A protein-free medium for the growth of hybridomas and other cells of the immune system

Summary A protein-free medium, termed ABC, has been developed which essentially eliminates the need for serum proteins. ABC supports the long-term growth of murine hybridomas as well as other transformed cells of the immune system. The requirement of hybridoma growth for transferrin has been met by substituting the soluble organo-iron compound, sodium nitroprusside. Substantial improvement in the growth of hybridomas was afforded by the inclusion of 18 trace elements complexed to disodium ethylene diaminetetraacetate (EDTA). The medium was further improved by the inclusion of components not found in Ham's F12 medium or by raising the concentrations of existing low molecular weight components. Murine hybridomas can be cultured routinely in this protein-free medium in an anchorage-independent manner with doubling times generally under 24 h. Visualized on electrophoretic gels, levels of monoclonal antibody taken from those cultures often exceeded 80% of the total protein. The medium was also able to support the growth of HuT 78 and H9 cells as well as certain other transformed cells of the immune system. In addition, normal human peripheral blood lymphocytes, activated with phytohemagglutinin and cultured with 50 U/ml recombinant interleukin 2, could be grown for 2 wk with a 50-fold expansion over input cell number.     

PHA激活的脐血T细胞条件培养液对人骨髓CFU-c的抑制

本文研究了PHA刺激18小时收获的脐血T细胞条件培养液(PHA-TCM)对正常人骨髓CFU-c的影响。结果显示PHA-TCM能够显著抑制CFU-c的生长,这种抑制与PHA-TCM浓度有关。并发现经PHA-TCM作用后M型集落比例明显降低。PHA-TCM中未检出IFN和IL-2活性。进一步研究证实,PHA-TCM中CFU-c抑制活性是一种对酸碱敏感对热相对不敏感的蛋白质,其分子量大于10,000道尔顿。     

l-[3H]Nitroarginine and l-[3H]Arginine Uptake into Rat Cerebellar Synaptosomes: Kinetics and Pharmacology

Abstract: Characteristics of the transport of the nitric oxide synthase substrate l -arginine and its inhibitor, N ^G-nitro- l -arginine ( l -NOARG), into rat cerebellar synaptosomes were studied. Uptake of both l -arginine and l -NOARG was linear with increasing amount of protein (up to 40 µg) and time of incubation (up to 5 min) at 37°C. Uptake of both compounds reached a steady state by 20 min. Maximal uptake of l -NOARG (650 pmol/mg of protein) was three to four times higher than that of l -arginine (170 pmol/mg of protein). l -NOARG uptake showed biphasic kinetics ( K _{m 1} = 0.72 m M , V _{max 1} = 0.98 nmol/min/mg of protein; K _{m 2} = 2.57 m M , V _{max 2} = 16.25 nmol/min/mg of protein). l -Arginine uptake was monophasic with a K _m of 106 µ M and a V _max of 0.33 nmol/min/mg of protein. l -NOARG uptake was selectively inhibited by l -NOARG, N ^G-nitro- l -arginine methyl ester, and branched-chain and aromatic amino acids. l -Alanine and l -serine also inhibited l -NOARG uptake but with less potency. Uptake of l -arginine was selectively inhibited by N ^G-monomethyl- l -arginine acetate and basic amino acids. These studies suggest that in rat cerebellar synaptosomes, l -NOARG is transported by the neutral amino acid carrier systems T and L with high affinity, whereas l -arginine is transported by the basic amino acid carrier system y⁺ with high affinity. These data indicate that the concentration of competing amino acids is an important factor in determining the rates of uptake of l -NOARG and l -arginine into synaptosomes and, in this way, may control the activity of nitric oxide synthase.     

