CD100在B淋巴细胞发育中的作用

CD100是第一个被确认的在免疫系统起重要作用的脑信号蛋白(semaphorin)家族成员。CD100在B淋巴细胞发育中起着重要作用,它通过封闭受体CD72的负调控信号而促进B细胞的活化,从而增强体液免疫应答。     

Development of Streptococcal L-Form Colonies

The development and architecture of L-form agar colonies produced from protoplasts and L-phase bodies were studied by both light and scanning electron microscopy. Agar blocks containing L-phase microcolonies of group A Streptococcus strains ADA and GL8 and group D Streptococcus strain F24 as well as longitudinal sections of mature colonies were used as samples. Initially, granules of about 0.5 mum in diameter were produced by multiple condensation and fragmentation of protoplasts and large bodies. Surface growth by granules ensued and infiltration into agar occurred only after 10 to 11 hr of incubation at 37 C. Club-shaped granules were noted and division seemed to take place by simple fission. The configuration of large bodies and granules in mature colonies suggested budding as another means of replication. Acellular spaces inside the colonies appeared to have been formed by lysis of large bodies or by the envelopment of space by the extending growth of minute granules. Whereas no significant strain variation was noted in colonies of less than 24 hr of incubation, fully mature colonies were differentiated on uniform media.     

Streptomyces Development in Colonies and Soils

Streptomyces development was analyzed under conditions resembling those in soil. The mycelial growth rate was much lower than that in standard laboratory cultures, and the life span of the previously named first compartmentalized mycelium was remarkably increased.Streptomycetes are gram-positive, mycelium-forming, soil bacteria that play an important role in mineralization processes in nature and are abundant producers of secondary metabolites. Since the discovery of the ability of these microorganisms to produce clinically useful antibiotics (2, 15), they have received tremendous scientific attention (12). Furthermore, its remarkably complex developmental features make Streptomyces an interesting subject to study. Our research group has extended our knowledge about the developmental cycle of streptomycetes, describing new aspects, such as the existence of young, fully compartmentalized mycelia (5-7). Laboratory culture conditions (dense inocula, rich culture media, and relatively elevated temperatures [28 to 30°C]) result in high growth rates and an orderly-death process affecting these mycelia (first death round), which is observed at early time points (5, 7).In this work, we analyzed Streptomyces development under conditions resembling those found in nature. Single colonies and soil cultures of Streptomyces antibioticus ATCC 11891 and Streptomyces coelicolor M145 were used for this analysis. For single-colony studies, suitable dilutions of spores of these species were prepared before inoculation of plates containing GYM medium (glucose, yeast extract, malt extract) (11) or GAE medium (glucose, asparagine, yeast extract) (10). Approximately 20 colonies per plate were obtained. Soil cultures were grown in petri dishes with autoclaved oak forest soil (11.5 g per plate). Plates were inoculated directly with 5 ml of a spore suspension (1.5 × 10⁷ viable spores ml⁻¹; two independent cultures for each species). Coverslips were inserted into the soil at an angle, and the plates were incubated at 30°C. To maintain a humid environment and facilitate spore germination, the cultures were irrigated with 3 ml of sterile liquid GAE medium each week.The development of S. coelicolor M145 single colonies growing on GYM medium is shown in Fig. ​Fig.1.1. Samples were collected and examined by confocal microscopy after different incubation times, as previously described (5, 6). After spore germination, a viable mycelium develops, forming clumps which progressively extend along the horizontal (Fig. 1a and b) and vertical (Fig. 1c and d) axes of a plate. This mycelium is fully compartmentalized and corresponds to the first compartmentalized hyphae previously described for confluent surface cultures (Fig. 1e, f, and j) (see below) (5); 36 h later, death occurs, affecting the compartmentalized hyphae (Fig. 1e and f) in the center of the colony (Fig. ​(Fig.1g)1g) and in the mycelial layers below the mycelial surface (Fig. 1d and k). This death causes the characteristic appearance of the variegated first mycelium, in which alternating live and dead segments are observed (Fig. 1f and j) (5). The live segments show a decrease in fluorescence, like the decrease in fluorescence that occurs in solid confluent cultures (Fig. ​(Fig.11 h and i) (5, 9). As the cycle proceeds, the intensity of the fluorescence in these segments returns, and the segments begin to enlarge asynchronously to form a new, multinucleated mycelium, consisting of islands or sectors on the colony surfaces (Fig. 1m to o). Finally, death of the deeper layers of the colony (Fig. ​(Fig.1q)1q) and sporulation (Fig. ​(Fig.1r)1r) take place. Interestingly, some of the spores formed germinate (Fig. ​(Fig.1s),1s), giving rise to a new round of mycelial growth, cell death, and sporulation. This process is repeated several times, and typical, morphologically heterogeneous Streptomyces colonies grow (not shown). The same process was observed for S. antibioticus ATCC 11891, with minor differences mainly in the developmental time (not shown).Open in a separate windowFIG. 