Molecular analysis of theArabidopsis pattern formation geneGNOM: gene structure and intragenic complementation

TheGNOM gene is required for pattern formation along the main body axis of the embryo in the flowering plantArabidopsis thaliana. Mutations in theGNOM gene alter the asymmetric division of the zygote and interfere with the formation of distinct apical-basal regions in the developing embryo. We have isolated theGNOM gene by positional cloning, characterised its structure and determined the molecular lesions in mutant alleles. Although the predicted 163 kDa GNOM protein has a conserved domain in common with the yeast secretory protein Sec7p, it is most closely related in size and overall similarity to the product of the yeastYEC2 gene, which is not essential for cell viability. Four fully complementinggnom alleles carry missense mutations in conserved regions, seven partially complementing alleles have premature stop codon mutations and two non-complementing alleles have splice-site lesions. Our results suggest that the GNOM protein acts as a complex of identical subunits and that partial complementation may involve low levels of full-length protein generated by inefficient translational read-through.Communicated by H. Saedler     

Trends in plant carbon concentration and plant demand for N throughout this century

 Atmospheric CO₂ concentration has increased by 25% over the preindustrial level. A parallel increase in C concentration and decreases in N concentration and δ¹³C of plants grown throughout this century have been observed in plant specimens stored in herbaria. We tested our previous results in a study of 12 more species collected in the western Mediterranean throughout this century (1920–1930, 1945–1955, and 1985–1990) and tree rings of Quercus pubescens from the same area. These changes were accompanied by apparent increases in condensed tannin concentration. A decreasing trend in δ¹⁵N both in herbarium material and tree rings was also found, indicating that ecosystems might cope with higher plant N demand by decreasing N losses and increasing N fixation and mineralization. These results may contribute to a better understanding of the effects of global change on carbon and nitrogen cycling. Received: 12 November 1995 / Accepted: 17 May 1996     

Eradication of established hepatic human neuroblastoma metastases in mice with severe combined immunodeficiency by antibody-targeted interleukin-2

 A major problem in the treatment of solid tumors is the eradication of established, disseminated metastases. Here we describe an effective treatment for established experimental hepatic metastases of human neuroblastoma in C. B.-17 scid/scid mice. This was accomplished with an antibody-cytokine fusion protein, combining the unique targeting ability of antibodies with the multifunctional activity of cytokines. An anti-(ganglioside GD2) antibody (ch14.18) fusion protein with interleukin-2 (ch14.18-IL2), constructed by fusion of a synthetic sequence coding for human interleukin-2 (IL-2) to the carboxyl end of the Cγ1 gene of ch14.18, was tested for its therapeutic efficacy against xenografted human neuroblastoma in vivo. The ch14.18-IL2 fusion protein markedly inhibited growth of established hepatic metastases in SCID (severe combined immunodeficiency) mice previously reconstituted with human lymphokine-activated killer cells. Animals treated with ch14.18-IL2 showed an absence of macroscopic liver metastasis. In contrast, treatment with combinations of ch14.18 and recombinant IL2 at dose levels equivalent to the fusion protein only reduced the tumor load. Survival times of SCID mice treated with the fusion protein were more than double that of control animals. These results demonstrate that an immunotherapeutic approach using a cytokine targeted by an antibody to tumor sites is highly effective in eradicating the growth of established tumor metastases. Received: 7 November 1995 / Accepted: 15 December 1995     

Effects of interleukin-12 on in vitro culture with interleukin-2 of regional lymph node lymphocytes from lung cancer patients

 In the present study, we carried out a functional analysis of regional lymph node lymphocytes (RLNL) from patients with lung cancer after in vitro activation by interleukin-2 (IL-2) and interleukin-12 (IL-12). IL-12 (100 U/ml) enhanced both the proliferation and cytotoxic activity of RLNL in a culture with low doses of IL-2 (5 – 10 JRU/ml). After comparing an RLNL culture with a low dose of IL-2 alone, a higher proportion of CD8⁺ cells and CD56⁺ cells and a lower proportion of CD4⁺ cells were found in the culture with both IL-12 and a low dose of IL-2. Such a combination of the cytokines effectively activated RLNL in terms of the expression of IL-2 receptors. In the culture condition of IL-12 and a low dose of IL-2, a synergistic effect was observed in the production of such cytokines as interferon γ, tumor necrosis factor α (TNFα), and TNFβ, as well as in tumor cytotoxicity. However, the addition of IL-12 inhibited the cytotoxicity of RLNL in the culture with a high dose of IL-2 (100 JRU/ml). This inhibition is considered to be partially due to the endogenous production of TNFα by lymphocytes, because the neutralization of TNFα bioactivity partially restored the cytotoxic activities of RLNL. Furthermore, in the presence of hydrocortisone, IL-12 synergistically enhanced the cytotoxic activity of RLNL cultured with a high dose of IL-2. These results provide useful information about the improvement of adoptive immunotherapy against cancer using RLNL. Received: 2 February 1996 / Accepted: 30 July 1996     

Induction of accessory cell function of human alveolar macrophages by inhalation of human natural interleukin-2

