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Biomedical subjects

J J Lightbody

Publications and source records attributed to J J Lightbody.

At least 19 recordsLinked to original sources
PubMed1993

The lethality of p60v-src in Saccharomyces cerevisiae and the activation of p34CDC28 kinase are dependent on the integrity of the SH2 domain.

The lethal effects of the expression of the oncogenic protein tyrosine kinase p60v-src in Saccharomyces cerevisiae are associated with a loss of cell cycle control at the G1/S and G2/M checkpoints. Results described here indicate that the ability of v-Src to kill yeast is dependent on the integrity of the SH2 domain, a region of the Src protein involved in recognition of proteins phosphorylated on tyrosine. Catalytically active v-Src proteins with deletions in the SH2 domain have little effect on yeast growth, unlike wild-type v-Src protein, which causes accumulation of large-budded cells, perturbation of spindle microtubules and increased DNA content when expressed. The proteins phosphorylated on tyrosine in cells expressing v-Src differ from those in cells expressing a Src protein with a deletion in the SH2 domain. Also, unlike the wild-type v-Src protein, which drastically increases histone H1-associated Cdc28 kinase activity, c-Src and an altered v-Src protein have no effect on Cdc28 kinase activity. These results indicate that the SH2 domain is functionally important in the disruption of the yeast cell cycle by v-Src.

CDC28 Protein Kinase, S cerevisiae↗
PubMed1988

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.

Animals↗
PubMed1986

Preparation and characterization of monoclonal antibodies to the extracellular hemoglobin of Lumbricus terrestris.

Murine monoclonal antibodies to the extracellular hemoglobin of Lumbricus terrestris were prepared by a modification of the method of Kohler and Milstein. 224 hybridomas were found to produce antibodies which bound to the hemoglobin; they were tested for binding to the four subunits of the hemoglobin: M (chain I, 16 kDa), D1 (chain V, 31 kDa), D2 (chain VI, 37 kDa) and T (50 kDa), a disulfide-bonded trimer of chains II, III and IV, each of about 17 kDa. 150 hybridomas bound to all four subunits and 40 hybridomas bound to various combinations of subunits. The remaining 34 hybridomas combined only with the hemoglobin. The twelve hybridomas obtained after subculturing and cloning were tested for their binding to the two fractions II and III, consisting of subunits D1 + D2 + T and M, respectively, obtained by dissociation at pH 9.5 and at pH 4.0 and to the reassociated whole molecules, obtained subsequent to return to neutral pH. Eight hybridomas which combined only with the hemoglobin also combined with all the reassociated molecules but not with any of the fractions: these monoclonal antibodies probably recognize conformation-dependent antigenic sites that are present only in the hexagonal bilayer structure characteristic of the native and reassociated hemoglobin molecules. Of the remaining four hybridomas, two bound to subunit T and two combined with subunits T and D2; they also combined with the reassociated molecules and with the fractions II. In addition, the hybridomas did not bind to the hemoglobins of Tubifex, Limnodrilus, Arenicola, Tylorrhynchus and Macrobdella or to the chlorocruorins of Myxicola and Eudistylia.

Amino Acids↗
PubMed1986

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.

Amino Acid Sequence↗
PubMed1983

A phenotypically normal revertant of an adenosine deaminase-deficient lymphoblast cell line.

Two lymphoblast lines from a patient with partial adenosine deaminase deficiency have been obtained. The patient has a T cell deficiency, with normal B cell function, and has been successfully treated by multiple partial exchange transfusions with normal erythrocytes. The patient's lymphocytes have about 8% of normal adenosine deaminase activity. The derived lymphoblast line that initially had low adenosine deaminase activity has undergone spontaneous reversion to normal enzyme activity. The HL-A types remain the same as the patient's. Both cell lines have the same HL-A types, and eight isoenzymes are identical. In addition, the isoenzymes of a fibroblast line derived earlier, GM-2445, are identical with those in our lymphoblast lines. The following characteristics of the enzyme in the cell lines are normal: Km, Vmax, inhibitor sensitivity, heat sensitivity, and m.w. This suggests, but does not prove, that the low adenosine deaminase activity in this patient is caused by underproduction of a normal enzyme, and the observed reversion to normal activity in one line is a correction of this regulation defect.