1.Confocal laser scanning fluorescence microscopy analysis of the development-related cell death of S. coelicolor M145 in surface cultures containing single colonies. Developmental culture times (in hours) are indicated. The images in panels l and n were obtained in differential interference contrast mode and correspond to the same fields as in panels k and m, respectively. The others are culture sections stained with SYTO 9 and propidium iodide. Panels c, d, k, l, p, and q are cross sections; the other images are longitudinal sections (see the methods). Panels h and i are images of the same field taken with different laser intensities, showing low-fluorescence viable hyphae in the center of the colonies that develop into a multinucleated mycelium. The arrows in panels e and s indicate septa (e) and germinated spores (s). See the text for details.Figure ​Figure22 shows the different types of mycelia present in S. coelicolor cultures under the conditions described above, depending on the compartmentalization status. Hyphae were treated with different fluorescent stains (SYTO 9 plus propidium iodide for nucleic acids, CellMask plus FM4-64 for cell membranes, and wheat germ agglutinin [WGA] for cell walls). Samples were processed as previously described (5). The young initial mycelia are fully compartmentalized and have membranous septa (Fig. 2b to c) with little associated cell wall material that is barely visible with WGA (Fig. ​(Fig.2d).2d). In contrast, the second mycelium is a multinucleated structure with fewer membrane-cell wall septa (Fig. 2e to h). At the end of the developmental cycle, multinucleated hyphae begin to undergo the segmentation which precedes the formation of spore chains (Fig. 2i to m). Similar results were obtained for S. antibioticus (not shown), but there were some differences in the numbers of spores formed. Samples of young and late mycelia were freeze-substituted using the methodology described by Porta and Lopez-Iglesias (13) and were examined with a transmission electron microscope (Fig. 2n and o). The septal structure of the first mycelium (Fig. ​(Fig.2n)2n) lacks the complexity of the septal structure in the second mycelium, in which a membrane with a thick cell wall is clearly visible (Fig. ​(Fig.2o).2o). These data coincide with those previously described for solid confluent cultures (4).Open in a separate windowFIG. 2.Analysis of S. coelicolor hyphal compartmentalization with several fluorescent indicators (single colonies). Developmental culture times (in hours) are indicated. (a, e, and i) Mycelium stained with SYTO 9 and propidium iodide (viability). (b, f, and j) Hyphae stained with Cell Mask (a membrane stain). (c, g, and l) Hyphae stained with FM 4-64 (a membrane stain). (d, h, and m) Hyphae stained with WGA (cell wall stain). Septa in all the images in panels a to j, l, and m are indicated by arrows. (k) Image of the same field as panel j obtained in differential interference contrast mode. (n and o) Transmission electron micrographs of S. coelicolor hyphae at different developmental phases. The first-mycelium septa (n) are comprised of two membranes separated by a thin cell wall; in contrast, second-mycelium septa have thick cell walls (o). See the text for details. IP, propidium iodide.The main features of S. coelicolor growing in soils are shown in Fig. ​Fig.3.3. Under these conditions, spore germination is a very slow, nonsynchronous process that commences at about 7 days (Fig. 3c and d) and lasts for at least 21 days (Fig. 3i to l), peaking at around 14 days (Fig. 3e to h). Mycelium does not clump to form dense pellets, as it does in colonies; instead, it remains in the first-compartmentalized-mycelium phase during the time analyzed. Like the membrane septa in single colonies, the membrane septa of the hyphae are stained with FM4-64 (Fig. 3j and k), although only some of them are associated with thick cell walls (WGA staining) (Fig. ​(Fig.3l).3l). Similar results were obtained for S. antibioticus cultures (not shown).Open in a separate windowFIG. 