Showing papers by "Kettering University published in 1976"

Properties of the K562 cell line, derived from a patient with chronic myeloid leukemia

Abstract: The K562 cell line derived from a CML patient in blast crisis was examined for properties of B and T lymphocytes and cell lines. K562 lacks the B markers of immunoglobulin, Epstein-Barr virus (EBV) genome and associated nuclear antigen, and receptors for EBV. A low proportion of cells form rosettes with sheep erythrocytes, the frequency of which is considerably increased after neuraminidase treatment. Unlike B lines but like T lines, K562 cells are lysed rapidly by C'/Fc receptor-positive human blood leukocytes and do not stimulate MLC reactions. On the other hand, K562 lacks T antigen, high radiosensitivity and sensitivity to growth inhibition by thymidine. The cells do not contain N-APase, an enzyme found in all lines derived from lymphoid cells and in lymphoproliferative diseases. By scanning electron microscopy, K562 cells were seen to be rounded and relatively smooth, with small numbers of short microvilli resembling undifferentiated leukemic cells. A few cells had narrow ridge-like profiles and small ruffles similar to granulocytic leukemic cells. K562 is strongly positive for immunoglobulin Fc receptors and pinocytosis, but does not phagocytose or mediate antibody-dependent phagocytosis or cytolysis. Among histochemical stains, K562 is positive for esterase, lipid, and acid phosphatase. There seems to be no doubt that K562 is not a B cell line. While it has some T cell properties, these are not exclusive. Some of its characteristics indicate that it is probably not lymphoid. Due to its low level of differentiation, its nature cannot be stated with certainty. On the basis of the possible presence of the cellular marker of chronic myeloid leukemia, the Ph chromosome, it may be regarded as belonging to the granulocytic series of cells. Propretes de la lignee k562, qui provient d'un sujet atteint de leucemie myeǐotde chronique La lignee K562, qui provient d'un sujet atteint de CML en crise blastique, a ete etudee du point de vue des proprietis des lymphocytes B et T et d'autres lignees cellulaires. Elk ne possede ni les marqueurs B de I'immunoglobuline, ni le genome du virus d'Epstein-Barr (EBV), ni l'antigene nucliaire qui h i est assocei, ni les recepteurs de I'EB V. Une faible proportion des cellules forment avec les erythrocytes de mouton des rosettes dont la frequence s'accrǐt considerablement aprds traitement a la neuraminidase. A la difkrence des lignees Bmais comme les ligntes T, les cellules K562 sont lysies rapidement par les leucocytes du sang humain portant des rkcepteurs C'IFc et ne stimulent pas les reactions en MLC. Par contre, elks nepossbdentpas l'antigbne Tet ne sont pas fortement radiosensibles; leur croissance n'est pas inhibke par la thymidine; elles ne contiennent pas de N-APase, une enzyme prisente dans toutes les lignies provenant de cellules lymphoides et dans les maladies Iymphoproliferatives. Au microscope eectronique ´ balayage, on constate que les cellules K562 sont arrondies, relativement lisses, avec quelques courtes microvillosites qui les font ressembler a des cellules leucemiques indifferenciees. Quelques cellules ont des protuberances en forme d'arětes etroites et de petites rides comme les cellules leucemiques granulocytaires. Pour les recepteurs Fc de l'immunoglobuline et la pinocytose, la lignee K562 est fortement positive. Elle ne phagocyte pas et ne declenche pas de phagocytose ou de cytolyse dependant de l'anticorps. Dans les tests de coloration histochimique, on a constate que la lignee K562 est positive pour l'esterase, les lipides et la phosphatase acide. Il ne s'agit incontestablement pas d'une lignee de cellules B. Si elle a quelques-unes des proprietes des cellules T, elle a aussi d'autres caracteristiques, dont certaines indiquent qu'elle n'est probablement pas lymphoide. Du fait de son faible niveau de differenciation, on ne peut pas determiner sa nature avec certitude. En raison de la presence possible du marqueur cellulaire de la leucemie myeloide chronique, le chromosome Ph, on peut considerer qu'elle appartient a la serie granulocytaire.

