Combined Cycloheximide and 8-Hydroxyquinoline Pretreatment for Study of Plant Chromosomes

The actions of cycloheximide and 8-hydmxyquinoline on dividing cells of root meristems of Zea mays L. have been studied during the development of a new cytological technique for sugar cane (Saccharum) root tips. The determination of mitotic phase indices revealed that combined treatment with cycloheximide (70 ppm) plus 8-hydroxyquinoline (250 ppm) was superior to treatments with either chemical separately. After the combined treatment, the preparations contained nearly ten times more cells in prophase and metaphase that were suitable for chromosome counting than those given a single pretreatment with 8-hydmxyquinoline. This new pretreatment has been developed especially for chromosome studies in tropical grasses with a large number of small chromosomes. However, both chemicals are active in a wide range of plant species.     

Air drying method using nitrous oxide for chromosome counting in maize

Nitrous oxide (N₂O), colchicine, trifluralin, amiprophos-methyl, 8-hydroxyquinoline, and temperature pretreatments (cold and cold-hot-cold) were compared for chromosome counting in maize (Zea mays L.). Pretreated root tips were prepared by enzymatic maceration and air drying, and the number of countable figures and mitotic indexes were recorded. N₂O treatment at 10 atm for 3 hr produced the largest number of countable chromosome figures (266.5 per preparation) and an average of 44.2 nonoverlapped chromosome figures per preparation. Treatment with 0.04% 8-hydroxyquinoline for 3 hr exhibited a moderate number of countable chromosome figures (53.9 per preparation). The effects of colchicine, trifluralin, amiprophos-methyl and temperature pretreatments were limited.     

Chromosome Contraction by o-Isopropyl-N-Phenylcarbamate (IPC)

A selective herbicide IPC (o-isopropyl-N-phenlycarbamate) causes contraction of chromosomes in the prophase, metaphase, and anaphase stages of mitosis in cells of treated root tips. Effective concentrations in aqueous solution lie between 2.5 and 50 ppm, and effective times between 1 and 4 hr, depending upon the species of plant. A suggested starting combination is 10 ppm for 2 hr. This compound is effective in causing contraction of chromosomes in a wide range of plant species, as well as enhancing separation in acetocarmine and aceto-orcein squashes in many cases. Possibly, it may produce similar results in species which have been found to be unaffected by colchicine, 8-hydroxyquinoline, p-dichlorobenzene, and other commonly used chemicals.     

高山榕染色体制片优化及核型分析

以高山榕种子的根尖为材料进行染色体常规压片,比较不同预处理方法和解离方法对高山榕染色体制片的影响,以选择最优的压片方法制片并进行核型分析。结果显示,预处理24 h的总体效果优于12 h;3种预处理液的总体作用效果为0.05%秋水仙素>混合液>0.002 mol.L-18-羟基喹啉,综合比较后认为混合液低温4℃处理24 h为最佳预处理方法。解离方法选择1 mol.L-1HCl中60℃水浴2~3 min较为适宜。首次报道了高山榕核型公式为2n=2x=30=22m(2SAT)+6sm+2T,核型不对称系数为61.54%,核型属于Stebbins核型分类中的2C类型。     

小水榕染色体数目及核型分析

以小水榕根尖为材料,通过取材时间,8-羟基喹啉、饱和对二氯苯2种药剂预处理,并以细胞中有丝分裂相的数目、染色体的清晰度、分散程度为指标,探索出适于小水榕染色体分析的方法.结果表明:小水榕有丝分裂中期在一天的16:00达到高峰期,取样时间确定在16:00.饱和对二氯苯水溶液预处理2 h,卡诺氏固定液固定2 h,1 mol/L盐酸60 ℃水浴中解离3 min～4 min,并以醋酸洋红染色效果较好.小水榕核型为2 n=2 x=30=12 m+14 sm+4 st,属于"2B"类型.     

