Organization and Ultra-Structural Components of Endothelial Surface Glycocalyx Revealed by Stochastic Optical Reconstruction Microscopy(STORM)

Introduction The endothelial cells(ECs)lining every blood vessel wall constantly expose to the mechanical forces generated by the blood flow.The EC responses to these hemodynamic forces play a critical role in the homeostasis of the circulatory system.In addition to forming a transport barrier between the blood and vessel wall,vascular ECs play important roles in regulating circulation functions.Besides biochemical stimuli,blood flow induced(hemodynamic)mechanical stimuli,such as shear stress,pressure and circumferential stretch,modulate EC morphology and functions by activating mechanosensors,signaling pathways,and gene and protein expressions.The EC responses to the hemodynamic forces(mechano-sensing and transduction)are critical to maintaining normal vascular functions.Failure in the mechano-sensing and transduction leads to serious vascular diseases including hypertension,atherosclerosis,aneurysms and thrombosis,to name a few[1].On the luminal surface of our blood vessels,there is a thin layer called endothelial surface glycocalyx(ESG)which consists of proteoglycans,glycosaminoglycans(GAGs)and glycoproteins.The GAGs in the ESG are heparan sulfate(HS),hyaluronic acid(HA),chondroitin sulfate(CS),and sialic acid(SA)[2].In order to play important roles in vascular functions,such as being a mechanosensor and transducer for the endothelial cells(ECs)to sense the blood flow,a molecular sieve to maintain normal microvessel permeability and a barrier between the circulating cells and endothelial cells forming the vessel wall,the ESG should have an organized structure at the molecular level.Due to the limitations of optical and electron microscopy,the ultra-structure and organization of ESG has not been revealed until recent development of a super high resolution fluorescence optical microscope,STORM(Stochastic Optical Reconstruction Microscopy).The diffraction of a single fluorescence molecule can be described as the point spread function(PSF).When the light of wavelengthλexcites the fluorophore(emitter),the intensity profile of the spot is defined as the PSF with the width^0.6λ/NA,NA is the numerical aperture of the objective.The diffraction-limited image resolution,for a high numerical aperture objective lens,is^200 nm in the lateral direction and^500 nm in the axial direction,for a conventional fluorescence microscope.The key idea of the single-molecule localization microscopy is to light the molecule,in turn,to achieve the nanometer-level accuracy of their position and reconstruction into a super-resolution image,such as STORM.STORM employs photo-switching mechanisms to stochastically activate individual molecules(photo-switchable or photoactivatable fluorophores)within the diffraction-limited region at different times.Then images with sub-diffraction limit resolution are reconstructed from the measured positions of individual fluorophores[3].To trade the super spatial resolution(accuracy),STORM sacrifices its temporal resolution(efficiency)by switching the state and sequentially exciting the emitters at a high density.Rust et al[3]employed organic dyes and fluorescent proteins as photo-switchable emitters to trade temporal resolution for a super spatial resolution(~20 nm lateral and^50 nm axial at present,can go down to a couple of nanometers if using smaller peptides or antibody fragments instead of currently used whole anti-bodies),which is an order of magnitude higher than conventional confocal microscopy.In the current study,we employed STORM to reveal the major ultra-structural components of the ESG,HS and HA,and their organization at the surface of the cultured EC monolayer[4].Materials and methods We used newly acquired Nikon-STORM system to observe the ESG on in vitro EC(bEnd3,mouse brain microvascular endothelial cells)monolayers.After confluency,the bEnd3 cells were immunolabeled with anti-HS,fol-lowed by an ATT0488 conjugated goat anti-mouse IgG,and with biotinylated HA binding protein,followed by an AF647 conjugated anti-biotin.The ESG was then imaged by the STORM with a 100x/1.49 oil immersed lens.Multiple Reporters of ATT0488 and AF647 with alternating illumination were used to acquire the 3D images of HS and HA.The field of 256×256(40×40μm2)of HS and HA at the surface of ECs was obtained based on totally 40,000 of EM-CCD captured images for each reporter at a capturing speed of 19 ms/frame.Results HA is a long molecule weaving into a network which covers the endothelial luminal surface.The diameter of the HA segments is 185.3±44.7 nm,155.5±57.2 nm,and 156.9±56.1 nm,respectively,at the top,middle and bottom regions of the cell luminal surface.In contrast,HS is a shorter molecule,perpendicular to the cell surface.HA and HS are partially overlapped with each other at the endothelial luminal surface.We quantified the length,diameter,orientation,and density of HS at the top,middle and bottom regions of the endothelial surface.The diameter of the observed HS is 191.0±46.0 nm,284.3±71.1 nm,and 184.2±59.6 nm,and the length of the HS is 621.0±75.7 nm,651.0±118.0 nm,and 575.2±105.6 nm,respectively,at the top,middle and bottom regions of the cell luminal surface.For the HS orientation,its angle with the cell surface is 92.9±1.9,88.7±8.2,and 96.2±10.9 degree,respectively,at the top,middle and bottom regions.The angle of 90 degree is perfectly perpendicular to the cell surface.For the HS distribution,the average density is0.398 elements/μm2,0.345 elements/μm2 and 0.665 elements/μm2,respectively,and the distance between the adjacent HS is 1 694.4±628.1 nm,1 844.8±758.5 nm,and 1 221.9±450.7 nm,respectively,at the top,middle and bottom regions.Conclusions Our results suggest that HS plays a major role in mechanosensing and HA plays a major role in the molecular sieve,due to their organization,ultra-structure and distribution.