Adenosine Deaminase↗
PubMed1979

The effect of nucleosides and deoxycoformycin on adenosine and deoxyadenosine inhibition of human lymphocyte activation.

Micromolar deoxyadenosine inhibits leucine uptake during the 1st day of proliferation in mitogen-stimulated lymphocytes if adenosine deaminase is inhibited. This inhibition occurs before DNA synthesis begins, suggesting that deoxyadenosine can affect mitogenesis by mechanisms that do not involve ribonucleotide reductase inhibition. If deoxyadenosine addition to mitogen-stimulated lymphocytes is delayed to the 2nd or 3rd day post-stimulation, inhibition of proliferation is markedly reduced. Although the time dependence of deoxyadenosine toxicity resembles that of adenosine, these compounds appear to inhibit early protein synthesis by different mechanisms: 1) deoxycoformycin markedly potentiates deoxyadenosine but not adenosine; 2) deoxycytidine and thymidine reverse deoxyadenosine toxicity but do not alter adenosine toxicity.

Adenosine↗
PubMed1978

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

The 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.

Animals↗
PubMed1978

Factors affecting the stimulating capacity of mitogen-transformed, glutaraldehyde-treated mouse spleen cells in the mixed lymphocyte reaction.

Mouse spleen cells transformed by certain mitogens and subsequently fixed with glutaraldehyde can be utilized as stimulators in the mixed lymphocyte reaction. In addition to bacterial lipopolysaccharide which has been shown previously to be effective, we have found the other B cell mitogens, dextran sulfate and Concanavalin A-Sepharose, to be functional. The ability of the treated cells to activate allogeneic cells in MLR depends upon the concentration of glutaraldehyde and the type of tissue culture medium utilized.

Aldehydes↗
PubMed1978

Cytotoxicity of human peripheral lymphocytes in cell-mediated lympholysis; antibody-dependent cell-mediated lympholysis and natural cytotoxicity assays after mixed lymphocyte culture.

Lymphocytes that have been purified by Ficoll-Hypaque centrifugation lose antibody-dependent and natural cytotoxic activities upon culture in tissue culture medium supplemented with human plasma. However, stimulation of peripheral lymphocytes in the mixed leukocyte culture (MLC) appears to enhance killer (K) and natural killer (NK) activities in addition to generating cytotoxic T ymphocytes. Enhancement of NK and antibody dependent activities appears to correlate with cell division as measured by 3H-thymidine uptake. However, elimination of dividing cells in the MLC by addition of 5-bromodeoxyuridine has no effect on NK and K cells activities. Since this treatment abolishes cell-mediated lympholysis mediated by cytotoxic T lymphocytes, it is a useful probe for determining the relative activities of NK, K, and cytotoxic T lymphocyte effector cells after lymphocyte stimulation.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1976

Isolation of a human lymphocyte mitogen from wheat germ with N-acetyl-D-glucosamine specificity.

A lectin, isolated from wheat germ by affinity chromatography on chitin, was mitogenic for purified human peripheral blood lymphocytes. Peak incorporation of 3H-thymidine was observed after incubation of lymphocyte cultures with wheat germ mitogen for 7 to 10 days. When lymphocytes were separated into two fractions based on their ability to form rosettes with unsensitized sheep erythrocytes, the mitogen induced a negligible proliferative response in either fraction. Mixing experiments demonstrated a strong response in the T lymphocyte fraction which required the collaboration, but not proliferation, of cells present in the nonrosetting fraction. Stimulation was specifically abolished by addition of N-acetyl-D-glucosamine at initiation of culture.

Acetylglucosamine↗
PubMed1975

Suppression of lymphocyte activation by a factor produced by Mycoplasma arginini.

Mycoplasma arginini, when grown in broth or in cultures of human lymphoblast cell lines, inhibits activation of lymphocytes by allogeneic cells of mitogens. This inhibition can also be produced by cell free media obtained after the growth of this organism. Inhibition is not species specific and inhibits both B and T cell activation in the mouse. The inhibitory factor is required during the first 3 days of the mixed leukocyte culture, but has no effect on the latter phase of this reaction when thymidine uptake is greatest. The factor is stable to treatment at 60 degrees, but not 90 degrees, for 30 min.

Animals↗