青杄愈伤组织在继代培养中的分化能力及染色体稳定性研究

青杄（Ｐｉｃｅａ ｗ ｉｌｓｏｎｉｉＭａｓｔ．）胚性愈伤组织在改良５９ 附加２, ４－Ｄ及Ｋｔ各１ ｐｐｍ 的培养基上继代３ 年（每月继代１ 次）,仍具有旺盛的增殖能力。在胚性愈伤组织转入１／２改良５９ 并附加ＡＢＡ １ ｐｐｍ 的分化培养基上,约３ 个月左右可分化出大量体细胞胚。体细胞胚分化率达９０％ 以上。经继代３ 年的胚性愈伤组织细胞的染色体倍性十分稳定,其染色体数及核型为２ｎ＝ ２４＝ １６ｍ （６ｓｃ）＋ ８ｓｍ ＋ ２Ｂ。这一结果与由实生苗根尖压片所得结果基本一致     

8-Hydroxyquinoline derivatives induce the proliferation of rat mesenchymal stem cells (rMSCs)

A series of 8-hydroxyquinoline derivatives with different substituted groups at 2- or 5-position have been synthesized and characterized. Their effects on the proliferation of the rat marrow-derived mesenchymal stem cells (rMSCs) have been evaluated by MTT assay and flow cytometry. We also analyzed the ability of these compounds to regulate the proliferation of rMSCs and the relationship with the structures of 8-hydroxyquinoline. Compounds 8-11, in which, the vinyl-substituents are on the 2-position of 8-hydroxyquinoline, appear to be able to induce the proliferation of rMSCs. These results show that compounds 8-11 provide a kind of new substances for regulating the proliferation of rMSCs.     

A squash technique for studying the cytology of maize endosperm and other tissues.

A new cytological procedure specifically suited to maize endosperms is presented. It uses 8-hydroxyquinoline with sucrose and aeration to pretreat the tissues. Glusulase is used to spread the cells. The procedure makes it possible to squash endosperms into a single cell layer and to photograph as many as 70 chromosomes in the same focal plane. It also allows identification of translocation chromosomes. With a slight modification the technique has been applied successfully to root tips and other tissues.     

A Squash Technique for Studying the Cytology of Maize Endosperm and Other Tissues

A new cytological procedure specifically suited to maize endosperms is presented. It uses 8-hydroxyquinoline with sucrose and aeration to pretreat the tissues. Glusulase is used to spread the cells. the procedure makes it possible to squash endosperms into a single cell layer and to photograph as many as 70 chromosomes in the same focal plane. It also allows identification of translocation chromsomes. with a slight modification the technique has been applied successfully to root tips and other tissues.     

石山苣苔属四种(含一变种)植物的染色体数目和倍性研究

石山苣苔属(苦苣苔科)约41种,主要分布于我国西南石灰岩地区。到目前为止,仅其中四种的染色体数目被研究和报道,其余绝大多数物种的染色体数目和倍性尚不清楚,染色体数目和倍性在该属及其姐妹属报春苣苔属中的演变历史及其对两属物种多样性分化的影响亦不清楚。该文以叶片水培生根法获取的四种(含一变种)石山苣苔属植物 [即石山苣苔原变种(Petrocodon dealbatus var. dealbatus)、齿缘石山苣苔(Petrocodon dealbatus var. denticulatus)、弄岗石山苣苔(Petrocodon longangensis)、石山苣苔未定名种(Petrocodon sp.)]的根尖细胞为材料开展染色体实验,探索了多种不同的实验条件对染色体制片效果的影响并获取染色体数目,在石山苣苔属和报春苣苔属的系统树上追踪了染色体数目和倍性的演变历史,同时探讨染色体数目尤其是倍性变化是否对两属物种多样性分化存在影响。结果表明:(1)长度为1～1.5 cm的根尖,0.002 mol·L^-1 8-羟基喹啉溶液预处理5 h,解离4 min为较适宜的染色体制备条件。(2)四种(含一变种)石山苣苔属植物染色体数目一致,均为二倍体(2n=2x=36)。(3)两属之间及两属各自的最近共同祖先染色体数目尚不能确定,除个别物种染色体条数或倍性有变化以外,其余已知染色体数目的物种均为2n=2x=36,在两属中高度一致,石山苣苔属与报春苣苔属物种多样性分化尤其两属物种多样性巨大差异与染色体数目和基因组倍性变化无关。综上结果为石山苣苔属植物及其近缘类群染色体制备提供了参考,也为进一步对该类群的分类、系统演化和物种形成等方面的研究提供了基础数据和启示。     

Phorbol ester induces manganese-superoxide dismutase in tumor necrosis factor-resistant cells.

