Germination Preventing Mechanisms in Iris Seeds

The different germination behaviour of the seeds of two irises,Iris lorteti and I. atropurpurea was found to be due to thedifferent mechanical resistance of the integument, at the micropylarend, to radicle protrusion. A pressure of 135 atm was necessaryin l. lorteti seeds for radicle protrusion, while in I. atropurpurea77 atm was sufficient. In contrast Pancratium maritimum requireda pressure of only 10 atm. The outer integument of seeds ofI. lorteti was found to contain a compound which was toxic tothe germinated embryo but did not act as a germination inhibitor.Extracts of the endosperm also had a slight germination inhibitingeffect. An interaction between this weak inhibitor and the effectof the testa could not be ruled out completely. A test assayfor germination using excised embryos was developed. A methodfor germination of Iris seeds, by cutting off the outer integumentat the micropylar end, was developed and is being exploitedcommercially. Iris lorteti, Iris atropurpurea, germination, germination inhibition, embryo culture, seed coat mechanical resistance     

鸢尾属植物种子休眠研究进展

鸢尾属(Iris L.)花卉具有较高的观赏性,极具开发利用价值,且主要以种子繁殖后代;但多数种子具有休眠特性,给育种工作带来了困难。其休眠原因包括种皮机械阻碍和种子中萌发抑制物存在等内外多重因素,单一或交互作用影响种子萌发。种子休眠解除的方法主要有环境条件处理、机械处理、化学试剂或激素处理等。本文对鸢尾属植物种子休眠原因、休眠解除方法及其机理等作了系统概述,旨在为鸢尾属花卉资源的合理开发利用及种质创新提供理论依据。     

Comparative effectiveness of lymphotoxin anticarcinogenic and tumor cell growth-inhibitory activities

Prevention of ultraviolet radiation- or chemical carcinogen-induced morphologic transformation and inhibition of tumor-producing transformed cell growth by lymphotoxin and by normal spleen leukocytes were quantitatively compared to define the antineoplastic activity spectra of these natural immune mediators. When Syrian golden hamster embryo cells seeded for colony formation in culture dishes were treated simultaneously with carcinogen and lymphotoxin, the number of morphologically transformed cell colonies was irreversibly reduced by 50% in the presence of 6 units of lymphotoxin/ml. Lymphotoxin inhibition of tumor cell growth, however, was reversible and 50% reduction in tumor cell growth in three transformed lines required 124, 330, and 477 units/ml. Thus, the anticarcinogenic activity of lymphotoxin can be 20-fold or more greater than its tumor growth-inhibitory activity. Similarly spleen leukocytes also were more effective as an anticarcinogen than as an inhibitor of tumor cell growth, consistent with previous observations that naturally occurring spleen leukocyte antineoplastic activity may result from lymphotoxin secretion.     

Detection of inhibitors of protein and nucleic acid synthesis using oocytes of Xenopus laevis microinjected with the herpes thymidine kinase gene

We have developed an assay that measures the inhibition of protein synthesis and can be used in conjunction with a whole embryo bioassay that detects the ability of a chemical to cause fetotoxicity, malformation and abnormal growth. The assay involves microinjecting the herpes thymidine kinase gene into stage 6 oocytes of Xenopus laevis then exposing the oocytes to a test compound for 18-24 h. The inhibition of thymidine kinase (TK) expression caused by an inhibitor is then measured by simple enzyme assay. Protein synthesis inhibitors such as cycloheximide, puromycin and emetine all inhibited TK synthesis. Concentrations of cycloheximide (1.4 X 10(-4) mg/ml) and puromycin (0.04 mg/ml) near the 96 h embryo LC50 inhibited thymidine kinase expression by 78% and 97%, respectively but emetine (0.01 mg/ml) had no effect. However, 0.1 mg/ml emetine inhibited TK synthesis by almost 50%. The RNA synthesis inhibitor, actinomycin D (0.013 mg/ml) inhibited TK expression by 61%. DNA synthesis inhibitors hydroxyurea (2.0 mg/ml), cytosine arabinoside (2.0 mg/ml) and ethidium bromide (0.02 mg/ml) failed to inhibit the expression of the TK gene even though these concentrations were near the 96 h embryo LC50. The whole embryo bioassay cannot differentiate the DNA synthesis inhibitors from the RNA and protein synthesis inhibitors but the oocyte assay can. This type of molecular test data can help separate classes of teratogens such as DNA synthesis inhibitors from nonteratogenic compounds such as protein synthesis inhibitors and allow the extrapolation of test data to other species.     