A T7 promoter-specific, inducible protein expression system forBacillus subtilis

The adaptation and application of theEscherichia coli T7 RNA polymerase system for regulated and promoter-specific gene expression inBacillus subtilis is reported. The expression cassette used inBacillus subtilis was tightly regulated and T7 RNA polymerase (T7 RNAP) appeared 30 min after induction. The efficiency of T7 promoter-specific gene expression inB. subtilis was studied using one secretory and two cytosolic proteins of heterologous origin. The accumulation ofE. coli -galactosidase, as well as a 1,4--glucosidase fromThermoanaerobacter brockii inB. subtilis after T7 RNAP induction was strongly enhanced by rifampicin inhibition of host RNAP activity. The-amylase ofThermoactinomyces vulgaris, a secretory protein, was found to accumulate in the culture supernatant up to levels of about 70 mg/l 10–20 h after T7 RNAP induction, but was also deposited in cellular fractions. The addition of rifampicin inhibited-amylase secretion, but unexpectedly, after a short period, also prevented its further (intra)cellular accumulation     

Lymphocyte subpopulations of regional lymph nodes in human colon and gastric adenocarcinomas

 In order to study the host immune response to tumours, previous knowledge of the cellular composition of regional draining lymph nodes is necessary. Enlarged regional lymph nodes are a common finding in colon and gastric adenocarcinomas. We have studied the cellular composition of normal non-reactive and of regional draining lymph nodes of colon and gastric adenocarcinomas. In normal non-reactive lymph nodes, T lymphocytes (CD2⁺, CD7⁺) constituted the largest fraction of the lymphoreticular cells. These lymphocytes were mainly CD4⁺, and there were more cells expressing the CD45RA isoform of the CD45 antigen than CD45RO. Reactive lymph nodes presented a decreased proportion of CD4⁺ CD45RA⁺ cells and an increased number of B cells. Although most of the T cells in the reactive nodes were CD4⁺ CD45RO⁺, their proportion was similar to that found in normal non-reactive nodes. We studied the presence of the molecules CD28 and CD80 involved in the processes of interaction and activation of T and B lymphocytes. The CD28 molecule was found in all the T lymphocytes, while the CD80 molecule was weakly expressed on the B lymphocyte membrane. Received: 4 January 1996 / Accepted: 28 May 1996     

Transient Expression of CD20 Antigen (Pan B Cell Marker) in Activated Lymph Node T Cells

In contrast to the case of peripheral T cells, the surface expression of CD20 antigen and the expression of CD20 mRNA in monkey lymph node (LN) T cells underwent a noticeable increase when they were cultured with mitogen and interleukin-2 (IL-2). To confirm in vivo regulation of CD20 expression during the activation of LN T cells, we examined LNs derived from monkeys experimentally inoculated with simian immunodeficiency virus (SIV). Significant expression of CD20 antigen was detected in the T cells of the LNs at the stage of lymphadenopathy. These findings suggest that lymphocyte activation in the LNs induced expression of the CD20 molecule in some T cells.     

小鼠卵母细胞减数分裂染色体的制备

PreparationofMeioticKarytypeofMouseOocyteLiChaojunYanLeipingZhangXiranChenYifeng(BiologyDepartmentofNanjingNormalUniversity,Nanjing210097)哺乳动物的卵母细胞的减数分裂过程中存在两次自发的停滞现象,第一次是在第一次减数分裂前期的双线期,这一静止期持续很长时间,一直到动物性成熟后卵母细胞进入发有周刎,在保住腺激素的作用下,卵母细胞的第一次减数分裂才重新启动．完成第一次减数分裂后．又停滞在第二次减数分裂的中期,在椅子或化学因素刺激的作用下,完成第二次减数分裂[4]．因此,对哺乳动物的卵母细胞在一…     