The effects of phorbol ester (TPA) and other known stimulators such as tumor necrosis factor (TNF), interleukin-1, and lipopolysaccharide on induction of mRNA for manganese-superoxide dismutase (Mn-SOD) were investigated in various cell lines. TPA enhanced Mn-SOD mRNA expression in TNF-resistant cell lines including HeLa cells, in which the other reagents also induced expression of the gene, but did not affect TNF-sensitive cells, in which the other stimulators did not alter expression of the gene. HeLa cells which had been desensitized to TPA by pretreatment with TPA for 24 h expressed Mn-SOD mRNA at a slightly higher level than the cells without TPA treatment. TPA-pretreated cells stimulated with TNF, however, expressed Mn-SOD mRNA at about twice the level of TNF-stimulated, TPA-untreated cells. When protein synthesis was inhibited by cycloheximide during TPA pretreatment, TNF no more enhanced the Mn-SOD mRNA accumulation. These data suggest that at least two separate signal-transducing pathways are involved in expression of this gene. One is triggered by protein kinase C activation itself in the absence of new protein synthesis. The other can be activated by stimulation with TNF, interleukin-1, or lipopolysaccharide and in which a protein factor that can be induced by TPA treatment is involved.     

The mechanism of action of lutropin on regulator protein(s) involved in Leydig-cell steroidogenesis

The dependence on lutropin of the synthesis of a proposed short-half-life protein regulator involved in Leydig-cell steroidogenesis was investigated. This was carried out by determining the effect of the protein-synthesis inhibitor cycloheximide, added before and during incubations with lutropin (and/or dibutyryl cyclic AMP), on the rate of testosterone production in suspensions of purified Leydig cells from adult rat testes. The Leydig cells were preincubated in Eagle's medium for 2.5h followed by 30min incubation with and without cycloheximide. The inhibitor was removed by washing the cells and then lutropin was added and testosterone concentrations were determined after incubation of the cells at 32 degrees C. No significant effect of cycloheximide pretreatment on lutropin-stimulated steroidogenesis was found during 60min incubation. This was in contrast with the complete inhibiting effect of cycloheximide when it was added with the lutropin. The pretreatment experiments with cycloheximide were repeated in the presence of dibutyryl cyclic AMP and elipten phosphate (to inhibit cholesterol side-chain cleavage) followed by incubation with lutropin. After 5, 10, 20 and 60min of incubation, testosterone concentrations were 61+/-3, 46+/-3, 27+/-4 and 18+/-4% lower than in the cells pretreated without cycloheximide respectively (means+/-s.e.m., n=4-6). In the cells not pretreated with cycloheximide and in the absence of lutropin, testosterone production increased from 1.36+/-0.5 to 36.5+/-1.0ng/10(6) cells during 20min of incubation, after which no further increase occurred. Pretreatment of the cells with cycloheximide decreased these testosterone concentrations by 65, 46, 42 and 36% in the 5, 10, 20 and 60min incubations respectively (mean values, n=2-4). It is apparent from these results that inhibition of steroidogenesis only occurs if protein synthesis is inhibited in the presence of lutropin or cyclic AMP. A new hypothesis is put forward to explain these findings: it is proposed that lutropin affects the stability of a precursor of a regulator protein by converting it from a stable (inactive) to an unstable (active) form with a short half-life.     

Evaluation of several holding solutions for prolonging vase-life and keeping quality of cut sweet pea flowers (Lathyrus odoratus L.)