Human mixed-lymphocyte culture reaction: genetics, specificity, and biological implications.

Abstract: Publisher Summary This chapter deals with the new and exciting developments in MLC (multi-level cell) typing in human histocompatibility studies. The genetics, specificity, and biological implications of human mixed-lymphocyte culture reaction are also discussed. The use of homozygous cells from specific delineates at least six different distinct MLC antigens that can be recognized by B-cell-specific alloantisera and clearly relate to the Ia antigens of the murine system. Certain disease associations and the genes involved in the complement components, appear more closely linked to the MLC genes than to the other components of the HLA (human leukocyte antigen) system. The MHC (major histocompatibility complex) in man HLA is defined by four distinct loci: (1) HLA-A, (2) B, (3) C, and (4) D. Three of these loci code for alloantigen are readily detectable by serological methods (HLA-A, B, and C). The fourth locus (HLA-D) controls lymphocyte responses in the in vitro mixed-lymphocyte culture reaction (MLR). Three important characteristics shared by the genes or gene products of each locus are also discussed. The study of genetic control of immune response and that of clinical transplantations emphasizes the significance of the HLA-D segment of the HLA complex. The chapter summarizes the major achievements in the clarification of the genetic control and polymorphism of the serologically detectable HLA-A, B, and C determinants.

Suppression of adenocarcinoma by the immunological consequences of calorie restriction.

Abstract: EARLIER extensive studies have indicated that calorie restriction as well as protein and amino acid restriction inhibit the spontaneous development of mammary or lung adenocarcinomas, hepatomas and certain chemical carcinogen-induced tumours in rodents1–4. Jose and Good5–7 and Cooper et al.8 have shown that chronic moderate protein deprivation in mice and rats dramatically depresses antibody production while increasing, or permitting maintenance of, vigorous cell-mediated immune responses. The latter include abilities to resist virus infection, reject skin allografts and develop killer-cell activity against syngeneic and allogeneic tumour cells. More profound chronic protein deprivation, however, depresses both cell-mediated and humoral immunity7,9. Walford et al.10,11 have shown that calorie restriction delays development of immune functions at an early age but prolongs maintenance of immunological capacity and survival in long lived, tumour-free mice. Further, Fernandes et al.12 have shown that dramatic prolongation of life of autoimmunity-prone, short lived mice was produced by life-long calorie restriction, and that tumours did not appear in these mice. Although some of the findings clearly link moderate protein and calorie restriction to heightened cellular immunity, no definitive efforts have yet been made to compare directly the influence of dietary restriction on spontaneous tumour development and immunological functions. This report presents observations on the influence of calorie restriction on development of spontaneous mammary adenocarcinoma in mice and relates the suppression of tumour development observed to alterations in immune functions produced by calorie restriction at weaning.

Chromosome 21 and the Cell Growth Inhibitory Effect of Human Interferon Preparations

Abstract: INTERFERONS constitute a class of biologically active glycoproteins1–3 which induce a spectrum of physiological changes in living cells, leading to the inhibition of virus replication4, of cell growth in vitro—both normal and tumour cells5,8—and of tumour growth in vivo9–15. The mechanism(s) underlying these effects is unknown. Gressor et al. have suggested that the same molecule in an interferon preparation is responsible for both the antiviral (AV) and cell growth inhibitory (CGI) effect5. This suggestion was based on the observation of a consistent and direct correlation between the two effects in an interferon preparation purified more than a millionfold. Gressor also reported that tumour cells treated with interferon had a lower capacity to form colonies in Agarose and were less tumorigenic when innoculated15, suggesting that the antitumour effect of interferon depended, in part, on the inhibition of tumour cell multiplication which can be demonstrated in vivo. I describe here a genetic approach to ascertain whether the CGI effect of human interferon preparations is mediated by chromosome 21-directed gene(s), known to govern the AV action of interferon16–19. Such an approach, should provide further insight into whether the different effects of interferon are governed by a cluster of genes on chromosome 21 or whether a single genetic locus on chromosome 21 codes for the pleiotropic effects of interferon.