3.Confocal laser scanning fluorescence microscopy analysis of the development-related cell death and hyphal compartmentalization of S. coelicolor M145 growing in soil. Developmental culture times (in days) are indicated. The images in panels b, f, and h were obtained in differential interference contrast mode and correspond to the same fields as the images in panels a, e, and g, respectively. The dark zone in panel h corresponds to a particle of soil containing hyphae. (a, c, d, e, g, i, j, and k) Hyphae stained with SYTO 9, propidium iodide (viability stain), and FM4-64 (membrane stain) simultaneously. (i) SYTO 9 and propidium iodide staining. (j) FM4-64 staining. The image in panel k is an overlay of the images in panels i and j and illustrates that first-mycelium membranous septa are not always apparent when they are stained with nucleic acid stains (SYTO 9 and propidium iodide). (l) Hyphae stained with WGA (cell wall stain), showing the few septa with thick cell walls present in the cells. Septa are indicated by arrows. IP, propidium iodide.In previous work (8), we have shown that the mycelium currently called the substrate mycelium corresponds to the early second multinucleated mycelium, according to our nomenclature, which still lacks the hydrophobic layers characteristic of the aerial mycelium. The aerial mycelium therefore corresponds to the late second mycelium which has acquired hydrophobic covers. This multinucleated mycelium as a whole should be considered the reproductive structure, since it is destined to sporulate (Fig. ​(Fig.4)4) (8). The time course of lysine 6-aminotransferase activity during cephamycin C biosynthesis has been analyzed by other workers using isolated colonies of Streptomyces clavuligerus and confocal microscopy with green fluorescent protein as a reporter (4). A complex medium and a temperature of 29°C were used, conditions which can be considered similar to the conditions used in our work. Interestingly, expression did not occur during the development of the early mycelium and was observed in the mycelium only after 80 h of growth. This suggests that the second mycelium is the antibiotic-producing mycelium, a hypothesis previously confirmed using submerged-growth cultures of S. coelicolor (9).Open in a separate windowFIG. 4.Cell cycle features of Streptomyces growing under natural conditions. Mycelial structures (MI, first mycelium; MII, second mycelium) and cell death are indicated. The postulated vegetative and reproductive phases are also indicated (see text).The significance of the first compartmentalized mycelium has been obscured by its short life span under typical laboratory culture conditions (5, 6, 8). In previous work (3, 7), we postulated that this structure is the vegetative phase of the bacterium, an hypothesis that has been recently corroborated by proteomic analysis (data not shown). Death in confluent cultures begins shortly after germination (4 h) and continues asynchronously for 15 h. The second multinucleated mycelium emerges after this early programmed cell death and is the predominant structure under these conditions. In contrast, as our results here show, the first mycelium lives for a long time in isolated colonies and soil cultures. As suggested in our previous work (5, 6, 8), if we assume that the compartmentalized mycelium is the Streptomyces vegetative growth phase, then this phase is the predominant phase in individual colonies (where it remains for at least 36 h), soils (21 days), and submerged cultures (around 20 h) (9). The differences in the life span of the vegetative phase could be attributable to the extremely high cell densities attained under ordinary laboratory culture conditions, which provoke massive differentiation and sporulation (5-7, 8).But just exactly what are “natural conditions”? Some authors have developed soil cultures of Streptomyces to study survival (16, 17), genetic transfer (14, 17-19), phage-bacterium interactions (3), and antibiotic production (1). Most of these studies were carried out using amended soils (supplemented with chitin and starch), conditions under which growth and sporulation were observed during the first few days (1, 17). These conditions, in fact, might resemble environments that are particularly rich in organic matter where Streptomyces could conceivably develop. However, natural growth conditions imply discontinuous growth and limited colony development (20, 21). To mimic such conditions, we chose relatively poor but more balanced carbon-nitrogen soil cultures (GAE medium-amended soil) and less dense spore inocula, conditions that allow longer mycelium growth times. Other conditions assayed, such as those obtained by irrigating the soil with water alone, did not result in spore germination and mycelial growth (not shown). We were unable to detect death, the second multinucleated mycelium described above, or sporulation, even after 1 month of incubation at 30°C. It is clear that in nature, cell death and sporulation must take place at the end of the long vegetative phase (1, 17) when the imbalance of nutrients results in bacterial differentiation.In summary, the developmental kinetics of Streptomyces under conditions resembling conditions in nature differs substantially from the developmental kinetics observed in ordinary laboratory cultures, a fact that should be born in mind when the significance of development-associated phenomena is analyzed.     

