Chromatic Regulation of Photosystem Composition in the Cyanobacterial Photosynthetic System: Kinetic Relationship between Change of Photosystem Composition and Cell Proliferation

Kinetics of the change of photosystem (PS) composition in cyanobacteriainduced by chromatic light were studied in relation to cellproliferation. The study was made for two unicellular strains,Synechococcus NIBB 1059 and Synechocystis (Aphanocapsa) PCC6714. We found that (1) the change to a higher or lower PS I/IIratio was due to acceleration or suppression of apparent PSI formation, and (2) it progressed on a similar time scale tothat of the cell proliferation. The apparent rate constant ofthe change in the PS I/II ratio was proportional to that ofcell proliferation, µ, when this was low, but at highvalues of µ the increase in the rate constant of the changein the PS I/II ratio became smaller, causing a deviation fromthe linear relationship. Results indicate that under autotrophicconditions, the photoregulated composition change occurs asa result of thylakoid development, which accompanies cell proliferation. (Received June 23, 1986; Accepted December 5, 1986)     

Nudeotide Pools and Purine Metabolism in Tissue Cultures of Wild-Type Datura innoxia and Adenine-Requiring Auxotroph

Cells of the auxotrophic mutant, Ad1, of Datura innoxia requiredadenine, adenosine, or inosine for their growth on solid agarmedium which contained Murashige-Skoog salts, 2,4-dichloro-phenoxyaceticacid, and sucrose. Thirteen purine and pyrimidine nucleotidesin extracts of wild-type and Ad1 cells were separated and quantifiedby HPLC. Levels of ADP-glucose and UMP were significantly higherin Ad1 than in wild-type cells, but those of other nucleotideswas found when Ad1 cells were transferred to fresh medium withoutadenine. The rate of the biosynthesis de novo of purines, asestimated from the rate of incorporation of ¹⁴C from [2-¹⁴C]-glycine and [¹⁴C]formate into adenine nucleotides, was reducedin Ad1 cells to 21 and 13% of the wild-type rate, respectively.The activities involved in the salvage of adenine and adenosinein Ad1 cells were similar to those in wild-type cells. Ad1 cellshad the capability to convert adenine to guanine nucleotidesand guanine to adenine nucleotides. ¹ Part 27 of the series, "Metabolic Regulation in Plant CellCulture". (Received March 7, 1988; Accepted August 3, 1988)     

Ig isotypes produced by EBV-transformed B cells as a function of age and tissue distribution

EBV can transform human B cells giving rise to lymphoblastoid cell lines that produce and secrete Ig. Herein B cells from various tissues of newborns and adults were transformed by EBV and their Ig products were analyzed with isotype-specific mAb. Although IgG- and IgA-bearing B cells were present in the newborn, EBV transformed IgM-producing cells almost exclusively in both newborn blood and breast milk. IgM-secreting cells were derived from IgM+ B cells and IgM- pre-B cells present in neonatal blood, but only from IgM+ cells in adult blood. Whereas in adults most EBV-transformed cells produced IgM, producers of IgG and of IgA were present in frequencies that varied according to the tissue source. Precursors of IgG-producing cells were relatively abundant in blood, spleen, and tonsil, and relatively infrequent in bone marrow and appendix. EBV-inducible IgA producers were relatively concentrated in the appendix and to a lesser extent in tonsils and blood. Differences in the subclass composition of EBV-transformed populations of IgG- and IgA-producers were also observed for the various adult lymphoid tissues. IgG1-producing cells predominated in most tissues, and precursors of IgG2 were largely confined to the circulation. Whereas IgA1-producing cells were predominant in all tissues, a marked enrichment in IgA2-producers was observed in the appendix. These results indicate a remarkable heterogeneity in the isotype distribution pattern of EBV-transformable B cells that is determined both by developmental age and tissue localization. We propose that EBV selectively transforms primed B cells, the isotype commitment of which varies according to tissue origin and age.     