Influence of Copper Stress on Ultra-structure of Wheat Seedlings

Transmission Electron Microscope (TEM) Technology was used to investigate the effect of 25,100 and 200 mg/kg copper on ultra-structure of root tip and leaf blade of wheat. Result showed that serious damage was found with Copper of 25,100 and 200 mg/kg. Plasmolysis,concentrated cytoplasm,chloroplast inflation,lamellar structure disturbance,capsule disappearance and disintegration,mitochondria structures ambiguity and vacuolization were all symptoms under Cu stress. There were positive correlation between concentration of coper stress and the degree of injury,and the degree of injury of copper were different in different organelles. Mitochondria were the most sensitive organelles,and there was patient difference in the same organelles of different parts.

Cd^(2+)污染对水花生生理生化及超微结构的影响

1 引言 自1935年日本发生因镉中毒而导致的骨痛病以来,镉污染越来越受到人们的重视.有关其对人类及其它生物的危害已进行了很多研究[3,8,12].对水生植物而言,多数工作集中在对生理生化毒害[2,7]或对细胞结构损伤[7]的个别研究.笔者认为把生理生化变化与结构的破坏联系起来.讨论两者变化的相互关系,探讨其本质,才符合此过程的实际,则更具现实意义.但这方面的研究在国内还不多见.

Induction and Characterization of Embryogenic Callus in Cotyledons Leaves of Tabebuia roseo-alba

Seeds from Tabebuia roseo-alba lose viability very fast. Moreover, the seed germination rate is very low, reaching approximately 40%. This study aimed at the in vitro induction of embryogenic callus. This technology allows subsequent plant regeneration as an alternative for the production of T. roseo-alba seedlings. Seeds were germinated in vitro and after 20 days, cotyledonary leaves, hypocotyls and root segments excised from these seedlings were used as explants. They were inoculated on MS medium supplemented with sucrose （30 g/L）, agar （5.0 g/L） and different auxins. The effect of 2,4-D, picloram and NAA at concentrations 0.0, 0.5, 1.0, 2.0 and 4.0 mg/L was evaluated. For the analysis of callus with embryogenic characteristics, ultra-structural study by scanning electron microscopy and cytochemical test with carmine were performed. The results showed that the culture medium supplemented with 4 mg/L NAA presented induction of callus with embryogenic characteristics in all explants used, with cotyledonary leaves showing the highest percentage （70% of explants with embryogenic characteristics）. The use of 2, 4-D and picloram was efficient for callus formation in different explants, but no embryogenic characteristics were observed. From the ultra-structural analysis of callus with embryogenic characteristics, it was found that cells from different explant sources had isodiametric format. This format is similar to somatic embryos in globular stage. The cytochemical analysis confirmed the presence of pro-embryogenic cells in callus mass. Callus induced from cotyledonary leaves presented 46% positive reaction to carmine acetic.