Azolla-Anabaena azollae Relationship: IV. Photosynthetically Driven, Nitrogenase-catalyzed H2 Production 1,2

Abstract: The water fern, Azolla caroliniana Willd., containing the symbiotic, heterocystous blue-green alga, Anabaena azollae , has been studied under various growth conditions to characterize its light-dependent production of H 2 . The response of H 2 production to N 2 and C 2 H 2 and the absence of a differential effect of m -chlorocarbonyl cyanide phenylhydrazone on H 2 production and C 2 H 2 reduction, coupled with the parallel inhibition of both processes by DCMU imply that the production of H 2 is nitrogenase-catalyzed and ATP-dependent. H 2 was produced by fronds grown under air-CO 2 in the presence or absence of combined nitrogen. When cultured under argon-O 2 -CO 2 , only those fronds provided with combined nitrogen remained viable and produced H 2 . Fronds grown on nitrate under air plus 2% CO also produced H 2 . In comparison to fronds grown on N 2 alone, fronds grown on nitrate had an increased rate of H 2 production relative to C 2 H 2 reduction, and the inhibition of H 2 production by air was less. CO in argon ± CO 2 resulted in a partial inhibition of H 2 production, whereas CO in argon-CO 2 -C 2 H 2 enhanced H 2 production in fronds grown without combined nitrogen. Our studies strongly indicate that H 2 production is nitrogenase-catalyzed but the possibility that the symbiont contains a hydrogenase cannot be totally excluded.

Mobility of sodium dodecyl sulphate - protein complexes.

Abstract: Reduced and unreduced lysozyme aggregates formed by formaldehyde cross-linking comprise a set of model compounds for studying the effects of protein conformation on the electrophoretic mobilities of sodium dodecyl sulphate-protein complexes. The reduced aggregates were indistinguisable from normal proteins, but the unreduced aggregates migrated anomalously fast by about 14%. Contrary to expectations, plots of logarithm Rf versus Kr (retardation coefficient) failed to reveal an unusual conformation for the unreduced aggregates. Thus the anomalous mobility caused by several intramolecular disulphide bonds escaped detection by the above two diagnostic plots. Also included in this paper is a discussion of the implications of these results with regard to current models for sodium dodecyl sulphate-protein complexes.

Variation in DNA swivel enzyme activity during the mammalian cell cycle

Abstract: Synchronized cells of a normal human lymphocytic cell line contain little swiven enzyme activity in G0 and G1 and high activity in Sphase. The level of activity in different growth phase appears to be related to the fraction of the population engaged in DNA replication. No endogenous inhibitor or activator of swiven activity could be demonstrated. The evidence implies that the enzyme may be present only during S phase; it is therefore a possible control factor for replication.

Studies of the presence of membrane receptors for complement, IgG and the sheep erythrocyte rosetting capacity on the same human lymphocytes

Abstract: Normal peripheral blood lymphocytes were tested by a mixed rosette method, employing different sized erythrocytes as indicators to identify lymphocytes simultaneously possessing membrane markers found commonly on B and T cells. Only small populations of these lymphocytes were detected regulary in normal lymphocyte preparations. One type of lymphocyte (ranged from 0.5%-8%) was shown to possess the following markers: receptors for human and rabbit IgG, receptors for the third complement component C3b and C3b inactivator-cleaved C3b (C3d), and the capacity to rosette spontaneously with uncoated sheep erythrocytes (SRBC). Another lymphocyte cell type was shown to possess both SRBC and IgG receptors but lack membrane immunoglobulins and complement receptors. This population was detected in lymphocyte preparations depleted of complement receptor cells, and an increased number of these cells was found in rosette preparations incubated with human serum. The possible presence of some lymphocytes possessing both complement and SRBC receptors and lacking other markers was considered. The possibility that these small populations of human lymphocytes are sub-populations of T cells with certain cytotoxic function is postulated.

