Cytotoxic T lymphocyte recognition of novel allodeterminants expressed on in vitro selected H-2Kb mutants

In the present study, in vitro derived H-2Kb mutants have been examined by alloreactive CTL. Two mutants, R8.24 and R8.246, have been shown to express novel determinants detected by CTL generated against some but not all in vivo derived Kb mutants. BDF1 anti-bm3, anti-bm11, anti-bm19, anti-bm23, and anti-bm6 CTL populations lyse the two R8 variants. The novel determinants expressed on the R8 mutants detected by the bm3 and bm23-specific CTL appear to differ from the determinant recognized by the bm6-specific CTL. No new serologically defined determinants were detected on any of 18 independent R8 variants. However, these results do not rule out the existence of new determinants which could be recognized by antibodies. Finally, the relationship between the T cell recognition of the in vivo and in vitro derived mutants and their use in understanding the structure/function relationships between the immune response and class I Ag based on recent crystallographic analyses is discussed.     

Induction and characterization of minor histocompatibility antigens. Specific primary cytotoxic T lymphocyte responses in vitro

A definite cytotoxic activity was developed in a BALB/c (H-2d) anti-DBA/2 primary mixed leukocyte culture (MLC), which received interleukin 2 (IL-2) on day 3 of culture. This cytotoxic activity was minor histocompatibility antigens (MIHA)-specific at the stimulator level, and was not developed in a syngeneic (BALB/c anti-BALB/c) MLC. The addition of IL-2 on day 3 of culture was crucial; no or very weak cytotoxic activity was developed in MLC receiving IL-2 on day 0 or on both day 0 and day 3. Only appropriate MIHA-allogeneic tumor cells were lysed as the target of the cytotoxic activity. The cytotoxic activity seemed MIHA-specific also at the target level; it lysed tumor cells of DBA/2 mouse origin but not those of BALB/c (syngeneic) origin. Phenotypes of the cytotoxic effector cell were Thy-1+ Lyt-2+. We concluded from these results that MIHA-specific cytotoxic T lymphocytes (CTL) were generated in the MIHA-allogeneic primary MLC. In this newly developed system, we studied genetic and antigenic requirements for primary anti-MIHA CTL responses in vitro. We demonstrated; among spleen cells (SC) of seven B10 H-2-congenic strains only SC of B10.D2 strain whose major histocompatibility complex (MHC) (H-2d) was compatible with the responder MHC effectively stimulated responder BALB/c (H-2d) SC for an anti-MIHA (DBA-C57BL-common) CTL response. Similarly, only SC of two out of seven C x B recombinant inbred strains (C x B.H and C x B.D), which were compatible at the MHC with responder SC, activated responder BALB/c SC for the response. The possibility that cells responding to H-2 alloantigens suppressed the anti-MIHA response was ruled out. Additional experiments showed that compatibility at the H-2K-end or the H-2D-end of the MHC was sufficient for a definite anti-MIHA response. These provided formal evidence that primary anti-MIHA CTL responses in vitro were MHC-restricted at the stimulator level. We then showed that sonication-disrupted SC or Sephadex G-10 column-passed nonadherent SC failed to stimulate responder SC for a primary anti-MIHA CTL response, whereas G-10-passed nonadherent SC responded well to adherent stimulator cells. Further study demonstrated that Ia+ adherent cells were the most active cell type as stimulator. Finally, we confirmed that the primary anti-MIHA CTL responses to adherent stimulator cells was MHC-restricted.     

