Abstract A method of assaying adenosine deaminase, using high-pressure liquid chromatography to isolate products, has been developed, which has several advantages over available procedures. The method has inherently low background values, affording high sensitivity. Ten picomoles of product can be reliably detected, so that as little as 0.008 unit of enzyme can be determined. Up to six samples per hour can be assayed. The procedure has been applied to erythrocytes and lymphocytes.

Stimulation in the mixed leukocyte culture and generation of effector cells in cell mediated lympholysis by a human T lymphoblast cell line

Abstract The lymphoblast cell lines CCRF-SB possessing B cell surface markers and CCRF-HSB-2 possessing T cell surface markers, both derived from the same individual, were tested for their ability to stimulate in the mixed leukocyte culture (MLC) and their ability to generate effector cells in cell mediated lympholysis (CML). Both the T and B cell lines were capable of stimulating human peripheral blood lymphocytes, however, the T cell line required four times as many cells for an equivalent response. Generation of effector cells in CML was achieved when both the T and B cell lines were used as stimulator cells but the T cell line was capable of generating a greater degree of 51 Cr release.

The role of plastoquinone in the photosynthetic reactions of Anabaena variabilis

Abstract The role of plastoquinone in the photosynthetic reactions of cell-free preparations of the blue-green alga, Anabaena variabilis, has been studied. Plastoquinone is required for the reduction of several oxidants in the Hill reaction, but is not required for the photoreduction of TPN+ when either reduced indophenol dye or N,N.N′,N′-tetramethyl-p-phenylenediamine is used as the electron donor instead of water. Also, plastoquinone is not required for the photooxidation of reduced cytochrome c. The synthesis of ATP elicited by PMS is dependent on the presence of plastoquinone in the preparations from A. variabilis. The light-triggered hydrolysis of ATP is apparently independent of plastoquinone while the light-dependent hydrolysis of ATP requires plastoquinone.

Comparison of 137cs irradiation and mitomycin c treatment of stimulator cells in the mixed lymphocyte culture reaction.

Abstract 137Cs-irradiation has been compared with mitomycin C treatment for the inhibition of DNA synthesis in the one-way mixed lymphocyte culture (MLC) reaction in mice and humans. 137Cs-irradiation was found to be as effective as mitomycin C in preventing cell proliferation without interfering with the ability of the cell to stimulate allogeneic cells in MLC.

The effect of adenosine on mitogenesis of ADA-deficient lymphocytes.

Abstract We have determined the effects of adenosine on intrinsically adenosine deaminase-deficient lymphocytes. These cells are circulating peripheral lymphocytes from a patient with T-cell immunodeficiency. The patient has been successfully treated with normal red cell transfusions. The lymphocytes contain about 8% of the normal adenosine deaminase level. Mitogen response is nearly normal but is inhibited by adenosine at one tenth the level required for inhibition of normal lymphocytes. Determination of the disappearance of exogenously added adenosine to lymphocytes cultured in horse serum suggest that (1) ADA-deficient cells are more sensitive to adenosine toxicity because of an impaired ability to deaminate adenosine and (2) that adenosine inhibits mitogenesis only if it is present during the first 24 hr of mitogen stimulation.

Suppression of lymphocyte proliferation by a nonspecific factor produced by the burkitt lymphoma-derived lymphoblast cell line

SummaryThe DAUDI lymphoblast cell line derived from a patient with Burkitt lymphoma was obtained from two different sources. One of these (DAUDI-I) produced a factor that inhibited lymphocyte proliferation in both human and mouse regardless of the stimulator, i.e. allogeneic lymphocytes or mitogens. Glutaraldehyde treatment eliminated production of the factor and demonstrated that DAUDI-I was capable of stimulating normal lymphocytes in MLR. A second DAUDI cell line (DAUDI-S) did not produce the inhibitory factor and was capable of MLR stimulation.

Immunological relatedness of annelid extracellular hemoglobins and chlorocruorins

1. The immunological relatedness of several annelid extracellular hemoglobins and chlorocruorins was investigated using ELISAs and Western blotting to determine the binding of purine polyclonal and monoclonal antibodies to Lumbricus terrestris hemoglobin with the hemoglobins of Tubifex tubifex, Tylorrhynchus heterochaetus, Arenicola marina and Macrobdella decora and the chlorocruoins of Myxicola infundibulum and Eudistylia vancouverii. 2. Polyclonal antibodies to Lumbricus terrestris hemoglobin bound to all the other hemoglobins and chlorocruorins. However, the titers were in all cases one to several orders of magnitude smaller than with Lumbricus terrestris hemoglobin. 3. Polyclonal antibodies to Eudistylia vancouverii chlorocruorin bound to the hemoglobins of Lumbricus terrestris, Tubifex tubifex, Arenicola marina, Tylorrhynchus heterochaetus and Macrobdella decora. 4. Of the nine monoclonal antibodies to Lumbricus terrestris hemoglobin isolated, two (No. 24 and No. 26) bound to the other hemoglobins and to Myxicola chlorocruorin, but the binding was again weaker than with Lumbricus hemoglobin. Antibody No. 26 also bound to Eudistylia chlorocruorin. Although antibody No. 24 appears to recognize a conformation-dependent epitope, antibody No. 26 recognizes a common epitope in each of the four subunits M, D1, D2, and T of unreduced Lumbricus hemoglobin. 4. An additional two monoclonal antibodies to Lumbricus hemoglobin (No. 21 and No. 25) bound also only to Tubifex hemoglobin. Antibody No. 21 recognizes subunits D1 and M of Lumbricus hemoglobin and No. 25 appears to recognize a conformation-dependent epitope.