A Thionin Stain for Visualizing Bone Cells, Mineralizing Fronts and Cement Lines in Undecalcified Bone Sections

A staining method is described using thionin, for undecalcified deacrylated bone sections. RNA is stained purplish violet, allowing still active osteoblasts to be distinguished from lining cells. Staining intensity of mineralized bone is related to the degree of mineralization. Mineralizing fronts and cement lines are visualized clearly. Lamellae show an alternate pattern. Histomorphometric parameters such as osteon thickness and interstitial bone thickness can be measured without using polarized light. The mineralizing front can be assessed and expressed as a percentage of the osteoblast-covered interface between osteoid and mineralized bone. The stain is also useful for qualitative assessment of metabolic bone disease. Thionin stained sections can be kept for at least one year when stored hi the dark at 7 C.     

The problem of bone lamellation: An attempt to explain different proposed models

Collagen texture and osteocyte distribution were analyzed in human woven‐ and lamellar‐bone using scanning and transmission electron microscopy. We provide data substantiating the concept that lamellar bone is made up of an alternation of dense‐acellular lamellae and loose‐cellular lamellae, all exhibiting an interwoven texture of collagen fibers. An attempt is also made to explain how the present findings might conform to those of authors whose models propose orderly, geometric arrangements of collagen fibers inside bony lamellae. Such a comparison is possible because the present investigation analyzes split loose lamellae and tangentially‐sectioned dense lamellae. It emerged that only loose lamellae can be dissected, revealing a loose interwoven collagen texture and halved osteocyte lacunae. Dense lamellae cannot be split because of their compactness. The analysis of tangentially sectioned dense lamellae demonstrates that they consist of a network of interwoven collagen fiber bundles. Inside each bundle, collagen fibers run parallel to each other but change direction where they enter adjacent bundles, at angles as described by other authors whose TEM investigations were performed at a much higher magnification than those of the present study. Consequently, what these authors consider to be a lamella are, instead, bundles of collagen fibers inside a lamella. There is discussion of the role played by the manner of osteocyte‐recruitment in the deposition of lamellar‐ and woven‐bone and how the presence of these cells is crucial for collagen spatial arrangement in bone tissues. J. Morphol., 2013. © 2013 Wiley Periodicals, Inc.     

Isolation and Compositional Analysis of Plant Cuticle Lipid Polyester Monomers

Terrestrial plants produce extracellular aliphatic biopolyesters that modify cell walls of specific tissues. Epidermal cells synthesize cutin, a polyester of glycerol and modified fatty acids that constitutes the framework of the cuticle that covers aerial plant surfaces. Suberin is a related lipid polyester that is deposited on the cell walls of certain tissues, including the root endodermis and the periderm of tubers, tree bark and roots. These lipid polymers are highly variable in composition among plant species, and often differ among tissues within a single species. Here, we describe a detailed protocol to study the monomer composition of cutin in Arabidopsis thaliana leaves by sodium methoxide (NaOMe)-catalyzed depolymerisation, derivatization, and subsequent gas chromatography-mass spectrometry (GC/MS) analysis. This method can be used to investigate the monomers of insoluble polyesters isolated from whole delipidated plant tissues bearing either cutin or suberin. The method can by applied not only to characterize the composition of lipid polymers in species not previously analyzed, but also as an analytical tool in forward and reverse genetic approaches to assess candidate gene function.     

Seminal roots of wild and cultivated barley differentially respond to osmotic stress in gene expression,suberization, and hydraulic conductivity

Wild barley, Hordeum vulgare spp. spontaneum, has a wider genetic diversity than its cultivated progeny, Hordeum vulgare spp. vulgare. Osmotic stress leads to a series of different responses in wild barley seminal roots, ranging from no changes in suberization to enhanced endodermal suberization of certain zones and the formation of a suberized exodermis, which was not observed in the modern cultivars studied so far. Further, as a response to osmotic stress, the hydraulic conductivity of roots was not affected in wild barley, but it was 2.5-fold reduced in cultivated barley. In both subspecies, osmotic adjustment by increasing proline concentration and decreasing osmotic potential in roots was observed. RNA-sequencing indicated that the regulation of suberin biosynthesis and water transport via aquaporins were different between wild and cultivated barley. These results indicate that wild barley uses different strategies to cope with osmotic stress compared with cultivated barley. Thus, it seems that wild barley is better adapted to cope with osmotic stress by maintaining a significantly higher hydraulic conductivity of roots during water deficit.     

Sources of plant-derived carbon and stability of organic matter in soil: implications for global change

Alterations in forest productivity and changes in the relative proportion of above‐ and belowground biomass may have nonlinear effects on soil organic matter (SOM) storage. To study the influence of plant litter inputs on SOM accumulation, the Detritus Input Removal and Transfer (DIRT) Experiment continuously alters above‐ and belowground plant inputs to soil by a combination of trenching, screening, and litter addition. Here, we used biogeochemical indicators [i.e., cupric oxide extractable lignin‐derived phenols and suberin/cutin‐derived substituted fatty acids (SFA)] to identify the dominant sources of plant biopolymers in SOM and various measures [i.e., soil density fractionation, laboratory incubation, and radiocarbon‐based mean residence time (MRT)] to assess the stability of SOM in two contrasting forests within the DIRT Experiment: an aggrading deciduous forest and an old‐growth coniferous forest. In the deciduous forest, removal of both above‐ and belowground inputs increased the total amount of SFA over threefold compared with the control, and shifted the SFA signature towards a root‐dominated source. Concurrently, light fraction MRT increased by 101 years and C mineralization during incubation decreased compared with the control. Together, these data suggest that root‐derived aliphatic compounds are a source of SOM with greater relative stability than leaf inputs at this site. In the coniferous forest, roots were an important source of soil lignin‐derived phenols but needle‐derived, rather than root‐derived, aliphatic compounds were preferentially preserved in soil. Fresh wood additions elevated the amount of soil C recovered as light fraction material but also elevated mineralization during incubation compared with other DIRT treatments, suggesting that not all of the added soil C is directly stabilized. Aboveground needle litter additions, which are more N‐rich than wood debris, resulted in accelerated mineralization of previously stored soil carbon. In summary, our work demonstrates that the dominant plant sources of SOM differed substantially between forest types. Furthermore, inputs to and losses from soil C pools likely will not be altered uniformly by changes in litter input rates.     