内蒙古野生马蔺种质农艺性状遗传多样性研究

以收集自内蒙古11个盟市的20个野生马蔺种质材料为研究对象,通过对其13个农艺性状指标进行主成分分析、相关性分析和聚类分析,探讨不同种质间的亲缘关系、遗传变异特性及其原因,为马蔺的开发利用、资源保护和育种应用提供理论依据。结果表明:(1)不同来源的马蔺种质农艺性状表现出不同程度的变异性,变异系数范围为9.72%~300.00%,变异系数较大的性状是千粒重、胚长和发芽率,变异系数较小的性状是株高和叶宽。(2)千粒重、发芽率、吸水率、胚长、胚乳长、种子长、生殖枝数、营养枝数、叶宽、株高10个主要性状是引起不同来源马蔺种质农艺性状分化的主要指标。(3)各农艺性状间存在不同程度的相关性,经度、纬度、海拔高度是引起马蔺种质变异的主要因素,胚长、千粒重、吸水率易受生态环境因子的影响。(4)来源不同的20个马蔺种质聚为4类,绝大多数种质材料表现出明显的地域性,经、纬度相近或小生境相似的种质聚为一类。     

Rethinking in vitro embryo culture: new developments in culture platforms and potential to improve assisted reproductive technologies

The preponderance of research toward improving embryo development in vitro has focused on manipulation of the chemical soluble environment, including altering basic salt composition, energy substrate concentration, amino acid makeup, and the effect of various growth factors or addition or subtraction of other supplements. In contrast, relatively little work has been done examining the physical requirements of preimplantation embryos and the role culture platforms or devices can play in influencing embryo development within the laboratory. The goal of this review is not to reevaluate the soluble composition of past and current embryo culture media, but rather to consider how other controlled and precise factors such as time, space, mechanical interactions, gradient diffusions, cell movement, and surface interactions might influence embryo development. Novel culture platforms are being developed as a result of interdisciplinary collaborations between biologists and biomedical, material, chemical, and mechanical engineers. These approaches are looking beyond the soluble media composition and examining issues such as media volume and embryo spacing. Furthermore, methods that permit precise and regulated dynamic embryo culture with fluid flow and embryo movement are now available, and novel culture surfaces are being developed and tested. While several factors remain to be investigated to optimize the efficiency of embryo production, manipulation of the embryo culture microenvironment through novel devices and platforms may offer a pathway toward improving embryo development within the laboratory of the future.     

Anti-hemostatic effects of a serpin from the saliva of the tick Ixodes ricinus

Serpins (serine protease inhibitors) are a large family of structurally related proteins found in a wide variety of organisms, including hematophagous arthropods. Protein analyses revealed that Iris, previously described as an immunomodulator secreted in the tick saliva, is related to the leukocyte elastase inhibitor and possesses serpin motifs, including the reactive center loop (RCL), which is involved in the interaction between serpins and serine proteases. Only serine proteases were inhibited by purified recombinant Iris (rIris), whereas mutants L339A and A332P were found devoid of any protease inhibitory activity. The highest Ka was observed with human leukocyte-elastase, suggesting that elastase-like proteases are the natural targets of Iris. In addition, mutation M340R completely changed both Iris substrate specificity and affinity. This likely identified Met-340 as amino acid P1 in the RCL. The effects of rIris and its mutants were also tested on primary hemostasis, blood clotting, and fibrinolysis. rIris increased platelet adhesion, the contact phase-activated pathway of coagulation, and fibrinolysis times in a dose-dependent manner, whereas rIris mutant L339A affected only platelet adhesion. Taken together, these results indicate that Iris disrupts coagulation and fibrinolysis via the anti-proteolytic RCL domain. One or more other domains could be responsible for primary hemostasis inhibition. To our knowledge, this is the first ectoparasite serpin that interferes with both hemostasis and the immune response.     

