Immunofluorescent Visualization of 100 Å Filaments in Different Cultured Chick Embryo Cell Types

Antibody prepared against the 55,000 dalton subunit of reconstituted chick gizzard 100 A filaments (anti-G55K) bound to the 100 Å filaments of chick smooth muscle, cardiac muscle, and skeletal muscle cells, and to the 100 Å filaments of Schwann cells and satellite glial cells of the peripheral nervous system. Anti-G55K did not bind to replicating presumptive myoblasts, fibroblasts, chondroblasts, pigment cells, neurons, or to central nervous system glial cells. This contrasted with the wider range of binding of antibody to the 58,000 dalton subunit of chick fibroblast 100 A filaments (anti-F58K) which bound to the 100 Å filaments of all cell types examined except hepatocytes and skin epithelial cells. Anti-G55K staining revealed a morphologically distinct distribution of 100 A filaments in the three types of muscle cells. Spindle shaped smooth muscle cells exhibited dense fluorescent staining near the poles of the cells, and also exhibited unique patches of fluorescent material after cytochalasin B and Colcemid treatment. In myotubes, the fluorescence was limited to longitudinal bundles of filaments between the striated myofibrils. Cardiac cells contained uniformly distributed fine filaments. Lastly, smooth muscle cells in various phases of mitosis bound the anti-G55K, whereas replicating presumptive skeletal myoblasts failed to bind the anti-G55K.     

Types of Neural Tissues Induced through the Presumptive Notochord of Newt Embryo

Neural induction through the presumptive notochord was tested by means of the sandwich method. The result disclosed that the notochord was a potent inducer of neural tissues not only in the ectoderm of gastrula but also in the ventral ectoderm of neurula and early tail-bud embryos. Structures formed by the induced neural tissue varied greatly. They can be classified into three types. (1) Tubular: the neural tissues induced in explants containing abundant mesenchymes always formed long tubular structures. The shapes of these neural tubes showed considerable variation; moreover, they were atypical and none formed the regular structure of the spinal cord. This type was most frequent, being found in about 50% of the explants. (2) Inverted: this type was produced when the explant contained mesenchymal component. Consequently, the epithelium of explants was missing. Nevertheless, a considerable mass of neural tissue was always induced. It was noticed that the induced neural tissues were invariably inside out; this type was found in about 30% of the explants. (3) Archencephalic: this was the only type to form the regular structure, i.e., the archencephalon. Formation of the archencephalon was limited solely to those explants containing only a few mesenchymes; this type was found in about 20% of the cases. As described above, it was found that the neural tissues induced by the same inducer of the notochord were not uniform but varied in type. Further, it was shown that the types of neural tissue differed according to different quantities of the surrounding mesenchyme. Based on these facts, it is to be concluded that it is not the inducer of notochord, but the surrounding mesenchyme that is of primary importance for the determination of the types of neural tissue.     

鸡miR-9不同组织表达差异及其功能预测分析

miRNA在动物生长发育过程中有重要作用. 本文采用定制茎环反转录引物,利用实时荧光定量PCR技术构建miR-9在鸡2个阶段11个组织中的表达谱,同时用TargetScan5.1与PicTar两种计算方法对其进行靶基因预测,交集的基因集合分别进行GO（gene ontology）富集分析和生物通路富集分析. 结果表明,采用实时定量PCR检测的miR-9在鸡下丘脑中的表达量和高通量测序结果一致;采用实时定量PCR在对不同组织定量结果表明,miR-9在0日龄鸡的肾脏、下丘脑、腿肌和大脑中高丰度表达,在成年鸡表达量较高为大脑、腿肌、心脏、小脑和下丘脑.在同一组织的0日龄和成年鸡中表达呈现时序性,除肝脏的表达量差异不显著,其他10个组织miR-9表达量差异显著（P<0.05）.预测到交集靶基因有160个.涉及到多个KEGG通路和GO富集中.GO分类结果显示,这些基因分布于63个群中,其中基因超过45个基因群集有26个群,与代谢有关的群有11个. 其它与发育、调控等过程有关. 在KEGG通路分析中,显著的通路有细胞骨架调控、细胞增殖和分化有关的通路（P<0.01）等5个通路. 表明miR 9基因的表达有组织和时序特异性,靶基因参与细胞代谢、生长发育和调控.这些结果为进一步验证miR 9基因在在脑中调控生长发育过程中的作用奠定了基础.     