细鳞鱼卵子发生过程中细胞器的形态变化与作用

通过组织学方法和透射电镜技术对细鳞鱼(Brachymystax lenok)卵子发生过程中细胞器的变化与作用进行了研究.结果表明,从刚分化的卵原细胞至成熟卵母细胞时期(Ⅰ～Ⅴ时相)胞质内均能观察到线粒体,其形态最初为圆形,随着其大量增殖,形态变为棒状、弯曲状或长形(Ⅱ时相),并导致线粒体簇形成,其嵴也由单个变为多个,电子密度呈由低到高的规律变化;但到Ⅲ时相末期线粒体又退化为圆形,个别线粒体还通过对分或牙分进行裂变,线粒体嵴被不断释放,形成空泡,其基质电子密度呈降低的规律变化;在此过程中线粒体主要参与各种囊泡的形成,为后期卵黄前体物质进入、积累创造条件.在Ⅱ时相卵母细胞早期的细胞核附近开始出现内质网和高尔基体,但数量少,结构简单,随着它们的大量增殖(Ⅲ～Ⅳ时相),这两种细胞器将对卵黄物质的合成与加工起到关键作用.内质网主要呈弓形,少数呈圆形或杯形,早期与高尔基体相伴出现,但随着内质网大量增殖,其合成功能也随之增强.早期高尔基体也呈弓形,但随着其高度发育,几个分散的高尔基体聚集形成高度发育的高尔基体复合体,其加工与修饰功能也不断增强,同时其周围伴有大量潴泡或电子密度不同的囊泡体(多层结构)出现,且这些多泡体常常与环形片层(annulate lamellae,AL)一同出现.AL与核膜结构相似,早期呈弧形排列,本研究推测环形片层起源于核膜,其主要作用可能是膜的储藏地.     

Characterization and Ectopic Expression of a Populus Hydroxyacid Hyd roxyci n nam oylt ra nsferase

Cutinized and suberized cell walls in plants constitute physiologically important environment interfaces. They act as barriers limiting the loss of water and nutrients and protecting against radiation and invasion of pathogens. The roles of cutin- and suberin polyesters are often attributed to their dominant aliphatic components, but the contri- bution of aromatic composition to their physiological function remains unclear. By functionally screening a subset of Populus trichocarpa BAHD/HXXXD acyltransferases, we identified a hydroxycinnamoyltransferase that shows specific transacylation activity on ~0-hydroxyacids using both feruloyl- and p-coumaroyl- CoA as the acyl donors. We named this enzyme P. trichocarpa hydroxyacid/fatty alcohol hydroxycinnamoyltransferase 1 （PtFHT1）. The ectopic expression of the PtFHT1 gene in Arabidopsis increased the incorporation of ferulate in root and seed suberins and in leaf cutin, but not that of p-coumarate, while the aliphatic load in both suberin and cutin polyesters essentially remained unaffected. The overaccumulation of ferulate in lipophilic polyester significantly increased the tolerance of transgenic plants to salt stress treatment; under sub-lethal conditions of salt stress, the ratios of their seed germination and seedling establishment were 50% higher than those of wild-type plants. Our study suggests that, although aromatics are the minor component of polyesters, they play important role in the sealing function of lipidic polymers in planta.     

Ultrastructure of pig tubal ova

Summary The unfertilized ova of the pig are characterized by the first polar body situated in the perivitelline space. The metaphase chromosomes of the ova are found free in a cortical area, predominantly inhabited by the spindle fibers. Mitochondria show morphological changes in the form of swelling of their matrices. Frequently, the membranes of the individual cristae mitochondriales meet each other, forming meeting points, at regular intervals. The endoplasmic reticulum increases in quantity when compared with that of the pig follicular oocytes (Norberg, 1972b). The Golgi complexes are sparse and scattered. Occasionally, remnants of the end bulbs of the corona radiata cell processes occur below the surface membrane of the ova.Usually, the sperm-penetrated ova contain the first and the second polar body within the perivitelline space. Intranuclear annulate lamellae are observed within the male and female pronucleoplasm, and of particular interest are extended linear structures in one of the pronuclei. These structures may be considered as precursor stage in the formation of the intranuclear annulate lamellae. The parapronuclear cytoplasm is rich in organelles, especially the cytoplasmic annulate lamellae. In contrast to the scarcity of Golgi complexes in the unfertilized ova, many newly formed Golgi vesicles and lamellae reappear in the pronuclear stage. The zona pellucida displays ultrastructural changes following sperm penetration of the ova.This work was supported by the Agricultural Research Council of Norway.