Analysis of lymphocytes reactive to histocompatibility antigens. II. Exponential alloantigen-dependent lymphocyte growth in vitro

Rat thoracic duct lymphocytes were maintained in continual blast transformation and cell division by repeated in vitro stimulation with allogeneic cells. This resulted in increases in responder cell numbers of up to 10,000-fold in 10-day periods. Growth of responder lymphocyte populations was dependent upon cell density, culture medium nutrients, and the presence of antigen in the form of allogeneic cells. A titration assay for mixed lymphocyte interactions (MLI) was used to relate absolute growth of cells in preparative cultures to [³H]thymidine incorporation in analytical MLI. Growth of lymphocyte populations derived by repeated stimulation with cells bearing a single foreign MHC haplotype was supported to lesser, variable degrees by stimulation with unrelated “third party” stimulator cells. The extent of this operational cross-reactivity was assessed by parallel line analysis of MLI titrations of responder lymphocytes enriched for specific alloreactivity.     

Interleukin-2 induces proliferation of T lymphocyte mutants lacking protein kinase C

We have identified a murine T lymphocyte clone that apparently lacks diacylglycerol- and phospholipid-activated protein kinase C (PKC): cell extracts do not display phosphatidylserine, Ca2+, or phorbol ester-dependent phosphotransferase activity; the enzyme was not detected in immunoblots with PKC-specific antibodies; phorbol ester binding sites are not detectable in intact cells; and activators of PKC do not stimulate proliferation or Na+/H+ exchange in intact cells. Only PKC beta mRNA was detected in normal murine T lymphocytes. The mutant T lymphocytes contained amounts of 4.4 kb PKC beta message similar to those in normal murine lymphocytes, but the 2.9 kb and 1.2 kb messages found in normal lymphocytes were barely detectable. No abnormalities were detected on Southern analysis, suggesting that the abnormality may be at the level of message splicing or stability. Since PKC-deficient cells proliferate in response to the T lymphocyte growth factor, interleukin-2, we conclude that activation of PKC is not essential for the growth-promoting action of interleukin-2.     

Monocyte function in mixed lymphocyte reactions

Subpopulations of human peripheral blood lymphocytes were isolated by sequential separation techniques. The stimulating and responding capacity of these cells together with the T-cell population remaining after the removal of other populations was studied in one-way allogeneic mixed lymphocyte culture. Incorporation of [³H]thymidine was used as a measure of response. Monocytes, present in the stimulating or responding cell population, were necessary for lymphocyte response. T cells stimulated responding T-cell populations containing monocytes but not B cells. Stimulation by T cells could be inhibited with DRW antisera. Response was also inhibited by sera detecting DRw antigens on the monocytes of the responding cell population. It is concluded that monocytes play an important functional role in mixed lymphocyte reactions. In addition, it appears that the combination of anti-DRw sera and monocytes influences mixed lymphocyte reactions by an active process in that inhibition of response cannot be explained entirely by blocking DRw determinants.     

Effect of CO2 on short-term human lymphocyte culture in vitro

There was no significant difference in the mitotic indices of the cultures maintained at different CO2 concentrations, i.e. 0 percent, 5 percent and 10 percent. However, considerable variation was recorded among different individuals.     

Reaction centers from three herbicide resistant mutants of Rhodobacter sphaeroides 2.4.1: Kinetics of electron transfer reactions