种子萌发过程中胚乳的突破性研究

胚乳将许多种子的胚完全包裹,是这些种子萌发的物理屏障,其破裂与否是决定种子萌发与否的最后开关。胚乳破裂是胚生长产生由内向外“顶”的机械力量以及胚乳组织本身机械强度下降（胚乳弱化）的共同结果,而胚乳弱化则包括细胞壁的酶促和非酶促松弛机制。本文综述胚生长产生的机械力量、胚乳破裂的部位和方式、胚乳的组织结构及其细胞壁的化学组成、各种细胞壁降解酶及非酶的扩展蛋白（expansin）和活性氧在胚乳弱化中的作用等方面的研究进展。     

Triiodobenzoic acid, an auxin polar transport inhibitor, suppresses somatic embryo formation and postembryonic shoot/root development in Eleutherococcus senticosus

The effect of auxin polar transport inhibitor on somatic embryo development and postembryonic growth in Siberian ginseng (Eleutherococcus senticosus) was examined. In the presence of 2,3,5-triiodobenzoic acid (TIBA), an auxin polar transport inhibitor, embryo formation from embryogenic cells was suppressed, while cell division was not affected. When globular embryos at different stages were transferred onto medium containing TIBA, development of axial and bilateral polarity was suppressed in a stagespecific manner. In abnormal embryos induced by TIBA, further development of shoot and root apical meristems and vascular differentiation was also suppressed. Thus, abnormal development of embryos induced by inhibition of auxin polar transport resulted in plantlets without shoots and roots.     

Requirement of Ethylene for Growth of Callus and Somatic Embryogenesis in Medicago sativa L.

The role of ethylene in the growth of callus and somatic embryogenesisin Medicago sativa was examined. The application of 2,5-norbornadiene,a competitive inhibitor of ethylene action, during a 10 d inductionperiod to medium containing 2,4-D and kinetin inhibited thegrowth of callus but did not affect somatic embryogenesis, nordid it affect ethylene production during the induction stage.The exposure of tissue, incubated on differentiation medium,without hormones, to an atmosphere of 2,5-norbornadiene, inhibitedboth growth and embryo maturation and stimulated pigmentation.The inhibition of embryo maturation was observed even in thepresence of norbornadiene at a concentration which did not affectgrowth of tissue. It is suggested that the action of endogenous ethylene is necessaryfor the growth of the callus and embryo maturation. Key words: Medicago sativa, ethylene, callus growth, somatic embryogenesis     

Effect of polyamine limitation on DNA synthesis and development of mouse preimplantation embryos in vitro

In-vitro treatment of preimplantation mouse embryos with spermine and spermidine biosynthesis inhibitor, methylglyoxal-bis-(guanylhydrazone) (MGBG), arrested embryo development at the 8-cell or morula stage. In addition, the embryo DNA synthetic rate, as measured by [3H]thymidine incorporation, was strongly inhibited. The inhibition of blastocyst formation and DNA synthesis by MGBG was readily reversible by an exogenous supply of spermine and/or spermidine to the culture medium. DL-alpha-Methylornithine or DL-alpha-difluoromethylornithine (alpha-DFMO), inhibitors of putrescine biosynthesis, had no effect on embryos cultured for 1 or 2 days, but on the 3rd day embryo DNA synthesis was significantly depressed in the presence of alpha-DFMO. These observations suggest that, during early development of the preimplantation mouse embryo, spermine and spermidine are involved in regulation of embryo growth and DNA synthesis. They may also indicate a role of putrescine at a later stage of mouse embryo development.     

Stimulation of root and somatic embryo production in Euonymus europaeus L. by an inhibitor of polyamine biosynthesis

In vitro formation of roots and somatic embryos is obtained from cotyledon explants of a Spindle tree (Euonymus europaeus L.) cultured on two different media: a medium inducing callus formation and the production of roots, and a medium inducing callus formation, root and somatic embryo production. We studied the effects of α-difluoromethylornithine (DFMO), a specific, irreversible inhibitor of ornithine decarboxylase (ODC) on root and somatic embryo production, growth and titers of putrescine in Euonymus explants and explant-derived calli. Early changes in putrescine levels were detected in both cultures before the visible emergence of roots or somatic embryos. DFMO rapidly inhibited putrescine accumulation and growth in non-embryogenic calli and highly stimulated rooting activity. DFMO partially inhibited putrescine accumulation in embryogenic calli. This inhibition had no effects on callus growth but significantly reduced the time of emergence of roots and highly stimulated somatic embryo production. The relationship among putrescine, putrescine metabolism, growth, root and somatic embryo formation is discussed.     

Stimulation of root and somatic embryo production in Euonymus europaeus L. by an inhibitor of polyamine biosynthesis

In vitro formation of roots and somatic embryos is obtained from cotyledon explants of a Spindle tree (Euonymus europaeus L.) cultured on two different media: a medium inducing callus formation and the production of roots, and a medium inducing callus formation, root and somatic embryo production. We studied the effects of -difluoromethylornithine (DFMO), a specific, irreversible inhibitor of ornithine decarboxylase (ODC) on root and somatic embryo production, growth and titers of putrescine in Euonymus explants and explant-derived calli. Early changes in putrescine levels were detected in both cultures before the visible emergence of roots or somatic embryos. DFMO rapidly inhibited putrescine accumulation and growth in non-embryogenic calli and highly stimulated rooting activity. DFMO partially inhibited putrescine accumulation in embryogenic calli. This inhibition had no effects on callus growth but significantly reduced the time of emergence of roots and highly stimulated somatic embryo production. The relationship among putrescine, putrescine metabolism, growth, root and somatic embryo formation is discussed.     