Enumeration by DNA Colony Hybridization of Virulent Yersinia enterocolitica Colonies in Artificially Contaminated Food

[This corrects the article on p. 441 in vol. 51.].     

单菌落PCR法直接快速鉴定重组克隆

利用单菌落PCR法直接筛选含有GFP、LTB-ST外源基因的重组克隆,阳性克隆可以扩增出目的条带,和质粒PCR扩增的结果一致。同时,单菌落PCR法也可应用于重组质粒转化后的农杆菌的筛选,单菌落PCR法的扩增结果和农杆菌液扩增的结果一致。结果表明,单菌落PCR法是一个有效简便的鉴定重组阳性克隆的方法。     

Lymphocyte electrotaxis in vitro and in vivo

Electric fields are generated in vivo in a variety of physiologic and pathologic settings, including penetrating injury to epithelial barriers. An applied electric field with strength within the physiologic range can induce directional cell migration (i.e., electrotaxis) of epithelial cells, endothelial cells, fibroblasts, and neutrophils suggesting a potential role in cell positioning during wound healing. In the present study, we investigated the ability of lymphocytes to respond to applied direct current (DC) electric fields. Using a modified Transwell assay and a simple microfluidic device, we show that human PBLs migrate toward the cathode in physiologically relevant DC electric fields. Additionally, electrical stimulation activates intracellular kinase signaling pathways shared with chemotactic stimuli. Finally, video microscopic tracing of GFP-tagged immunocytes in the skin of mouse ears reveals that motile cutaneous T cells actively migrate toward the cathode of an applied DC electric field. Lymphocyte positioning within tissues can thus be manipulated by externally applied electric fields, and may be influenced by endogenous electrical potential gradients as well.     

Studies of Chloroplast Development in Euglena: XV. Factors Influencing the Decay of Photoreactivability of Green Colony Formation

When UV-treated cells of Euglena gracilis var. bacillaris are incubated in the dark in a nutrient medium which permits cell division, they lose the ability to be photoreactivated. The rate of this loss increases with the UV dose. For any given UV dose, the rate of decay increases with increasing growth rate. The same phenomena are observed in light-grown and in dark-grown cells, although the sensitivity to UV of the light-grown cells is smaller by a factor of 1.7. The kinetics of photoreactivation (PR) change during the decay of photoreactivability only if the cells are incubated in growth medium. A UV-inactivation curve for cells photoreactivated only after appreciable PR shows the same slope as that for untreated cells (number of UV-sensitive targets). These results are discussed from the point of view of possible models.     

Human tumor — Lymphocyte interaction in vitro

Summary Biopsy derived tumor cells have been tested for capacity to induce DNA synthesis in autologous blood lymphocytes in vitro (ATS test). In the present series 5/20 biopsies were ATS positive when mechanically separated cells were used. Preincubation of the tumor cells at 37° C, but not at 0° C, markedly increased the stimulating capacity. Parallel experiments gave 7/9 positive tests with preincubated and 3/9 positive tests with cells used directly. When the biopsy cells were brought into suspension by enzyme treatment they did not stimulate unless incubated at 37° C before confronting the lymphocytes. In accordance with previous results, the presence of autologous serum during ATS inhibited the reaction.     