 Accessory function allows antigen-presenting cells to produce sufficient secondary signals for optimum T cell proliferation and interleukin-2 (IL-2) production. Alveolar macrophages are inferior accessory cells compared to monocytes (PBM). We report here that the accessory index (AI) of alveolar macrophages and PBM of patients with lung metastases of solid tumors treated with inhalations of human natural IL-2 (hnIL-2) increased following its administration (P<0.005). The accessory index was significantly elevated from baseline values after 2 weeks of inhalation of 300 000 IU hnIL-2/day (8.2±10.2 compared to 1.1±1; P<0.001). The inhalation of 150 000 IU also induced increases in the index (AI = 2.3±1.9), however, without reaching statistical significance. In addition at 300 000 IU IL-2/day a significant increase in the accessory index was observed for PBM (4±2.5; P<0.05). The indices of PBM and alveolar macrophages prior to inhalation showed a significant negative correlation with the age of the patients (r _s =  – 0.5; r _s =  – 0.8, respectively; P<0.03 for all comparisons). Our data demonstrate that the inhalational application of hnIL-2 enhances the accessory function of alveolar macrophages and, to lesser extent, the accessory index of PBM, indicating the occurrence of pharmacological immunostimulation. Received: 16 August 1995 / Accepted: 4 January 1996     

白细胞介素－2中枢镇痛作用途径的探讨

抗ＩＬ－２受体α亚基的单克隆抗体不能阻断ＩＬ－２的中枢镇痛作用,以及丧失与ＩＬ－２受体β亚基结合能力的ＩＬ－２突变体仍具有提高大鼠痛阈的能力,这表明ＩＬ－２的中枢镇痛作用并不是通过ＩＬ－２受体所介导,亦表示ＩＬ－２的免疫和镇痛作用是通过不同的受体途径实现的。加之内源性阿片肽与ＩＬ－２分子有着共同的抗原决定基和结构相似性,提示ＩＬ－２可以与阿片受体直接结合产生中枢镇痛效应。从放射免疫法测定的ＩＬ－２侧脑室注射后不同时间大鼠脑内不同核团的内源性阿片肽含量,推测ＩＬ－２的中枢镇痛作用可能还与弓状核、室旁核、蓝斑等核团的β－ＥＰ和ＬＥＫ有关。     

Is the Mg2+-ATP-dependent proton pumping activity of the synaptic vesicles a factor involved in the cerebral hypoxia?

The changes in the Mg²⁺-dependent V-type ATPase activity and the Mg²⁺-ATP-dependent H⁺ pumping activity of the synaptic vesicles from the cerebral cortex of rats submitted to intermittent chronic (4 weeks) mild or severe hypoxia were evaluated. The adaptation to the chronic severe hypoxia increases both the ATPase and the H⁺ pumping activities which are inhibited by NEM with an exponential relationship between the IC₅₀ values and the in vivo O₂ concentration. The Mg²⁺-dependent increase in H⁺ pumping activity of synaptic vesicles from the rats subjected to in vivo chronic hypoxia may be antagonized by nigericin (dissipating ΔpH) and by FCCP (dissipating ΔpH and Δ_ΨSV). In contrast, valinomycin (dissipating the Δ_ΨSV and facilitating an enhancement in ΔpH) increases in vitro the H⁺ pumping activity that is inhibited by the addition of high concentration of K gluconate (reducing the rate of K⁺ efflux). The preincubation of vesicles from hypoxic rats with FCCP, but not with nigericin, inhibits the valinomycin-increased H⁺ pumping activity.l-glutamate increases the H⁺ pumping activity in synaptic vesicles from the cerebral cortex of chronic hypoxic rats, whereas other amino acids (i.e.,l-aspartate andl-homocysteate) and glutamate analogs (i.e., quisqualate and ibotenate) are ineffective. The adaptation to both chronic intermittent severe hypoxia and in vivo treatment with posatireline causes a decrease in the Mg²⁺-ATPase activity consistent with the decrease in the H⁺ pumping one of the synaptic vesicles. The addition of nigericin into incubation medium magnifies the decrease in the H⁺ pumping activity, while the addition of FCCP is ineffective, suggesting that the treatment with posatireline interferes with the Δ_ΨSV component in the of the synaptic vesicles from rats submitted to chronic hypoxia. The results of the in vivo and in vitro experiments suggest that in the synaptic vesicles from hypoxic rats the Δ_ΨSV component in may be most effective in increasing the Mg²⁺-ATP-dependent H⁺ pumping activity.     

Ecophysiology and distribution of the endemic leafless spurgeEuphorbia aphylla and the introducedE. tirucalli (Euphorbiaceae,Euphorbia sect.Tirucalli) in the Canary Islands

The leafless spurgeEuphorbia aphylla (Euphorbiaceae), an endemic species restricted to several of the Canary Islands where it inhabits coastal and arid localities, expresses Crassulacean Acid Metabolism (CAM) when it is subjected to summer drought. A flexible CAM is consistent with the general ecology of the species. It is the only member of sect.Tirucalli native to the Canary Islands and is at the north-western edge of the section's biogeographical range. The other members of the section have a paleotropic distribution and are found throughout Africa. Many of them are regarded as obligate CAM plants, includingE. tirucalli which was used as a comparison in ecophysiological experiments examining the response ofE. aphylla to drought and temperature.     

Augmentation of Interferon Production after Cell-Differentiation of U937 Cells by TPA

Persistent infections with mumps virus were established in several human lymphoid cells of T-cell origin (Molt-4, TALL-1, and CCRF-CEM) and human monocyte cells (U937 and THP-1). 2′,5′-Oligoadenylate synthetase (2–5AS) activity was demonstrated to be only slightly induced by interferon (IFN) or TPA (12-O-tetradecanoyl-phorbol-13-acetate) treatment in these cells. Treatment of the persistently infected cells with IFN or TPA did not stimulate an increase in the amount of synthetase mRNA. Induction of cell differentiation and augmentation of IFN production by TPA were demonstrated in U937 cells persistently infected with mumps virus (U937-MP). Similar results for IFN production were obtained from differentiated U937 cells. It is suggested that cell differentiation of U937 cells might be associated with the development of IFN inducibility.