Electron transfer rates were measured in RCs from three herbicide-resistant mutants with known amino acid changes to elucidate the structural requirements for last electron transfer. The three herbicide resistant mutants were IM(L229) (Ile-L229 Met), SP(L223) (Ser-L223 Pro) and YG(L222) (Tyr-L222 Gly). The electron transfer rate D⁺Q_A ^-Q_BD⁺Q_AQ_B (k _AB) is slowed 3 fold in the IM(L229) and YG(L222) RCs (pH 8). The stabilization of D⁺Q_AQ_B ^- with respect to D⁺Q_AQ_B ^- (pH 8) was found to be eliminated in the IM(L229) mutant RCs (G⁰ 0 meV), was partially reduced in the SP(L223) mutant RCs (G⁰=–30 meV), and was unaltered in the YG(L222) mutant RCs (G⁰=–60 meV), compared to that observed in the native RCs (G⁰=–60 meV). The pH dependences of the charge recombination rate D⁺Q_AQ_B ^-DQ_AQ_B (k _BD) and the electron transfer from Q_A ^- (k _QA ^-Q_A) suggest that the mutations do not affect the protonation state of Glu-L212 nor the electrostatic interactions of Q_B and Q_B ^- with Glu-L212. The binding affinities of UQ₁₀ for the Q_B site were found in order of decreasing values to be native IM(L229) > YG(L222) SP(L223). The altered properties of the mutant RCs are used to deduce possible structural changes caused by the mutations and are dicscussed in terms of photosynthetic efficiency of the herbicide resistant strains.Abbreviations Bchl bacteriochlorophyll - Bphe bacteriopheophytin - cholate 3,7,12-trihydroxycholanic acid - D donor (bacteriochlorophyll dimer) - EDTA ethylenediamine tetraacetic acid - Fe²⁺ non-heme iron atom - LDAO lauryl dimethylamine oxide - PS II photosystem II - Q_A and Q_B primary and secondary quinone acceptors - RC bacterial reaction center - Tris tris(hydroxymethyl)aminomethane - UQ₀ 2,3-dimethoxy-5-methyl benzoquinone - UQ₁₀ ubiquinone 50     

丙型肝炎病毒E2糖蛋白糖基化作用的研究

本研究构建丙型肝炎病毒(HCV)糖蛋白E2的N-糖基化位点定点突变体。采用高保真性的Pfx DNA聚合酶,设计两对引物,分别引入两个突变位点,通过PCR体外定点突变,使E2第535、583位核苷酸由A突变为T,从而使AAC编码的天冬酰氨突变为TAC编码的酪氨酸,使得N-糖苷化位点NNT、NST突变为YNT、YST.结果得到两个单位点以及一个双位点突变体,并将突变型E2连接到真核表达载体peDNA3．1(-)／Myc—HisB上。成功获得的3个HCVE2糖蛋白糖基化位点定点突变体,为进一步进行HCVE2糖蛋白糖基化位点与分子伴侣之间的相互关系以及突变体对机体的免疫功能的影响的研究奠定了基础。     

In vivo and in vitro investigation of salt tolerance in three anthocyaninless mutants in tomato (Lycopersicon esculentum Mill.)

Growth responses of a tomato cultivar Ailsa Craig and the ah-, aw- and bls-isogenic/near isogenic lines (IL/NIL) from it were evaluated and compared at cotyledons stage under salt treatment in vivo and in vitro experiments. No differences in hypocotyl and root growth responses were detected between the anthocyanin-containing and the anthocyaninless lines within the in vivo experiments. The anthocyaninless mutants, (except in some cases the bls mutant), exhibited higher callogenic and shoot-forming capacity on both, control and salinized media. It was concluded that for this reason it would be difficult to determine the relationship between the in vivo and in vitro responses of the lines studied and as well as to evaluate the usefulness of the in vitro method in testing these lines for salt tolerance.     

Temperature-sensitive plant mutants isolated in vitro

Summary Conditional-lethal, temperature-sensitive plant mutants have been isolated using a simple protoplast cloning method. The leaf protoplasts used were obtained from sterile, haploid shoot cultures of Nicotiana plumbaginifolia. Recessive mutations are described at three loci: ts1, ts2 and ts3. The mutations are lethal when either tissue cultures or plants are incubated at 33°C but not at 26°C.     

Growth studies of three phi chi 174 mutants in tsDNA mutants Escherichia coli.

Three mutants of phi chi 174 were examined for their abilities to grow in temperature-sensitive dna,A, dnaC, dnaE, or dnaG mutants of Escherichia coli. The results indicate that the phage mutants have acquired the ability to grow in some tsDNA mutants that normally block the replication of wild-type phi chi 174. Evidence is presented indicating that the phage mutants contain one or more altered structural proteins. Several models are presented to explain how altered phage structural proteins could affect phi chi 174 replication.