Chronic Administration of Sildenafil Citrate (Viagra) on the Frontal Cortex of Adult Male Rats: An Ultrastructural Study

Objectives: Sildenafil citrate (Viagra) is widely used as an aphrodisiac drug and for the treatment of erectile dysfunction. The present study was planned to study the morphological changes that might occur in the frontal cortex of the adult rat in response to chronic intake of Viagra and discover whether these changes are reversible or irreversible. Material and Methods: Forty adult male rats were used where they were classified into three groups: A control group (10 rats), B experimental treated group (20 rats), and C recovery group (10 rats). The treated and recovery groups received therapeutic dose of Viagra for 30 days. The control and the treated group were sacrificed at the first day after the designated period (30 days), the recovery group was sacrificed two weeks after the end of experiment. Results: The pyramidal cell neurons are little in number;some of them are atrophic, degenerated. Their cytoplasm showed varied degree of cellular degenerative changes with vaculation of their myelinated axons. Pyramidal neurons of recovery animals showed nearly complete recovery. Conclusion: Chronic intake of Viagra produced reversible morphological changes in pyramidal neurons of the frontal cortex of adult male rats.

Effects of Methanol on Wettability of the Non-Smooth Surface on Butterfly Wing

The contact angles of distilled water and methanol solution on the wings of butterflies were determined by a visual contact angle measuring system. The scale structures of the wings were observed using scanning electron microscopy, The influence of the scale micro- and ultra-structure on the wettability was investigated. Results show that the contact angle of distilled water on the wing surfaces varies from 134.0° to 159.2°. High hydrophobicity is found in six species with contact angles greater than 150°. The wing surfaces of some species are not only hydrophobic but also resist the wetting by methanol solution with 55% concentration. Only two species in Parnassius can not resist the wetting because the micro-structure （spindle-like shape） and ultra-structure （pinnule-like shape） of the wing scales are remarkably different from that of other species. The concentration of methanol solution for the occurrence of spreading/wetting on the wing surfaces of different species varies from 70% to 95%. After wetting by methanol solution for 10 min, the distilled water contact angle on the wing surface increases by 0.8°-2.1°, showing the promotion of capacity against wetting by distilled water.

Structural and Functional Changes of Immune System in Aging Mouse Induced by D-Galactose

Objective To investigate the role of D-galactose, especially in the structural and functional changes of the immune system in aging. Methods Serum levels of advanced glycation end-products （AGE） were determined by ELISA method. Ultra-structures of thymus and spleen were detected by transmission electron microscopy. MTT method was used to determine the lymphocyte proliferation. IL-2 activity was determined by bioassay. Northern blot was used to detect the IL-2 mRNA levels. Results Serum AGE levels of D-galactose- （P〈0.01） and AGE-treated （P〈0.05） mice （n=8） were increased significantly. The ultra-structures of thymus and spleen in D-galactose- and AGE-treated mice showed regressive changes similar to those in the aged control group. The lymphocyte mitogenesis and IL-2 activity of spleen were also decreased significantly （P〈0.01, n=8）. The change of IL-2 activity shown by Northern blot resulted from the change of mRNA expression. The AGE plus aminoguanidine group, however, showed no significant change in these parameters in comparison with the young control group （P〈0.01 or P〈0.05, n=8）. Conclusion D-galactose and AGE lead to a mimic regression change of aging in the immune system in vivo.