Ultrastructural Studies of Microconidium Formation in Neurospora crassa

Microconidiating cultures of “peach-fluffy” (pe, fl; Y8743m, L; FGSC #569) were fixed at various times after the initiation of growth and examined with an electron microscope. Hyphae from which microconidia form are markedly vacuolated and show a much more extensive system of rough endoplasmic reticulum than young vegetative hyphae. A bulge in the hypha presages the start of microconidium formation, followed by the rupture of the outermost wall layers. A thick collar forms around the protruding microconidium due to extensive thickening of the inner wall layer of the parent hypha. At this stage, the cytoplasm of the developing microconidium is still continuous with that of the microsporophore cell from which it arises and is contained by a wall which is derived from the thickened collar. The microconidium is finally isolated from the cytoplasm of the microsporophore by a centripetal extension of the collar. Microconidia differ from macroconidia in having a more extensive endoplasmic reticulum and fewer mitochondria, in addition to being smaller and having a single nucleus.     

Augmented B Lymphocyte Response to Antigen in the Absence of Antigen-Induced B Lymphocyte Signaling in an IgG-Transgenic Mouse Line

IgG-containing B cell antigen receptor (IgG-BCR), the BCR mostly expressed on memory B cells, contains a distinct signaling function from IgM-BCR or IgD-BCR expressed on naïve B cells. Because naïve B cells transgenic for IgG exhibit augmented response to antigens similar to memory B cells, the distinct signaling function of IgG-BCR appears to play a role in augmented antibody responses of memory B cells. However, how IgG-BCR signaling augments B cell responses is not yet well understood. Here we demonstrate that B cells from IgG-transgenic mice are anergic with defect in generation of BCR signaling upon BCR ligation. However, these IgG-transgenic B cells generate markedly augmented antibody response to a T cell-dependent antigen, probably due to hyper-responsiveness to a T cell-derived signal through CD40. Both BCR signaling defect and augmented response to CD40 ligation are partially restored in xid IgG-transgenic mice in which BCR signaling is down-modulated due to a loss-of-function mutation in the tyrosine kinase Btk crucial for BCR signaling. Thus, IgG-BCR induces augmented B cell responses in the absence of antigen-induced BCR signaling probably through high ligand-independent BCR signaling that may “idle” B cells to make them ready to respond to T cell help. This finding strongly suggests a crucial role of ligand-independent signaling in receptor function.     

Studies of Chloroplast Development in Euglena: XIV. Sequential Interactions of Ultraviolet Light and Photoreactivating Light in Green Colony Formation

Photoreactivation (PR) of green colony-forming ability in Euglena is pH-insensitive from pH 6.0 to 8.0 and temperature-sensitive with a maximum rate at 35°C. There is no PR at 0°C. The rate of PR varies with the growth stage of the cells; PR of exponential phase cells is slower than that of stationary phase cells. The reciprocity rule holds for PR over a 6-fold range of intensity. The shape of PR curves is a function of the UV dose; there appears to be a progressive increase in multiplicity until a limiting multiplicity is reached as indicated by the fact that curves for high doses are superposable. Dark-grown and light-grown cells give the same PR response for comparable UV doses. UV inactivation of cells which have been treated with UV and then with PR light shows that, if the PR dose is sufficiently large, the same UV-inactivation curve is obtained as for nonpretreated control cells. Doses of PR lower than the saturating dose produce UV-inactivation curves, the ultimate slopes of which are parallel to the slope of the control curve, but which show reduced multiplicity. The multiplicity of these curves increases with increasing PR dose. The UV inactivation of green colony-forming ability in Euglena is completely photoreactivable at the doses studied, in contrast with the UV inactivation of colony-forming ability, which occurs at considerably higher UV doses and behaves like most other photoreactivable systems, showing a photoreactivable sector of 0.32.     

