Death of stoma guard cells in leaf epidermis under disturbance of energy provision

Cyanide is an apoptosis inducer in stoma guard cells from pea leaf epidermis. Unlike CN-, the uncoupler of oxidative and photosynthetic phosphorylation carbonyl cyanide m-chlorophenylhydrazone (CCCP), the combination of CCCP, 3-(3 ,4 -dichlorophenyl)-1,1-dimethylurea (DCMU), benzylhydroxamate (BH), myxothiazol, antimycin A, and a glycolysis inhibitor 2-deoxyglucose (DG) did not induce destruction of guard cell nuclei for 20 h of incubation of epidermal peels in the light. DCMU prevented the effect of CN- as a programmed cell death (PCD) inducer. CCCP, the combination of DCMU and CCCP, or the combination of DCMU, CCCP, BH, myxothiazol, antimycin A, and DG supplemented by CN- caused destruction of cell nuclei; the number of the cells lacking nuclei in this case was higher than with CN- alone. DG and CCCP caused cell destruction after longer incubation of the isolated epidermis - after 2 days and to a greater degree after 4 days. The effect of DG and CCCP was reduced by illumination. Cell destruction during long-term incubation was prevented by the combination of DG and CCCP. From data of electron microscopy, DCMU and dinitrophenyl ester of iodonitrothymol (DNP-INT) prevented apoptotic changes of the nuclear ultrastructure induced by CN-. The suppression of the destruction of the guard cell nuclei under combined action of DG and CCCP was apparently caused by switching of cell death from PCD to necrosis. Thus, the type of cell death - via apoptosis or necrosis - is controlled by the level of energy provision.     

Microtubules spatial alterations in root cells of Brassica rapa under clinorotation

Organization of tubulin cytoskeleton in epidermis and cortex cells in different root growth zones in Brassica rapa L. 6-day-old seedlings under clinorotation has been investigated. It was shown that changes in cortical microtubules orientation occur only in the distal elongation zone. In control, cortical microtubule arrays oriented transversely to the root long axis. Whereas under clinorotation an appearance of shorter randomly organized cortical microtubules was observed. Simultaneously, a significant decrease in a cell length in the central elongation zone under clinorotation was revealed. It is suggested that the decline of anisotropic growth, typical for central elongation zone cells, is connected with cortical microtubules disorientation under clinorotation.     

Nitrogen metabolism in cultured cotyledon explants of Pinus radiata during de novo organogenesis

Nitrogen metabolism was investigated under shoot-forming (SF) and non-shoot-forming (NSF) conditions in cultured cotyledon explants of Pinus radiata by following the incorporation of [¹⁴C]-l,2-acetate into various metabolites. Early in culture, the lipid fraction contained the most ¹⁴C; however, this percentage decreased in favor of increased label in the amphoteric fraction. Label in the amphoteric fraction of SF cultures decreased by day 21 but plateaued in NSF cultures at this time. Radioactive labeling of the principle nitrogen metabolites, glutamate and glutamine, which made up the majority of the amphoteric fraction, paralleled labeling patterns in the amphoteric fraction. Percentage label in glutamate remained at similar levels throughout the 21-day culture period for both SF and NSF cultures. Specific activity of glutamate (kBq mg^-1) was significantly greater during promeristemoid formation in SF compared to that in NSF tissues. Glutamine labeling increased during shoot bud initiation in SF cultures, but dropped to lower levels during shoot bud development. In contrast, in NSF cultures, there was a continual and substantial increase in glutamine labeling throughout the 21-day culture period. These trends were similar when the specific activities of glutamine were determined, as there was a continual decrease from culture initiation to the end of shoot bud differentiation in SF cultures. In NSF cultures, in contrast, specific activity of glutamine increased substantially from day 5 to 21 relative to that in SF cultures. The nitrogen assimilation enzymes glutamate synthase and glutamine synthase increased in activity from day 0 to 21 for both SF and NSF tissues. Enzyme activities for glutamate dehydrogenase were similar in both treatments to day 10 in culture but subsequently diverged, with activities in NSF cultures being substantially greater than those of SF cultures by day 21. Taken together, labeling and enzyme data indicate that nitrogen metabolism is enhanced during culture, especially in SF tissues at the time of promeristemoid formation, and in non-organ-forming tissue senescence-like metabolism was exhibited later in culture.     