Differential Response of Various Tissues4Asparagus officinalis to Sodium Chloride

Tolerance of Asparagus officinalis tissues of different levelsof organization to 0–2% NaCl was studied in a tissue culturesystem. The following tissues (organs) were examined: friablecallus with no organogenesis, compact callus exhibiting organogenesis,one-bud shoot segments, and plantlets. Growth of friable callus,the less organized tissue, was progressively inhibited by risingconcentrations of NaCl, whereas growth of compact callus wassomewhat less sensitive. NaCl at concentrations of 1% or highercaused an increase in the mortality of one-bud shoot segmentsand inhibited growth. Concentrations up to 2% NaCl did not causeany mortality of plantlets, the most organized tissue tested;root production declined progressively in response to salinitywhile shoot growth was inhibited only at 2% NaCl. Moderate concentrationsof NaCl (0.5–1.0%) stimulated growth and induced phyllocoidproduction in both shoot segments and plantlets. With increasedNaCl there was a massive uptake of sodium and chloride, principallyinto the shoots, while uptake and accumulation of potassiumdecreased somewhat in shoots, but not in roots and rhizomes.Rooted and unrooted plantlets exhibited similar levels of tolerance.It is, therefore, inferred that salt tolerance of asparagusin culture is dependent on tissue organization. Key words: Asparagus officinalis, salt tolerance, callus, shoot segment, plantlet     

Simultaneous Expression of Two Different Types of Intermediate Sized Filaments in Mouse Keratinocytes Proliferating in vitro

The intermediate-sized filaments present in epidermal keratinocytes derived from mouse skin and in an established cell line (HEL) derived from spontaneous transformation of murine keratinocytes grown in vitro, have been examined by immunofluorescence microscopy, using antibodies directed against subunit proteins of different classes of intermediate-sized filaments, as well as by electron microscopy and gel electrophoresis of cytoskeletal preparations highly enriched in intermediate-sized filaments. The keratinocytes derived from neonatal skin, which are capable of only limited replication in vitro, show only a single type of intermediate-sized filaments, i.e., the tonofibril-like arrays of filaments containing prekeratin. HEL cells, which proliferate indefinitely in vitro, retain the tonofilament-like structures typical of differentiated epidermal cells but in addition display intermediate-sized filaments of the vimentin type, i.e., the filament system typically found in mesenchymal and mesenchyme-derived cells. We discuss the possibility that (i) the advent of vimentin-type filaments in epidermal cells in culture is related either to the transformed state or the in vitro growth conditions as such and (ii) other differentiated epithelial cells proliferating in vitro may have more than one system of intermediate-sized filaments.     

鉴别超氧化物歧化酶类型的定位染色法

介绍一种鉴别SOD类型的方法─—聚丙烯酰胺凝胶电泳的定位染色法. 由于不同类型的SOD对抑制剂的表现各异, 电泳后的凝胶经不同的抑制剂处理, 染色, 结果展示在凝胶上, CuZn-SOD酶带在H₂O₂或CN^-的作用下消失, Mn-SOD在CHCl₃-CH₃OH作用下消失, Fe-SOD在H₂O₂或. CHCl₃-CH₃CH₂OH作用下失活, 从酶带消失或存活的情况, 可以判断SOD的类型.     

Organization of Actin Filaments and Zonula Adherens during Somitogenesis in the Chick Embryo

The organization of F-actin during somitogenesis in the chick embryo was studied by use of rhodamine-conjugated phalloidin and transmission electron microscopy (TEM). Separation of a somite from the segmental plate proceeded simultaneously with the organization of segmental plate cells into a hemispherical epithelial sheet whose open side was directed antero-laterally. At the same time, intense staining of F-actin appeared in the apical surface of the epithelial sheet. Observations by TEM showed that zonulae adherentes associated with many actin filaments increased in the apical region of cells being organized into an epithelial sheet while this junctional apparatus was only sparsely distributed in the segmental plate cells. The hemispherical sheet subsequently closed to form an epithelial vesicle, with increase in curvature of its apical surface, and narrowing of cellular apices. At the same time, the zonulae adherentes and actin filaments in the cellular apices further increased, and many cellular processes formed on the apical surface of the epithelial somites. These findings suggest that segmentation involves organization of zonulae adherentes and a contractile process caused by acin filaments anchored to the zonulae adherentes.     