Phytochrome control of short-day-induced bud set in black cottonwood

In trees and other woody perennial plants, short days (SDs) typically induce growth cessation, the initiation of cold acclimation, the formation of a terminal bud and bud dormancy. Phytochrome control of SD-induced bud set was investigated in two northern clones of black cottonwood (Populus trichocarpa Torr. & Gray) by using night breaks with red light (R) and far-red light (FR). For both clones (BC-1 and BC-2), SD-induced bud set was prevented when R night breaks as short as 2 min were given in the middle of the night. When night breaks with 2 min of R were immediately followed by 2 min of FR, substantial reversibility of bud set was observed for BC-1 but not for BC-2. By comparing the effects of the R night breaks on bud set and the length of specific internodes, we determined that the R night breaks influenced internode elongation in two opposing ways. First, the addition of a R night break to the SD treatment prevented the cessation of internode elongation that is associated with bud set. Those internodes that would not have elongated under SDs (and would have been found within the terminal bud) elongated in the R treatment. Second, the R night breaks decreased internode length relative to the long-day (LD) control. In contrast to the clonal differences in reversibility that we observed for bud set, the decrease in internode length (i.e. the second effect of R) was R/FR reversible in both clones. Based on these results, we conclude that internode elongation is influenced by two distinct types of phytochrome-mediated response. The first response is a typical response to photpperiod, whereas the second response is a typical “end-of-day” response to light quality. Our results demonstrate that SD-induced bud set in black cottonwood is controlled by phytochrome but that clonal differences have an important influence on the R/FR reversibility of this response. The availability of an experimental system in which SD-induced bud set is R/FR reversible will be valuable for studying the physiological genetics of photoperiodism in trees.     

RFLP-based maps of the homoeologous group-6 chromosomes of wheat and their application in the tagging of Pm12, a powdery mildew resistance gene transferred from Aegilops speltoides to wheat

Genetic maps of the homoeologous group-6 chromosomes of bread wheat, Triticum aestivum, have been constructed spanning 103 cM on 6A, 90 cM on 6B and 124 cM on 6D. These maps were transferred to a Chinese Spring (CS) x line #31 cross to locate a dominant powdery mildew resistance gene, Pm12, introgressed into line #31 from Aegilops speltoides. Pm12 was shown to lie on the short arm of translocation chromosome 6BS-6SS.6SL in line #31, but could not be mapped more precisely due to the lack of recombination between the 6S Ae. speltoides segment and chromosome 6B. Possible strategies to reduce the size of the alien segment, which probably encompasses the complete long arm and more than 82% of the short arm of chromosome 6B, are discussed.     

T cells in murine lupus: propagation and regulation of disease

MRL/Mp-lpr/lpr mice develop a spontaneous lupus syndrome, including hypergammaglobulinemia, autoantibodies, glomerulonephritis, and lymphadenopathy. To investigate the role of lymphocyte subsets in the pathogenesis of disease, lupus-prone MRL mice deficient in T cells, T cells, or both were generated. Mice deficient in T cells developed a partially penetrant lupus syndrome, characterized by lymphadenopathy, elevated levels of class-switched immunoglobulins, an increased incidence of antinuclear antibodies, and immune deposits in kidneys which progressed to renal insufficiency over time. In comparison to wild type animals, T cell-deficient animals developed an accelerated and exacerbated disease phenotype, characterized by accelerated hypergammaglobulinemia and enhanced autoantibody production and mortality. Repertoire analysis of these latter animals identified polyclonal expansion (V) of CD4+B220-cells. Mice lacking both and T cells failed to generate class-switched autoantibodies and immune complex renal disease. First, these findings demonstrate that murine lupus in the setting of Fas-deficiency does not absolutely require the presence of T cells, and they also suggest that a significant basis for MRL/lpr disease, including renal disease, involves T cell-independent, T cell dependent, polyreactive B cell autoimmunity, upon which T cell-dependent mechanisms aggravate specific autoimmune responses. Second, these data indicate that T cells partake in the regulation of systemic autoimmunity, presumably via their effects on CD4+B220-T cells that provide B cell help. Finally, these results demonstrate that MRL/lpr B cells, despite their intrinsic abnormalities, cannot per se cause tissue injury without T cell help.Abbreviations snRNPs small nuclear ribonucleoprotein particles