Calcium balance changes in tip growing plant cells under clinorotation

Calcium is known to play an important role in the regulation of cellular processes as a secondary messenger in signal transduction to a specific response in all eucaryotic cells. Its messenger role is realized by transient changes in the Ca2+ cytosolic concentration induced by variety of external stimuli such as light, hormones and gravity. Recent findings claim the modifications in a calcium balance in plant cells in microgravity and under clinorotation reproducing partially the microgravity effect. Based on these data, the hypothesis is proposed that Ca2+ level changes can trigger the rearrangements in cell metabolism occurring in the conditions of altered gravity. However, the methods used in previous works permit only to determine the relative quantity of intracellular membrane-bound calcium and to observe the localization of free Ca2+. Therefore, it is of essential interest to measure the concentration of free calcium ions ([Ca2+]i) in plant cells under the influence of altered gravity. In this paper results from measurements of the [Ca2+]i in plant cells under clinorotation will be reviewed and discussed in an effort to examine their effectiveness, causes and consequences.     

Effects of clinorotation on the structure of xylem in Arabidopsis roots]

Lignin, which is the most important component of vascular tissue, is essential material for the structural support of higher plants and considered to play a critical role in evolution of land plants. It has been postulated that development of secondary wall is mediated by gravity. However, effects of gravity on the development and the morphology of secondary cell wall have not been well investigated. In this study, effects of averaging of gravity vector using 2-D clinostat rotation on the structure of xylem was examined. The results indicated that the morphology of xylem is regulated by the orientation of gravity vector.     

瘤足蕨科及其近缘类群植物叶表皮微形态扫描电镜观察

瘤足蕨科是一个自然的群,但由于各类群之间的形态差异及种内变异比较大,其分类和系统位置一直有争议。该研究利用扫描电子显微镜对瘤足蕨科10种及其近缘类群桫椤科2种、蚌壳蕨科1种共13种植物,即瘤足蕨(Plagiogyria adnata)、耳形瘤足蕨(P.stenoptera)、镰羽瘤足蕨(P.falcata)、峨嵋瘤足蕨(P.assurgens)、密羽瘤足蕨(P.pycnophylla)、灰背瘤足蕨(P.glauca)、华东瘤足蕨(P.japonica)、华中瘤足蕨(P.euphlebia)、日本瘤足蕨(P.matsumureana)、美洲瘤足蕨(P.pectinata)、桫椤(Alsophila spinulosa)、西亚黑桫椤(A.khasyana)和金毛狗(Cibotium barometz)的叶表皮微形态进行了观察和比较,探讨其分类学意义,并据此编制了供试10个种类的分类检索表。结果表明:13种瘤足蕨科及其近缘类群植物气孔均仅分布在叶下表皮,气孔微下陷,气孔器椭圆形至圆形;叶上下表皮角质层纹饰复杂,类型多样,少数类群叶下表皮具毛状物;多数类群气孔外拱盖凹陷;气孔外拱盖内缘浅波状或齿状;保卫细胞两极大多数有明显的"T"型加厚。不同种间叶表皮扫描电镜特征表现出一定差异,对种的划分有一定的分类学鉴定意义。     

Mll fusions generated by Cre-loxP-mediated de novo translocations can induce lineage reassignment in tumorigenesis

Chromosomal translocations are primary events in tumorigenesis. Those involving the mixed lineage leukaemia (MLL) gene are found in various guises and it is unclear whether MLL fusions can affect haematopoietic differentiation. We have used a model in which chromosomal translocations are generated in mice de novo by Cre-loxP-mediated recombination (translocator mice) to compare the functionally relevant haematopoietic cell contexts for Mll fusions, namely pluripotent stem cells, semicommitted progenitors or committed cells. Translocations between Mll and Enl or Af9 cause myeloid neoplasias, initiating in pluripotent stem cells or multipotent myeloid progenitors. However, while Mll-Enl translocations can also cause leukaemia from T-cell progenitors, no tumours arose with Mll-Af9 translocations in the T-cell compartment. Furthermore, Mll-Enl translocations in T-cell progenitors can cause lineage reassignment into myeloid tumours. Therefore, a permissive cellular environment is required for oncogenicity of Mll-associated translocations and Mll fusions can influence haematopoietic lineage commitment.     