鸡胚三类组织体外培养细胞的生物学特性分析

采用组织培养的方法获取鸡胚不同组织细胞,利用M199培养基进行原代、传代培养,经形态学观察、生长曲线绘制、分裂指数测定等进行生物学特性分析。实验表明,鸡胚不同组织细胞具有不同的生物学特性,从形态结构到生长周期都有明显差异。获得的躯体来源细胞、心来源细胞为成纤维型,肺来源细胞为上皮型;其中,躯体来源细胞生长能力最强,心来源细胞次之,肺来源细胞最慢,躯体来源细胞倍增时间最短;核型分析表明,该实验体外培养的鸡胚细胞染色体数目为78条。上述结果可为今后鸡胚不同组织细胞的深入研究提供实验材料和依据。     

Specific Levels of DNA Methylation in Various Tissues, Cell Lines, and Cell Types of Daucus carota

The level of DNA methylation in Daucus carota was found to be tissue specific, but no simple correlation between developmental stage or age of tissue and the level of DNA methylation was found. Among three different suspension culture lines from the same variety grown under identical conditions, large differences in the level of DNA methylation were observed. The highest and lowest levels were found in two embryogenic cell lines originating from the same clone. Suspension cells from one of the embryogenic cell lines were fractionated into three morphologically defined cell types using Percoll gradient density centrifugation, and the uniformity of these fractions was evaluated by image analysis. The three cell types showed different levels of DNA methylation. The lowest level was found in the fraction containing the precursor cells of somatic embryos.     

Bacterial Responses to Different Dietary Cereal Types and Xylanase Supplementation in the Intestine of Broiler Chicken

Several studies were carried out to investigate the influence of dietary cereals differing in soluble non starch polysaccharides (NSP) content and a xylanase preparation on selected bacterial parameters in the small intestine of broiler chicken. Compared to a maize diet colony forming units (CFU) of mucosa associated bacteria were higher in a wheat/rye diet, most notably for enterobacteria and enterococci. Xylanase supplementation to the wheat/rye diet generally led to lower CFU, especially in the first week of life. However, xylanase supplementation also displayed higher in vitro growth potentials for enterobacteria and enterococci. Bacterial growth of luminal samples in minimal media supplemented with selected NSP showed that the wheat/rye diet enhanced bacterial capacities to utilize NSP only in ileal samples. The xylanase application generally shifted respective maximum growth to the proximal part of the small intestine. The presence of soluble NSP from wheat or rye in the diet per se did not enhance bacterial NSP hydrolyzing enzyme activities in the small intestine, but xylanase supplementation resulted in higher 1,3-1,4- g - glucanase activity. Compared to a maize diet the activity of bacterial bile salt hydrolases in samples of the small intestine was not increased due to inclusion of wheat/rye or triticale to the diet. However, xylanase supplementation led to a reduction with a corresponding increase of lipase activity. It was concluded that dietary cereals producing high intestinal viscosities lead to increased overall bacterial activity in the small intestine. The supplementation of a xylanase to cereal based diets producing high intestinal viscosity, changes composition and metabolic potential of bacterial populations and may specifically influence fat absorption in young animals.     

Differential Accumulation of Salicylic Acid and Salicylic Acid-Sensitive Catalase in Different Rice Tissues