Redox titration of fluorescence yield of photosystem II

Abstract: The variable fluorescence yield of photosystem II is dependent on the redox state of the fluorescence quencher molecule or the primary electron acceptor of the system. We have carried out redox titrations of fluorescence yield of a photochemically active photosystem-II reaction-center particle and have measured the redox potential of the photosystem-II primary acceptor.During reductive titrations using dithionite as the reductant, only a single quenching transition was observed. For instance, at pH 7.0, the midpoint potential of the fluorescence transition is -325 mV, and those at a pH between 6.0 and 7.5 are consistent with a pH dependence of about 60 mV/pH unit. At a given pH, the midpoint potential of the transition closely corresponds to that of the most negative transition previously measured in unfractionated chloroplasts (both by chemical reductive titration). Oxidative titrations using ferricyanide as the oxidant yielded hysteresis in the titration curves.Similar changes in fluorescence yield were observed in redox titrations by electrochemical reduction or oxidation. Electrochemical reductive and oxidative titrations yielded reversible transitions, contrary to the hysteresis observed during chemical oxidative titration. From coulometric-titration data, we have estimated that most likely one electron is involved in the redox transition of the fluorescence-quencher or primary-electron-acceptor molecule of photosystem II. These findings are consistent with the current proposal that a membrane-bound plastoquinone functions as the primary acceptor of photosystem II.

The utilization of in vitro mutagenesis techniques to explain strain, age and sex related differences in dimethylnitrosamine tumor susceptibilities in mice.

Abstract: The carcinogen dimethylnitrosamine (DMNA) is known to exhibit a high degree of strain, organ, age, and sex related tumor specificity in mice. Using microbial mutagenesis assays coupled with mouse tissue microsomal enzyme activation systems, evidence has been obtained that demonstrated a close relationship between the level of in vitro DMNA activation to a mutagen and in vivo tumor susceptibility. DMNA activation by liver, lung, and kidney microsomes from several mouse strains was compared by measuring the rate of mutagenic metabolites formed during incubation of the carcinogen in mutation assays using Salmonella typhimurium G-46 as the indicator microorganism.

IgG on Infants' B Lymphocytes: Enhanced Binding of IgG by IgM‐Bearing Lymphoid Cells in Early Childhood

Abstract: IgM/IgD-bearing lymphocytes (B cells) from children in the first few weeks of life were found to also have surface IgG, unlike most normal adult B cells. The IgG was loosely bound to the lymphocyte surface and was partially or completely removed by incubation at 37 degrees C or by trypsinization. When F(ab')2 antisera were employed, very few infant B cells had surface IgG, although the IgM staining was similar to that obtained with the native antisera. IgM/IgD-positive cells bound IgG anti-Rh-coated (Ripley) erythrocytes, unlike most adult B lymphocytes. Capping experiments suggested that an Fc receptor on the cells could be redistributed by the anti-IgM-surface IgM complex. These data indicate that, during infancy, B-lymphocyte receptors for IgG are of altered affinity, frequency, or availability compared with adult B-lymphocyte Fc receptors and resemble the Fc receptors found on "third population" (Fc + Ig-) mononuclear cells and monocytes.

125I-labelling = 125I-Labelling.

Abstract: Erythropoietin, the hormone that regulates erythropoiesis in mammals, was 125I-labelled by using the catalytic properties of lactoperoxidase and the H2O2-generating properties of glucose oxidase. This methodology, both rapid and simple, not only produced hormone preparations with high specific radioactivity but also did not substantially alter the biological integrity of erythropoietin when it was assayed in vivo.

Immunoassay for thymopoietin

Abstract: An immunoassay for the polypeptidic thymic hormone thymopoietin (thymin) is described. Determination of thymopoietin levels in biological fluids provides a useful diagnostic test for myasthenia gravis (elevated levels), immune deficiency diseases (reduced levels), immunologically mediated diseases such as rheumatoid arthritis, systemic lupus erythematosus and allergy (reduced levels) cancer (reduced levels), malnutrition (reduced levels) and infections (reduced levels). The present assay is also useful as a monitor of the effectiveness of therapy in each of the aforementioned types of disease states.