Quantitative analysis of microtubule orientation in interdigitated leaf pavement cells

Leaf pavement cells are shaped like a jigsaw puzzle in most dicotyledon species. Molecular genetic studies have identified several genes required for pavement cells morphogenesis and proposed that microtubules play crucial roles in the interdigitation of pavement cells. In this study, we performed quantitative analysis of cortical microtubule orientation in leaf pavement cells in Arabidopsis thaliana. We captured confocal images of cortical microtubules in cotyledon leaf epidermis expressing GFP-tubulinβ and quantitatively evaluated the microtubule orientations relative to the pavement cell growth axis using original image processing techniques. Our results showed that microtubules kept parallel orientations to the growth axis during pavement cell growth. In addition, we showed that immersion treatment of seed cotyledons in solutions containing tubulin polymerization and depolymerization inhibitors decreased pavement cell complexity. Treatment with oryzalin and colchicine inhibited the symmetric division of guard mother cells.     

Regulation of de novo purine biosynthesis in Chinese hamster cells

Regulation of de novo purine biosynthesis was examined in two Chinese hamster cell lines, CHO and V79. De novo purine biosynthesis is inhibited at low concentrations of adenine. The mechanism of inhibition was studied using the RNA and protein synthesis inhibitors actinomycin D, cycloheximide, and azacytidine. Although all three inhibitors rapidly inhibited de novo purine biosynthesis in vivo, neither adenine nor the RNA and protein synthesis inhibitors could be found to have an effect in vitro on either phosphoribosylpyrophosphate (PRPP) synthetase or amido phosphoribosyltransferase, the first enzymes of the de novo pathway. However, in the presence of actinomycin D, cycloheximide, and azacytidine, there was a 50% or greater reduction in PRPP concentrations. This reduction in PRPP levels is correlated with a 2-fold increase in purine nucleotides in the acid-soluble pool. It is proposed that in the presence of the metabolic inhibitors there is an increase in nucleotide pools due to degradation of RNA, with a resulting feedback inhibition on de novo purine biosynthesis. In contrast to a previous report (Martin, D. W., Jr., and Owen, N. T. (1972) J. Biol. Chem. 247, 5477-5485), we could find no evidence for a repressor type mechanism in these cells.     

CD4+ T cells generated de novo from donor hemopoietic stem cells mediate the evolution from acute to chronic graft-versus-host disease

Acute and chronic graft-versus-host disease (GVHD) remain the major complications limiting the efficacy of allogeneic hemopoietic stem cell transplantation. Chronic GVHD can evolve from acute GVHD, or in some cases may overlap with acute GVHD, but how acute GVHD evolves to chronic GVHD is unknown. In this study, in a classical CD8+ T cell-dependent mouse model, we found that pathogenic donor CD4+ T cells developed from engrafted hemopoietic stem cells (HSCs) in C57BL/6SJL(B6/SJL, H-2(b)) mice suffering from acute GVHD after receiving donor CD8+ T cells and HSCs from C3H.SW mice (H-2(b)). These CD4+ T cells were activated, infiltrated into GVHD target tissues, and produced high levels of IFN-gamma. These in vivo-generated CD4+ T cells caused lesions characteristic of chronic GVHD when adoptively transferred into secondary allogeneic recipients and also caused GVHD when administered into autologous C3H.SW recipients. The in vivo generation of pathogenic CD4+ T cells from engrafted donor HSCs was thymopoiesis dependent. Keratinocyte growth factor treatment improved the reconstitution of recipient thymic dendritic cells in CD8+ T cell-repleted allogeneic hemopoietic stem cell transplantation and prevented the development of pathogenic donor CD4+ T cells. These results suggest that de novo-generated donor CD4+ T cells, arising during acute graft-versus-host reactions, are key contributors to the evolution from acute to chronic GVHD. Preventing or limiting thymic damage may directly ameliorate chronic GVHD.     

中国桑寄生科植物叶表皮微形态

通过扫描电镜对中国桑寄生科桑寄生亚科8属18种和槲寄生亚科1属2种植物成熟叶的上、下叶表皮内表面和下表皮外表面进行了研究。内面观发现桑寄生科植物叶上、下表皮形状为多边形,垂周壁式样平直或稍弓形,常具有角质增厚,平周壁常覆盖厚角质或颗粒状、丝状角质增厚;气孔存在于上下表皮,通常下表皮较多,气孔的形状,特别是保卫细胞的形态在亚科间、属间或种间都具有一定的差异,气孔器类型为平列型或单圈型。下表皮表面观察了的角质膜和蜡质纹饰、气孔的形状,外部气孔缘及外部气孔缘内缘的特征。这些特征在亚科或属级水平上较为稳定,有的也表现出种间差异,有一定的分类价值。从气孔形态和外部气孔周围角质膜来看,两亚科显示出明显的不同:桑寄生亚科上、下表皮均具有内部气孔缘,而槲寄生亚科没有此结构;桑寄生亚科外部气孔周围角质膜增厚成环状,其上具增厚的条纹,而槲寄生亚科外部气孔周围角质膜增厚成脊状,不具条纹。这些特征支持槲寄生亚科作为独立1个科来处理。     