We previously proposed that salicylic acid (SA)-sensitive catalases serve as biological targets of SA in plant defense responses. To further examine the role of SA-sensitive catalases, we have analyzed the relationship between SA levels and SA sensitivity of catalases in different rice (Oryza sativa) tissues. We show here that, whereas rice shoots contain extremely high levels of free SA, as previously reported (I. Raskin, H. Skubatz, W. Tang, B.J.D. Meeuse [1990] Ann Bot 66: 369-373; P. Silverman, M. Seskar, D. Kanter, P. Schweizer, J.-P. Metraux, I. Raskin [1995] Plant Physiol 108: 633-639), rice roots and cell-suspension cultures have very low SA levels. Catalases from different rice tissues also exhibit differences in sensitivity to SA. Catalase from rice shoots is insensitive to SA, but roots and cell-suspension cultures contain SA-sensitive catalase. The difference in SA sensitivity of catalases from these different tissues correlates with the tissue-specific expression of two catalase genes, CatA and CatB, which encode highly distinctive catalase proteins. CatA, which encodes a catalase with relatively low sequence homology to the tobacco SA-sensitive catalases, is expressed at high levels exclusively in the shoots. On the other hand, in roots and cell-suspension cultures, with northern analysis we detected expression of only the CatB gene, which encodes a catalase with higher sequence homology to tobacco catalases. The role of catalases in mediating some of the SA-induced responses is discussed in light of these results and the recently defined mechanisms of catalase inhibition by SA.     

Vimentin Expression Pattern is Different between the Flank Region and Limb Regions of Somatopleural Mesoderm in the Chicken Embryo

It is known that the chicken flank somatopleure also has a limb-forming potential at early stages of development, but loses this potential later. Molecular changes during this process is, however, not well known. We obtained a monoclonal antibody which reacts to the flank somatopleure, but not to the wing bud, the leg bud and the neck somatopleure in the stage 22 chicken embryo. Further study revealed that this antibody is specific to vimentin. Time course of vimentin expression in the somatopleural mesoderm during the development was studied. It was revealed to be biphasic. Somatopleural mesoderm expressed vimentin at stage 10, but not at stage 16. Flank somatopleural mesoderm began to express vimentin again at stage 18, whereas limb bud mesenchymal cells did not until stage 27. The earlier re-expression of vimentin at the flank somatopleure suggests that certain physiological changes take place in cells at this region.     

Xenotransplantation of Human Stem Cells into the Chicken Embryo

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the behavior of cells in a standardized in vivo environment. The main advantages of the chicken embryo include low cost, high accessibility, ease of surgical manipulation and lack of a fully developed immune system. Xenotransplantation into chicken embryos can provide valuable information about cell proliferation, differentiation and behavior, the responses of cells to signals in defined embryonic tissue niches, and tumorigenic potential. Transplanting cells into chicken embryos can also be a step towards transplantation experiments in other animal models. Recently the chicken embryo has been used to evaluate the neurogenic potential of human stem and progenitor cells following implantation into neural anlage^1-6. In this video we document the entire procedure for transplanting human stem cells into the developing central nervous system of the chicken embryo. The procedure starts with incubation of fertilized eggs until embryos of the desired age have developed. The eggshell is then opened, and the embryo contrasted by injecting dye between the embryo and the yolk. Small lesions are made in the neural tube using microsurgery, creating a regenerative site for cell deposition that promotes subsequent integration into the host tissue. We demonstrate injections of human stem cells into such lesions made in the part of the neural tube that forms the hindbrain and the spinal cord, and into the lumen of the part of the neural tube that forms the brain. Systemic injections into extraembryonic veins and arteries are also demonstrated as an alternative way to deliver cells to vascularized tissues including the central nervous system. Finally we show how to remove the embryo from the egg after several days of further development and how to dissect the spinal cord free for subsequent physiological, histological or biochemical analyses.     

Differential Glycosylation of the 5A11/HT7 Antigen by Neural Retina and Epithelial Tissues in the Chicken