The Dynamics of Changes in Starch and Lipid Droplets and Sub-Cellular Localization of β-Amylase During the Growth of Lily Bulbs

The ultra-structure of mother and outer daughter scales of Lilium Oriental hybrid Sorbonne were studied using transmission electron microscope to examine the sub-cellular localization of starch and lipid droplets during growth and development from shoot emergence to senescence.The contents of starch granules and lipid droplets in the cell of the mother scales decreased significantly from shoot emergence to anthesis,indicating that these scales served as a source for growth and development.After flowering,the number of starch granules and lipid droplets increased dramatically,and finally the cells were filled with the above molecules indicating that the bulb becomes a major sink during bulb enlargement.Ultrastructure observation also showed that symplastic pathway is the main pathway in cells in the exchange and transportation of material during bulb development.The activity of β-amylase,one of the key enzymes catalyzing starch breakdown,showed a similar trend.The enzyme sub-cellular localization via immune-gold electron-microscopy showed that βamylase was predominantly located together with starch granules,while the gold particles were scarcely found in other sub-cellular compartments.The result suggested that this enzyme is compartmented together with its functional substrate supporting its function in catalyzing starch breakdown in living plant cells.

Antennal epicuticular structure of camel crickets (Orthoptera: Rhaphidophoridae) for identifying the prey of Mustella sibrica Pallas

Camel crickets (Orthoptera: Rhaphidophoridae) are a group of wingless and humpbacked Orthopteran insects. They are closed relatives to katydids and crickets;they were called spider crickets somewhere because of their long legs. Camel cricket plays an important role in the food web in various ecosystems. In the forest ecosystem, they live in the ground level of forestry under litters and fallen leaves, and some of them live beneath the log and hid themselves under loosen tree barks. They feed on decomposing organic substances and serve as decomposers in the food chain. It is a very common insect prey to insectivorous animals in natural and artificial vegetation. Guandaushi (GDS) is the experimental forest station of National Chung Hsing University, as the study site of long term ecological research (LTER) in Taiwan. And the camel cricket became an important subject and a dominant insect group in the LTER studies. They are the major parts of the insect preys of vertebrates, especially to the insectivorous mammals. According to our previous publication, 32.2% of the insect preys of Mustela sibirica Pallas are camel crickets in GDS forest ecosystem. There were 5 morpho-species of camel crickets by the characters of antennal epicuticular characters. The camel cricket was collected from various ways according to the LTER study during 1995-1998. The morphology of antennal epicuticular structure from totally 60 individuals was observed by using SEM. The epicuticular sculpture was described and classified into Type I and Type II by the SEM photographs. Type I has 8 patterns and Type II has 5 patterns in this study. The illustration and the pictorial key were provided here as the tool for further application. The morpho-species could not be identified due to the remainder being fragmented while examined. After the study, the SEM antennal epicuticular structures were classified and identified based upon the antennae sample of the morphospecies from the specimens of the 5 camel crickets which were collected from GDS forest ecosystem in this study. This is a part of the fundamental study for the protocol of ecosystem study. The protocol for identifying the insectivorous animal’s prey by the insect fragment of food remainders is necessary. One more, there were 7 of the 13 patterns of antennal epicuticular structure applied to analysis the 5 morpho-species provided as the case study for taxonomy.