Lymphocyte in vitro cytotoxicity: mechanism of human lymphotoxin-induced target cell destruction

These in vitro studies were conducted in an attempt to elucidate the mechanism of how cell-free supernatant fluids obtained from PHA-stimulated human lymphocytes cause destruction of cells. The undiluted supernatant fluids with high activity exerted a nonspecific cytotoxic effect on many different continuous cell lines. However, upon dilution, a wide spectrum of cell sensitivities was observed. These studies suggest human lymphotoxin acts by first absorbing to receptors on the target cell plasma membrane. The next effect is shut-down of cellular DNA synthesis, followed later by a decrease in cell numbers and finally, cellular destruction. Once sufficient LT has bound to the target cell surface, the cytopathic effect is irreversible. A role for LT in lymphocyte-mediated tissue destruction is discussed.     

Mitosis, Cytokinesis and Colony Formation in Pediastrum boryanum

Uninucleate cells of Pediastrum become multinucleate by a seriesof synchronous mitoses. Mitotic nuclei are enclosed by a perinuclearenvelope of endoplasmic reticulum. Cytoplasmic cleavage of themultinucleate cells leads to the production of uninucleate,biflagellate zoospores (zooids) which are subsequently releasedinto a lenticular vesicle through a rupture in the outer layerof the parental cell wall. Within the vesicle, presumably derivedfrom part of the inner layer of parental wall, the zooids swarmactively before aggregating in a planar array. Bands of microtubulesunderlie the plasmalemma of the zooids which, when the zooidsaggregate, are usually coplanar with the newly formed colony.The role of microtubules in patterned colony formation and inthe development of the characteristic horns on peripheral cellsof colonies of Pediastrum is discussed.     

Notch 信号通路与淋巴细胞发育

Notch 信号通路为一广泛应用且高度保守的信号转导途径,决定多能祖细胞的分化方向,其中在共同淋巴祖细胞向 T 淋巴细胞或 B 淋巴细胞分化选择中具有决定性作用 . Notch 信号通路参与淋巴细胞的发育过程,促进 Tαβ细胞的形成、诱导处女型 T 细胞变为调节型 T 细胞、阻止 CD4+T 细胞向 Th1 类型分化,以及增加外周免疫器官边缘区 B 细胞的数量 . 在分析 Notch 蛋白结构的基础上,综合最新进展,系统阐明了 Notch 信号通路的组成、作用机制、参与的淋巴细胞发育过程以及所起的作用 .     

银杏大孢子形成的超微结构研究

银杏 (GinkgobilobaL .)的大孢子母细胞在减数分裂前变成近长圆形 ,细胞核移向珠孔端 ,造粉质体围绕细胞核分布。线粒体主要分布在细胞的偏向合点端。细胞的偏向珠孔端存在大量的粗面内质网 ,而线粒体和质体较少。到了减数分裂前期Ⅰ时 ,细胞中液泡增加 ,向珠孔端的内质网减少。减数分裂的第一次分裂形成二分体细胞后 ,更表现出明显的极性分化。偏向珠孔端的细胞 (A)相对较小 ,细胞中除环状内质网和少量线粒体外 ,几乎看不到质体 ,而偏向合点端的细胞 (B)体积增大 ,各种细胞器的含量也较丰富。减数分裂第二次分裂时 ,这两个二分体细胞 (A和B细胞 )的分裂时间也不相同。形成直立四分体大孢子细胞时 ,最向合点端的细胞 (B2 )最大 ,成为具功能大孢子。其余 3个大孢子细胞陆续退化 ,但是细胞间差别很大。偏向珠孔端的两个细胞 (A1和A2 )首先退化。后来B1和B2 细胞之间形成了厚壁。由于减数分裂时极性的变化也可能形成T字型四分体大孢子细胞或只产生 3个大孢子细胞 ,最后只有最下面的一个细胞 (B2 )成为具功能大孢子。