Abstract: The 5A11/HT7 antigen, a member of the immunoglobulin supergene family, has been implicated in heterotypic cell-cell interactions during retina development. Immunopurified 5A11 antigen isolated from Nonidet P-40-solubilized retina membranes had two components as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), a 45.5-kDa doublet and a 69-kDa polypeptide. Immunoreactive bands of 46-50 kDa were recognized following SDS-PAGE of detergent-solubilized membrane proteins from liver, kidney, and erythrocytes. Treatment with N-glycosidase F (EC 3.2.2.18) converted the 45.5–50-kDa immunoreactive polypeptides from all tissues to 32 kDa, indicating that the observed differences in molecular mass were due to differences in glycosylation. N-Glycosidase Ftreatment also converted the 69-kDa form from retina to 46 kDa, indicating a different polypeptide core than the 32-kDa species. Treatment with endo-β-N-acetylglucosaminidase H (EC 3.2.1.96) resulted in modest increases in electrophoretic mobility due to hydrolysis of high mannose or hybrid oligosaccharides and lack of hydrolysis of complex oligosaccharides resistant to endo-β-N-acetylglucosaminidase H digestion. Immunoreactivity was retained after deglycosylation. Much of the difference in molecular weight could be attributed to variations in sialylation. The higher molecular mass species of the 45.5-kDa doublet from retina and the polypeptides from other tissues were susceptible to neuraminidase (EC 3.2.1.18) and O-glycosidase (endo-α-N-acetylgalactosaminidase; EC 3.2.1.97) digestion. Labeling with elderberry bark lectin (specific for α2, 6-linked sialic acid) was confined to the higher molecular mass species of the 45.5-kDa doublet and was considerably greater in antigen derived from epithelia rather than neural retina. In paraffin sections of chick retina, elderberry bark lectin staining was confined to the retinal pigmented epithelium, photoreceptor cells, and bipolar cells with no staining of the Müller cells, which bear the bulk of the 5A11 antigen. These results indicate tissue-specific posttranslational modifications, particularly differences in sialylation of antigen-bearing polypeptides.     

Appearence of LH-Immunoreactive Cells in the Rathke's Pouch of the Chicken Embryo

The differentiation of the pituitary of the chicken embryo was studied by means of an immunohistochemical technique using antisera to turkey and chicken pituitary hormones. Immunoreactive LH-cells are detected in 4-day embryos (stage 23 of Hamburger and Hamilton) when the primordium of the anterior pituitary, the Rathke's pouch is only composed of a single-layer epithelium lined with an undifferentiated mesenchyme. A few immunoreactive cells are observed grouped on the posterior aspect of the pouch. As development proceeds, a strip of positive cells is detected encircling the Rathke's pouch. Prolactin-, growth hormone-, and ACTH-immunoreactive cells are detected in 6- and 7-day embryos, only after the pituitary has acquired its characteristic structure with cords in which different cell types become progressively recognizable. The early appearance of immunoreactive LH-cells following a precise distribution shows that secretory properties and differentiation capacities are acquired simultaneously in the epithelium of the Rathke's pouch and may be induced by the same stimulus.     

不同品系小鼠的体外受精、胚胎冷冻及移植的比较研究

目的　探讨不同品系小鼠的体外受精、胚胎和精子的低温保存效果。方法　本实验分别在中国科学院上海实验动物中心 (SLAC)和日本熊本大学动物资源开发中心 (CARD)对 13个品系小鼠 (C57BL 6J、BALB c、C3H HeJ、ICR、KM、FVB、MRL、NOD、CBA、DBA 2、CD 1、BDF1、B6C3F1)的体外受精 (IVF)率、胚胎培养及移植成绩进行了比较研究。结果　各品系小鼠新鲜精子的IVF率 15 1%～ 87 9% ,冻融精子的IVF率 8%～ 80 % ;冷冻胚胎的复苏率4 2 6 %～ 83 9% ;冻融胚胎移植后的产仔率在 17 8%～ 5 1 8%。结论　遗传背景不同的小鼠体外受精率、冷冻胚胎复苏率和胚胎移植的产仔率差异有显著性。但同一品系两个实验室间的新鲜精子的IVF率、冷冻胚胎的复苏率及移植产仔率差异无显著性 (P >0 0 5 ) ;冻融精子的体外受精率CARD明显高于SLAC(P <0 0 1)。     

一种新的与细胞分化相关的基因在不同组织及细胞系中表达与定位

目的　研究一种新的可能与细胞分化相关的基因在正常组织、肿瘤组织及细胞系中的表达与定位 ,初步探讨该基因的作用机制。方法　利用Northern杂交方法检测 10种人胎儿组织、6种人肿瘤细胞系、4种人的肿瘤组织及癌旁组织中该基因的表达。利用免疫荧光实验检测该基因在细胞中定位。结果　该基因在人的胎儿组织及肿瘤组织和肿瘤细胞中均有高表达 ,在正常组织及癌旁组织中表达明显减弱。癌旁组织和癌组织中的表达差异有显著性 (P <0 0 5 )。该基因在K56 2 细胞中主要定位在膜上。结论　该基因可能在细胞分化及肿瘤发生中起着重要作用