Comparative studies on histological and ultra-structure of the pituitary of different ploidy level fishes

The histological and ultra-structure of the pituitary in diploid red crucian carp(Carassius auratus red var.),triploid crucian carp and allotetraploid hybrids within and after the breeding season were comparatively studied.The result showed that there were six endocrine cell types in the pituitary of these three kinds of fishes,and there was an obvious difference in cell size among different ploidy level fishes.As for the same type of pituitary cells,the cell size was increased gradually with the in-creasing ploidy level.In the breeding season,the allotetraploid hybrids had higher proportion of go-nadotropin cells(GTH)than triploids,and the triploids had higher proportion of GTH than diploids.The results were related to the earlier sexual maturity of allotetraploid hybrids and sterility of triploid cru-cian carp.On the other hand,among the three kinds of fishes,the proportion of somatotropin(STH)cells in triploids crucian carp was the highest,whereas that in allotetraploid hybrids was the lowest.The results might be connected with the faster growth rate of triploids and slower growth rate of al-lotetraploid hybrids.In addition,in GTH cells of meso-adenohypophysis after the breeding season,there were many endocrine particles in triploids,while those endocrine particles were released from the cells in allotetraploids and diploids.This result showed that the sterility of triploid crucian carp might be related to the hormone which was not released from the GTH cells.In a word,the present study indicated that the differences in the structure of pituitary among different ploidy level fishes contributed to their difference in the growth rate and gonadal development.

Comparative Proteomic Analysis of Rice Shoots Exposed to High Arsenate

Consumption of arsenic contaminated water and cereals is a serious threat to humans all over the world. Rice （Oryza sativa ＂Nipponbare＂）, as a main cereal crop, can accumulate arsenic more than 10-fold that of in other cereals. To gain a comprehensive understanding of the response of rice subjected to 100 μM arsenate stress, a comparative proteomic analysis of rice shoots in combination with morphological and biochemical investigations have been performed in this study. The results demonstrated that arsenate suppressed the growth of rice seedlings, destroyed the cellular ultra-structure and changed the homeostasis of reactive oxygen species. Moreover, a total of 38 differentially displayed proteins, which were mainly involved in metabolism, redox and protein-metabolism, were identified. The data suggest the arsenic can inhibit rice growth through negatively affecting chloroplast structure and photosynthesis. In addition, upregulation of the proteins involved in redox and protein metabolism might help the rice to be resistant or tolerant to arsenic toxicity. In general, this study improves our understanding about the rice arsenic responsive mechanism.

A REVIEW ON RESEARCH ON DEVELOPMENT OF LIGAMENT CONSTITUTIVE RELATIONS ON MACRO, MESO, AND MICRO LEVELS

Ligaments are densely connective soft tissues capable of maintaining stability and function of knee joint.As an important factor,the constitutive relation of ligament would affect its biomechanics and further play an essential role in the research on ligament injury,healing and treatment.The objective of this paper is to provide an overview of the current research on ligament constitutive relations on the macro,meso,and micro levels as well as the anatomy and histological structure of ligament.Some studies of biomechanical behaviors during ligament injury and healing periods have also been investigated.Based on the research on ligament constitutive relation in the past three decades,a discussion of some research perspectives is also presented,such as a validated accurate measuring method of in situ strain in ligament,a new constitutive relation involving the distribution of ultra-structural properties,and a rational estimation of ligament injury and healing process by the change of its ultra-structural or histological characteristics.

Effect of Cistanche Desertica Polysaccharides on Learning and Memory Functions and Ultrastructure of Cerebral Neurons in Experimental Aging Mice

To observe the effects of Cistanche desertica polysaccharides (CDP) on the learning and memory functions and cerebral ultrastructure in experimental aging mice. Methods: CDP was administrated intragastrically 50 or 100 mg/kg per day for 64 successive days to experimental aging model mice induced by D-galactose, then the learning and memory functions of mice were estimated by step-down test and Y-maze test; organelles of brain tissue and cerebral ultrastructure were observed by transmission electron microscope and physical strength was determined by swimming test. Results: CDP could obviously enhance the learning and memory functions (P＜0.01) and prolong the swimming time (P＜0.05), decrease the number of lipofuscin and slow down the degeneration of mitochondria in neurons(P＜0.05), and improve the degeneration of cerebral ultra-structure in aging mice. Conclusion: CDP could improve the impaired physiological function and alleviate cerebral morphological change in experimental aging mice.