Differential effects of recombinant human interferon-gamma and interleukin 2 on natural killer cell activity of peripheral blood in early human development

Ontogenic development and the lymphokine responsiveness of human NK cell activity against K562 target cells in peripheral blood lymphocytes were evaluated in fetuses, premature infants, and term neonates by using a 4-hr 51Cr-release assay. Basal NK activity and NK boosting by lymphokines were comparatively assayed after an 18-hr incubation with medium alone, recombinant human IFN-gamma (1000 U/ml), and recombinant human IL 2 (25 U/ml), respectively. Lymphocytes from 20-wk-old fetuses lacked NK cell activity even after the pretreatment with IFN-gamma. Low, but significant levels of NK activity and NK boosting by IFN-gamma were observed in premature infants after 27 wk of gestation, with a progressive intrauterine maturation of these activities. Both basal NK activity and NK boosting by IFN-gamma in term neonates were still lower than those of adult controls. The grade of NK boosting by IFN-gamma appeared to depend on the development of basal NK activity. Contrary to IFN-gamma, IL 2 could induce marked NK activity even in 20-wk-old fetuses who lacked both basal and IFN-gamma inducible NK activities. NK boosting by IL 2 was much more efficient than that by IFN-gamma at any period of human life. The facts that IL 2-induced NK boosting could occur without any appreciable production of IFN-gamma in neonatal lymphocytes, and that ample neutralizing doses of anti-IFN-gamma antibody hardly suppressed IL 2-mediated NK boosting even in adult lymphocytes, indicated that the effect of IL 2 on NK boosting might be independent of IFN-gamma production. On the basis of the ontogenic differences in the development of the lymphokine responsiveness of NK cell activity and on the different NK boosting mechanisms of these lymphokines it was suggested that so-called human "pre-NK cells" might be divided into IFN-gamma sensitive and IL 2-sensitive cells. Whether these cell populations belong to different cell lineages or different maturation stages of the same cell line, however, remains unsettled.     

Interferon-gamma can augment expression ability of HLA-DR antigens on pokeweed mitogen-stimulated human T lymphocytes

The effect of natural interferon (IFN)-gamma on HLA-DR molecule expression of pokeweed mitogen (PWM)-stimulated T cells from cord blood and adult peripheral blood was assessed by direct immunofluorescence with fluorescein-labeled monoclonal anti-HLA-DR antibody on a flow cytometer. Although cord blood T cells showed only weak expression of HLA-DR antigens on PWM stimulation, IFN-gamma could enhance HLA-DR expression of PWM-stimulated cord blood T cells to levels comparable to those of adult ones. A similar, but slight, increase in HLA-DR expression was inducible in PWM-stimulated adult T cells by the addition of IFN-gamma, but at higher doses. This increased expression of HLA-Dr antigens on PWM-stimulated T cells was almost completely abolished by both acid treatment of IFN-gamma and neutralization of IFN-gamma with specific antiserum. In contrast to IFN-gamma, neither recombinant IFN-alpha nor IFN-beta showed any effect on HLA-DR expression of PWM-stimulated T cells. These results suggested a possible function of IFN-gamma that might modulate HLA-DR expression ability of T cells in their activation process.     

Cyclosporin A does not prevent expression of Tac antigen, a probable TCGF receptor molecule, on mitogen-stimulated human T cells