Cut spikes of sweet pea (Lathyrus odoratus L.) were kept in 2% sucrose, 200 ppm 8-hydroxyquinoline sulfate (8-HQS), pulsing treatment with 200 ppm 8-HQS in combination with 2% sucrose for 12 h, pulsing the spikes with 0.2 mM silver thiosulfate (STS) for 1 h and pulsing with 0.2 mM STS for 1 h followed by 2% sucrose solution. Therefore, this study aimed to see their effects on keeping quality and vase-life of the cut flowers. A control (deionized water) and a standard preservative were also included in the experiment. The results showed that all treatments had improved the keeping quality and vase-life of the cut flowers comparing to control ones. Among all these treatments, the 8-HQS combined with 2% sucrose showed the best water uptake, water balance, percentage of maximum increase in fresh weight of the cut flower stems and vase-life which was extended up to 17 days. Moreover, this keeping solution retarded the chlorophyll as well as carbohydrate degradation. However, anthocyanin concentrations were increased by treatments with sucrose alone or STS followed by sucrose during the postharvest life. It has been concluded that 200 ppm 8-HQS combined with 2% sucrose solution has the potential to be used as a commercial cut flower preservative solution to delay flower senescence, enhance post harvest quality and prolong the vase-life of sweet pea cut flowers.     

Parthenogenetic development of bovine oocytes matured in vitro for 24 hr and activated by ethanol and cycloheximide

This research was undertaken to improve development of parthenogenetic embryos following various combined treatments of ethanol and cycloheximide. In Experiment 1 in vitro matured oocytes (IVM, 24 hr) were treated with 7% ethanol for 5 min followed by incubation in 10 μg/ml cycloheximide in Medium 199 for 0 (control), 5, 10, and 20 hr. Development to 2–8 cells following culture for 3 days was similar among treated groups (32–41%; P > 0.05), which was higher than that of controls (6%; P < 0.05). Experiment 2 compared pre-ethanol exposures for 0, 1, 2.5, and 5 min, followed by 5 hr cycloheximide treatment on activation development. One- to 5-min groups resulted in 42–44% cleavage contrasted to 1–12% for controls (P < 0.05). Experiment 3 examined the effect on oocyte development of ethanol and different concentrations of cycloheximide (0, 1, 5, and 10 μg/ml). Cleavage to 2–8 cells was similar among the 5 and 10 μg/ml cycloheximide groups (36% and 42%, P > 0.05) but lower (P < 0.05) for the 1 μg/ml group (24%) and the controls (2–13%). When 5 μg/ml cycloheximide was used (Experiment 4), pre-exposure to ethanol (1, 2.5, and 5 min) resulted in more oocytes cleaved (38–41%) than in the cycloheximide alone group (0%) or the control (0%, P < 0.05). Experiment 5 tested blastocyst development of the activated oocytes with or without cytochalasin B treatment. Oocytes developed to blastocyts were 0%, 14%, 3%, and 3% (P < 0.05), respectively, for control, treatment with ethanol and cycloheximide in the presence, or absence of cytochalasin B, or electrical pulse plus cycloheximide. In conclusion, the combined ethanol and cycloheximide treatment supported high rates of parthenogenetic development using 24 hr IVM bovine oocytes. Blastocyst rate was significantly higher when cytochalasin B was added to the combined activation regimen. © 1994 Wiley-Liss, Inc.     