Cytokinin-Inhibitor Antagonism in the Hormonal Control of α-Amylase Synthesis and Growth in Barley Seed

The effect of cytokinins and gibberellic acid on the inhibition of growth and α-amylase synthesis by germination inhibitors was investigated in intact and embryoless seed halves. The cytokinins, kinetin and benzyladenine, effectively reversed the inhibition of coleoptile growth and α-amylase synthesis by abscisic acid and courmarin in barley seed. An antagonism between cytokinins, kinetin and benzyladenine, effectively reversed the inhibition of coleoptile growth and α-amylase synthesis by abscisic acid and coumarins in barley seed. An antagonism between cytokinins and germination inhibitors was also shown in root growth. Abscisic acid inhibited coleoptile growth to a greater extent than the root growth while the opposite held true in the case of coumarin. The apparent increase in coleoptile growth and α-amylase synthesis by gibberellic acid plus abscisic acid (or coumarins) over abscisic acid (or coumarin) appears to be a result of the overall stimulation of growth and metabolism by exogenous gibberellic acid and probably does not involve an interaction of gibberellic acid with the inhibitors. Gibberellic acid reversed root inhibition to some extent. Abscisic acid inhibition of gibberellic acid induced α-amylase synthesis in the embryoless endosperm was not reversed by excess gibberellic acid or kinetin Cytokinin reversal of inhibition of growth and enzyme synthesis probably depends on some factor(s) in the embryo. Cytokinin reversal of inhibitor action leading to enzymen synthesis and growth may be at the level of genome or at the site protein assembly.     

Phosphatidylinositol 3-kinase activity is critical for glucose metabolism and embryo survival in murine blastocysts

The phosphatidylinositol 3-kinase (PI3K) signal transduction pathway is a well known mediator of cell growth, proliferation, and survival signals. Whereas the expression and function of this pathway has been documented during mammalian development, evidence demonstrating the physiologic importance of this pathway in murine preimplantation embryos is beginning to emerge. This study demonstrates that inhibition of the PI3K pathway leads to the induction of apoptosis in both murine blastocysts and trophoblast stem cells. The apoptosis induced in both model systems correlates with a decrease in the expression of the glucose transporter GLUT1 at the plasma membrane. In addition, blastocysts cultured in the presence of the PI3K inhibitor LY-294002 display a decrease in both 2-deoxyglucose uptake and hexokinase activity as compared with control blastocysts. To determine the impact of PI3K inhibition on pregnancy outcome, embryo transfer experiments were performed. Blastocysts cultured in the presence of LY-294002 demonstrate a dramatic increase in fetal resorptions as compared with control embryos. Finally, we demonstrate that impairment of glucose metabolism via iodoacetate, a glyceraldehyde-3-phosphate dehydrogenase inhibitor, is sufficient to induce apoptosis in both blastocysts and trophoblast stem cells. Moreover, blastocysts treated with iodoacetate result in poor pregnancy outcome as determined by embryo transfer experiments. Taken together these data demonstrate the critical importance of the PI3K pathway in preimplantation embryo survival and pregnancy outcome and further emphasize the importance of glucose utilization and metabolism in cell survival pathways.     

Determination of embryonic polarity in a regulative system: evidence for endogenous inhibitors acting sequentially during primitive streak formation in the chick embryo

Avian embryos have a remarkable capacity to regulate: when a pre-primitive streak stage embryo is cut into fragments, each fragment can spontaneously initiate formation of a complete embryonic axis. We investigate the signalling pathways that initiate primitive streak formation and the mechanisms that ensure that only a single axis normally forms. As reported previously, an ectopic primitive streak can be induced by misexpression of Vg1 in the marginal zone. We now show that Vg1 induces an inhibitor that travels across the embryo (3 mm distance) in less than 6 hours. We provide evidence that this inhibitor acts early in the cascade of events downstream of Vg1. We also show that FGF signalling is required for primitive streak formation, in cooperation with Nodal and Chordin. We suggest that three sequential inhibitory steps ensure that a single axis develops in the normal embryo: an early inhibitor that spreads throughout the embryo (which can be induced by Vg1), a second inhibition by Cerberus from the underlying hypoblast, and finally a late inhibition from Lefty emitted by the primitive streak itself.     