Results of recent studies indicated that a monoclonal anti-Tac antibody might recognize the receptor sites or closely related structures for T cell growth factor (TCGF) on activated human T cells. In the present study, we examined the effect of cyclosporin A (CsA) on the expression of Tac antigen by mitogen-stimulated T cells. CsA inhibited the proliferative response of T cells to Con A and PHA in a dose-dependent manner. Both Con A- and PHA-induced cellular proliferation were decreased to about 10% of controls at 5 micrograms/ml of CsA. When T cells were stimulated with these mitogens, many of them expressed Tac antigen on their surfaces, assessed by the immunoperoxidase method. The appearance of Tac-positive cells occurred earlier than a rise of cellular DNA synthesis. Characteristically, CsA showed no inhibitory effect on the expression of Tac antigen by mitogen-stimulated T cells, even at a relatively high concentration of 5 micrograms/ml, whereas the expression of other "activation" antigens reactive with monoclonal anti-Ia, OKT9, or OKT10 antibodies by T cells was blocked completely by CsA. Morphologically, the majority of Tac-positive cells in culture with mitogens alone showed the characteristics of blastoid cells; Tac-positive cells in the culture containing CsA mainly consisted of medium-sized cells, indicating these cells probably accumulated at a stage of partial activation. T cells, once stimulated with Con A or PHA for 3 days whether in the presence or in the absence of CsA, were able to absorb TCGF activity from TCGF-containing media similarly. In addition, T cells, even stimulated in the presence of CsA with these mitogens for 24 hr, were capable of responding to TCGF with the same grade of proliferation as did T cells stimulated with mitogen alone. CsA showed no appreciable inhibition in a TCGF-dependent proliferation of such prestimulated cells. These functional properties of activated T cells might be correlated with their ability to express Tac antigen. These experimental findings present some evidence that CsA might not prevent the expression of probable functional receptor sites for TCGF in mitogen-dependent activation of human T cells.     

THE EFFECT OF IRON NUTRITION ON PHOTOSYNTHESIS AND NITROGEN FIXATION IN CULTURES OF TRICHODESMIUM (CYANOPHYCEAE)1

Cultures of Trichodesmium NIBB 1067 were grown in the synthetic medium AQUIL with a range of iron added from none to 5 × 10^?7 M Fe for 15 days. Chlorophyll-a, cell counts, and total cell volume were two or three times higher in medium with 10^?7 M Fe than with no added Fe. Oxygen production rate per chlorophyll-a was over 60% higher with higher iron. Increased iron stimulated photosynthesis at all irradiances from about 12–250 μE · m^?2· s^?1. Nitrogen fixation rate, estimated from acetylene reduction, for 10^?7 and 10^?8 M Fe cultures was approximately twice that of the cultures with no added Fe. The range of rates of O₂ production and N₂ fixation in cultures at the iron concentrations we used were similar to the rates from natural samples of Trichodesmium from both the Atlantic, and the Pacific oceans. This similarity may allow this clone to be used, with some caution, for future physiological ecology studies. This study demonstrates the importance of iron to photosynthesis and nitrogen fixation and suggests that Trichodesmium plays a central role in the biogeochemical cycles of iron, carbon and nitrogen.     

1-Acylglycerophosphocholine O-acyltransferase in maturing safflower seeds

The activity of 1-acylglycerophosphocholine (1-acyl-GPC) O-acyltransferase (EC 2.3.1.23) varied during maturation of safflower (Carthamus tinctorius L.) seeds, and activity per seed was highest in the middle period of seed development when triacylglycerol (TAG) is most rapidly synthesized. The specific activity of acyl transfer in a 20000·g particulate preparation exceeded 500nmol·min^-1·(mg protein)^-1 and was higher than those of any other enzymes involved in TAG synthesis (K. Ichihara et al., 1993, Plant Cell Physiol. 34, 557–566). This suggested the presence of a large flux of acyl-CoA to phosphatidylcholine in the cell. The reaction was specific to C₁₆ and C₁₈ acyl-CoAs with a double bond at position 9. Lauroyl- and erucoyl-CoA were completely ineffective, while ricinoleoyl- and elaidoyl-CoA were utilized efficiently. The relative order of specificity for native acyl-CoA species was linoleoyl > oleoyl stearoyl = palmitoyl. When acyl-CoA mixtures were presented, preference for the unsaturated species rather than the saturated species was even more apparent. The enzyme preferentially utilized 1-C₁₆-acyl- and 1-C₁₈-acyl-GPC molecular species, and 1-palmitoyl-, 1-stearoyl-, 1-oleoyl-and 1-linoleoyl-GPC equally served as acyl acceptor. No activity was detected with 1-octanoyl-GPC, and 1-erucoyl-GPC produced little effect. The effectiveness of 1-alkyl-GPC was comparable to that of 1-acyl-GPC. It was thus concluded that the enzyme recognizes the chain lengths of the acyl donor and acceptor, and the double bond at position 9 of the acyl donor.Abbreviations DAG diacylglycerol - DTNB 5,5-dithiobis(2-nitrobenzoic acid) - GP sn-glycerol 3-phosphate - GPC sn-glycero-3-phosphocholine - GPE sn-glycero-3-phosphoethanolamine - GPI sn-glycero-3-phosphoinositol - PC phosphatidylcholine - TAG triacylglycerol     