The photosensitized oxidation of tyrosine reaction under homogeneous conditions derivatives in the presence of alginate‐i

Abstract: —The rates of dye-sensitized photooxidation of tyrosine and tyramine to brown products were compared in the presence and the absence of the anionic polysaccharide, alginate. The polyelectrolyte did not affect the reaction when it was sensitized by monochromatic light absorbed mainly by the monomeric form of the dye. In white light, the rate of oxidation sensitized by thionine or phenosafranine was increased in the presence of alginate for tyramine, but not for tyrosine. In the thionine sensitized reaction, the ratio of brown product formation to tyramine consumption increased with decreasing wavelength of monochromatic excitation. These and other phenomena are believed related to the formation of complexes between the dyes and some of the oxidation products, and to association between some of the oxidation products and alginate. A mechanism for oxidation of phenols is proposed, based on the addition of O2 (1Δ9) across a double bond ortho to the phenolate oxygen. Dyes bind to alginate in monomeric and in aggregated forms; only the monomeric forms of thiazine dyes are photochemically active, but both the monomeric and the aggregated forms of crystal violet are active.

Synergetic Cultures of Glycine max Root Cells and Rhizobia Separated by Membrane Filters.

Abstract: When suspension cultures of actively growing soybean (Glycine max L.) root cells were separated by two or three membrane filters from suspension cultures of the bacteria, a synergetic (cooperative) activation of nitrogenase was observed in the Rhizobium japonicum used in the bacterial side. Either plant cells or plant cell-conditioned medium was needed for this activation to take place. Both acetylene reduction and hydrogen evolution by the activated R. japonicum persisted for several days after removal from the apparatus when (a) a suitable carbon source was provided, (b) oxygen supply was limited, and (c) growth of bacteria was suppressed by lowering of ammonia and nitrate concentrations. Activation could also take place when the bacteria were placed in media to which plant cell-conditioned medium was added. The advantages of this method for studies on symbiosis are discussed.

Arrangement and Regulation of the Nitrogen Fixation Genes in Klebsiella pneumoniae Studied by Derepression Kinetics

Abstract: Events underlying derepression of the nitrogen fixation (nif) genes in Klebsiella pneumoniae M5A1 were analyzed in vivo by comparing the effects of selective inhibitors of transcription and translation on subsequent nitrogenase activity (rate of acetylene reduction) When batch cultures were induced for derepression, an 87-min lag separated ammonium ion/oxygen removal and the appearance of activity To prevent eventual activity by adding inhibitors during this period, it was found necessary to add rifampin, ammonium ion, or chloramphenicol more than 50, 20, or 10 min, respectively, before activity appeared in a parallel control When these inhibitors were added to cultures in which nitrogenase activity had already appeared, further increase in activity was not stopped by rifampin or ammonium ion until 45 or 20 min, respectively, after addition Chloramphenicol stopped further increase in nitrogenase activity almost immediately These data indicated a nif operon whose transcription/translation consumes 40 min When the kinetics of β-galactosidase induction were analyzed under similar conditions, it was found that 7 min separated the initiation of transcription and the first completions of translation Extrapolating from this, we find the nif operon to occupy 17 average gene lengths It is argued that the nif structural genes in K pneumoniae are contained on one operon, and further, that the disparity between the kinetics of inhibition by rifampin and those by ammonium ion suggests regulation at a locus other than the operator

The photosensitized oxidation of tyrosine derivatives in the presence of alginate. II: Reaction under heterogeneous conditions.

Abstract: —The thionine sensitized photooxidations of several tyrosine derivatives have been compared in a system in which the dye, bound to the anionic polysaccharide alginate, is separated by a dialysis membrane from a much larger external phase containing most of the substrate. Brown oxidation products appear both inside the bag and outside, but at considerably greater concentration inside. Oxidation products inside the bag retard the oxidation of the original substrate by competing with it for photooxidant. Substrates were divided into two groups, related to tyrosine and tyramine respectively, according to their rates of oxidation. The groups differ as to whether the prevailing phenolate form of the substrate is a zwitterion or an anion. In case of the latter, the Donnan equilibrium set up by the polyelectrolyte reduces its concentration in the polymer phase, and thereby the rate of oxidation. Titration and dialysis data support this interpretation.