Inhibition of cell growth by bafilomycin A1, a selective inhibitor of vacuolar H+-ATPase

Summary Bafilomycin A₁, a potent selective inhibitor of vacuolar H⁺-ATPase, inhibited the growth of a variety of cultured cells dose-dependently, including golden hamster embryo and NIH-3T3 fibroblasts, whether or not they were transformed, and PC12 and HeLa cells. The concentration of bafilomycin A₁ for 50% inhibition of cell growth ranged from 10 to 50 nM. The dose response was nearly parallel with that of the bafilomycin A₁-induced lysosomal pH increase. The degree of pH increase for growth inhibition produced by bafilomycin A₁ was similar to that produced by NH₄Cl in which little difference was recognized in effect among cell types.     

Impaired mitochondrial function in the preimplantation embryo perturbs fetal and placental development in the mouse

The preimplantation embryo is sensitive to its environment and, despite having some plasticity to adapt, environmental perturbations can impair embryo development, metabolic homeostasis, fetal and placental development, and offspring health. This study used an in vitro model of embryo culture with increasing mitochondrial inhibition to directly establish the effect of impaired mitochondrial function on embryonic, fetal, and placental development. Culture in the absence of the carbohydrate pyruvate significantly increased blastocyst glucose oxidation via glycolysis to maintain normal levels of ATP and tricarboxylic acid (TCA) cycle activity. This culture resulted in a significant reduction in blastocyst development, trophectoderm cell number, and respiration rate but, importantly, did not impair implantation rates or fetal and placental development. In contrast, increasing concentrations of the mitochondrial inhibitor amino-oxyacetate (AOA) impaired glycolysis, TCA cycle activity, respiration rate, and ATP production; incrementally reduced blastocyst development; and decreased blastocyst inner cell mass and trophectoderm cell numbers. Importantly, AOA did not affect implantation rates; however, 5 μM AOA significantly reduced placental growth but not fetal growth, increasing the fetal:placental weight ratio. Furthermore, 50 μM AOA significantly reduced both placental and fetal growth but not the fetal:placental weight ratio. Hence, this study demonstrates that a threshold of mitochondrial function is required for normal development, and despite developmental plasticity of the embryo, impaired mitochondrial function in the embryo affects subsequent fetal and placental growth. These results highlight the importance of mitochondrial function in regulating pre- and postimplantation development; however, the effect on offspring health remains unknown.     

CHANGES IN GLUCOSE OXIDATION DURING GROWTH OF EMBRYONIC HEART CELLS IN CULTURE

A rapid and convenient method has been utilized to investigate glucose oxidation during growth of chick embryo heart cells in tissue culture. Primary isolates of chick embryo heart cells showed exponential growth when plated at low densities and exhibited density-inhibited growth as cultures became confluent. The density-dependent growth inhibition of chick embryo heart cells is associated with a marked decrease in the specific activity of glucose oxidation to CO₂. This decrease in glucose oxidation was observed as density increased as either a function of time in culture or as related to initial plating density. The decrease in ¹⁴CO₂ production associated with density-dependent inhibition of growth is due to a marked decrease in activity of the pentose phosphate pathway.     

IGFBP-1, an insulin like growth factor binding protein, is a cell growth inhibitor

A novel cell growth inhibitor, IDF45 (inhibitory diffusible factor), was recently purified to apparent homogeneity. It is a bifunctional molecule: able to bind Insulin like growth factor (IGF) and to 100% inhibit DNA synthesis stimulated by serum in fibroblasts. It was of interest to verify whether other members of the IGF-binding protein (IGFBP) family show the same bifunctional growth inhibitory properties. In this paper we show that purified IGFBP-1 derived from amniotic fluid is a cell growth inhibitor. In chick embryo fibroblasts, it inhibited DNA synthesis stimulated by serum. However the stimulation was maximally 60% inhibited and half of the inhibition was observed with 100ng/ml IGFBP-1. So the specific activity of IGFBP-1 is lower than that of IDF45. IGFBP-1 also reversibly prevented the CEF growth. In the same cells IGFBP-1 inhibited DNA synthesis stimulated by IGF-I. We demonstrated that the same protein IGFBP-1 is able to inhibit DNA synthesis stimulated by serum and by IGF-I. The possibility that IGFBP-1 is a bifunctional molecule is discussed.