Mitogenic responsiveness and monocyte-lymphocyte interaction of early and late rosette-forming cell populations of human peripheral blood lymphocytes.

Adherent cells in human peripheral blood mononuclear cells were removed by the attachment to the plastic surface of tissue culture dishes. After removal of adherent cells, early rosette-forming cells (early RFC), which were characterized by early (5 min) rosette formation with sheep blood cells (SRBC) at an SRBC to lymphocyte ratio of 8:1, were separated from nonrosetting cells by sedimentation on Ficoll-Hypaque gradient. Total (60 min) rosette formation was carried out with the early RFC-depleted cell population on the gradient interface by the use of neuraminidase-treated SRBC at an SRBC to lymphocyte ratio of 20:1 and the resulting rosette-forming cells (late RFC) were sedimented by gradient centrifugation. These T cell subpopulations, early RFC-enriched and late RFC-enriched, were reasonably pure with respect to the ability to bind SRBC and contained less than 0.5% monocytes. Monocyte preparations, which were obtained after vigorous washing of the adherent cell layers on tissue culture dishes, responded to phytohemagglutinin P (PHA-P) or concanavalin A (Con A) with negligible incorporation of 3H-thymidine. There was no significance difference in the responsiveness to PHA-P between early RFC-enriched and late RFC-enriched populations either in the absence or in the presence of graded numbers of additional autologous monocytes. However, the response of early RFC-enriched population to Con A was significantly poor as compared with that of late RFC-enriched one unless additional monocytes were added. In the presence of 20% autologous monocytes in the culture, the Con A-induced response of early RFC-enriched population was markedly enhanced to reach close to that of late RFC-enriched population. These results suggest that early RFC and late RFC might be different from each other in their responsiveness and in their need for monocytes on the stimulation with Con A.     

Photoreversible absorption changes of guanidine-HCltreated phycocyanin and allophycocyanin isolated from the blue-green alga Tolypothrix tenuis

In the search for the photoreceptor in photocontrolled phycoerythrinformation, photoreversible absorption changes of chromoproteinsin vivo and in vitro were studied with the blue-green alga Tolypothrixtenuis. Neither intact cells nor crude extracts of soluble proteinsshowed any significant absorption changes which were reversiblyinduced by green and red light. However, the photoresponse wasobservable when the crude protein extracts were treated withthe chaotropic reagent guanidine-HCl (0.4 M, for 1 hr in thedark). Isolated phycocyanin and allophycocyanin also showedthe same photoresponse after the guanidine-HCl treatment. Thedifference spectrum (green minus red) of guanidine-HCl-treatedphycocyanin was almost identical with that shown by phycochromea of Bj?rn and Bj?rn (3), and the allophycocyanin showed thesame difference spectrum as those of phycochrome c of Bj?rnand Bj?rn and the photoreversible pigment isolated by Scheibe(7). Urea at a concentration higher than 1 M or alkaline incubation(pH 8.5) also showed the same effect. The results were interpretedas indicating that phycocyanin and allophycocyanin obtain theability for photoresponsiveness when their protein conformation,probably around the chromophore site, is modified. (Received October 30, 1978; )