Demonstratioin of de novo synthesis of RNase in Rhoeo leaf sections by deuterium oxide labeling

The increase in RNase activity that occurs in fresh cut Rhoeodiscolor leaf sections was followed in sections aged in wateror deuterium oxide for 24 hr. The increase in RNase activityis probably due to de novo synthesis, since the buoyant densityof the RNase from the deuterium labelled tissue was significantlyhigher (1.10 to 1.88%) than that of the controls. (Received June 29, 1973; )     

Ultrastructure of statocytes and cells of distal elongation zone of Arabidopsis thaliana under clinorotation

Results of the electron-microscopic investigation of root apices of Arabidopsis thaliana 3-, 5- and 7-days-old seedlings grown in the stationary conditions and under clinorotation are presented. It was shown the similarity in the root apex cell ultrastructure in control and under clinorotation. At the same time there were some differences in the ultrastructure of statocytes and the distal elongation zone under clinorotation. For the first time the sensitivity of ER-bodies, which are derivatives of GER and contain beta-glucosidase, to the influence of simulated microgravity was demonstrated by increased quantity and area of ER-bodies at the cell section as well as by higher variability of their form under clinorotation. A degree of these changes correlated with the duration of clinorotation. On the basis of experimental data a protective role of ER-bodies in adaptation of plants to microgravity is supposed.     

Activation and de novo synthesis of transglutaminase in cultured glioma cells

The activity of transglutaminase (TGase) was measured in cultured C6 glioma cells after their stimulation by either isoproterenol and isobutyl-methylxanthine or by a serum-containing medium. The activity fluctuated in a biphasic manner, with the peaks at 2-3 hr and 7-8 hr poststimulation. The first peak of TGase activity was affected neither by cycloheximide nor by actinomycin D, which inhibited protein synthesis. The second peak, on the other hand, was completely eliminated by cycloheximide and was reduced by actinomycin D. Immunological procedures were employed to find out whether or not the activity of TGase corresponded with the presence of the TGase antigen in the cultured cells. Indirect immunofluorescent staining and radioimmunoblot techniques suggested that unstimulated cells contained an inactive enzyme. This inactive, or cryptic, enzyme had the same molecular weight as its active counterpart. Activation of the enzyme was mediated by cell stimulation, probably by its release from the membrane. This step did not require protein synthesis, unlike the second step, which was dependent on de novo protein synthesis.     

Synthesis of wheat leaf nitrite reductase de novo following induction with nitrate and light

Nitrite reductase has been purified almost 3000-fold, in 35% yield, to a specific activity of 77 units (mg protein)-1 from wheat leaves using a multi-step procedure with affinity chromatography on ferredoxin-Sepharose as the final step. The purified enzyme, although not homogeneous, exhibited absorption maxima at 278, 390, 568 and 687 nm. Minor contaminants were removed by gel filtration in the presence of sodium dodecyl sulphate to yield a single polypeptide of Mr 60 500 as judged by polyacrylamide gel electrophoresis. Antibodies raised against this polypeptide were shown to cross-react with native nitrite reductase and were used to study the synthesis of nitrite reductase in vivo and in vitro. The increase in nitrite reductase activity following exposure of dark-grown plants to nitrate and light was shown by immunodecoration of Western blots to be due to synthesis de novo. Poly(A)-rich RNA isolated from plants actively synthesising nitrite reductase was shown to direct the synthesis in a rabbit reticulocyte lysate of a polypeptide of Mr 64000 which was immunoprecipitated by antibodies to nitrite reductase.     

蜘蛛抱蛋属植物叶表皮微形态的扫描电镜观察

在扫描电子显微镜下,观察9种蜘蛛抱蛋属植物的叶表皮微形态。结果表明,9种植物叶下表皮的细胞都为长方形,垂直壁为直的,垂周界限为凸的,外层周壁凹陷。气孔椭圆形至卵圆形,气孔外拱盖表面多平滑,其内缘近平滑、浅波状或锯齿波状;角质膜多为脊状条纹、有的外层周壁有横纹或颗粒。气孔大小与染色体的倍性有一定的正相关关系。不同种间叶表皮特征表现出一定差异,对种的划分有一定的分类鉴定意义。