急性臭氧暴露对大鼠肺部细胞遗传毒性的影响*

目的: 探讨不同浓度臭氧急性暴露对大鼠肺部细胞的遗传毒性的影响。方法: 36只wistar大鼠随机分为对照组(过滤空气暴露)、臭氧暴露组(0.12 ppm、0.5 ppm、1.0 ppm、2.0 ppm、4.0 ppm)共6组,每组6只。以不同浓度的臭氧对大鼠进行动态染毒4 h后,取肺组织并分离单细胞,采用酶联免疫吸附法检测8-羟基脱氧鸟苷(8-OHdG),利用彗星实验、微核试验和DNA-蛋白质交联实验进行DNA和染色体损伤分析。结果: 与对照组相比,肺组织中8-OHdG含量从臭氧暴露浓度为0.12 ppm起即显著增加,在0.5 ppm时达到最高值。随着臭氧暴露浓度升高,彗星拖尾率逐渐上升,且存在明显的剂量-效应关系;DNA-蛋白质交联率有先升高后下降的趋势,且在2.0 ppm时达到最大值;而肺部细胞微核率尽管呈现出上升趋势,但与对照组相比无显著性差异。结论: 急性臭氧暴露在较低浓度(0.12 ppm)时即可导致大鼠肺部细胞的DNA损伤;而在较高浓度(4 ppm)时却未见显著的染色体损伤。     

Genetic differences in phosphorus absorption among white clover populations

Summary Eight semi-natural white clover populations and two cultivars were grown in culture solutions containing 10 ppm and 0.01 ppm phosphorus (P). The rate of P uptake by the intact plants was then measured in solutions containing 10 ppm P.Phosphorus uptake per unit root length was twice as great by plants previously grown at 0.01 ppm P than those grown at 10 ppm P. Large differences in total P uptake were found among populations regardless of the pretreatment; most of this variation was accounted for by differences in root length. Only small differences were found between populations for P uptake per unit root length, and then only after pretreatment with 10 ppm P; this variation was largely accounted for by relative growth rate and shoot %P.     

Study on the Hybrid Endosperm Culture of Wheat-Rye In Vitro

The endosperm culture of wheat-rye hybrid was studied in order to explore a new pathway of chromosome engineering. The preliminary results were obtained to show that the endosperm callus formation could be induced from the young endosperm within 7–14 days after crossing on the medium supplemented with 2 ppm 2,4-D, 0.5 ppm kinetin and 3%–8% sucrose. The induction frequency of callus amounts to 35.3%. When the calli were transfered onto an auxin step-down medium containing 0.5 ppm IAA and 1 ppm kinetin, both shoots and roots were formed. 4 endosperm plantlets were obtained. The chromosome number in somatic cells of endosperm plantlets was very unstable. The numbers varied from 6—42, but there is no 49 to be found. The chromosome number with 1—4 times of 7 can be found in higher percentage.     

Nitric oxide as a critical factor for perception of UV-B irradiation by microtubules in Arabidopsis

Influence of ultraviolet-B (UV-B) as an abiotic stress factor on plant microtubules (MTs) and involvement of nitric oxide (NO) as a secondary messenger mediating plant cell response to environmental stimuli were investigated in this study. Taking into account that endogenous NO content in plant cells has been shown to be increased under a broad range of abiotic stress factors, the effects of UV-B irradiation and also the combined action of UV-B and NO donor sodium nitroprusside (SNP) or NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO) on the MTs organization in different root cells of Arabidopsis thaliana were tested. Subsequently, realization of the MT-mediated processes such as root growth and development was studied under these conditions. Arabidopsis thaliana seedlings expressing the chimeric gene gfp-map4 were exposed to the enhanced UV-B with or without SNP or c-PTIO pretreatment. The UV-B irradiation alone led to a dose-dependent root growth inhibition and to morphological alterations of the primary root manifested in their swelling and excessive root hair formation. Moreover, dose-dependent randomization and depolymerization of MTs in both epidermal and cortical cells under the enhanced UV-B were found. However, SNP pretreatment of the UV-B irradiated A. thaliana seedlings recovered the UV-B inhibited root growth as compared to c-PTIO pretreatment. It has been shown that in 24 h after UV-B irradiation the organization of MTs in root epidermal cells of SNP-pretreated A. thaliana seedlings was partially recovered, whereas in c-PTIO-pretreated ones the organization of MTs has not been distinctly improved. Therefore, we suppose that the enhanced NO levels in plant cells can protect MTs organization as well as MT-related processes of root growth and development against disrupting effects of UV-B.