Effects of dimethylsulfoxide on the ultrastructure of fixed cells

It has been reported that the use of dimethylsulfoxide (DMSO) as a solvent for fixatives enhances preservation of cellular ultrastructure. By contrast, we have shown that DMSO alters the ultrastructural integrity of glutaraldehyde fixed cells. The cell membrane, nuclear envelope, endoplasmic reticulum, ribosomes, microtubules and intracytoplasmic organelles are most susceptible to the action of DMSO. We hypothesize that DMSO exerts intracellular alterations via its interaction with remnant interfacial water in fixed cells. DMSO-induced alterations of these and related cellular components may result in the formation of artefactual structures and networks. Thus, it appears that DMSO containing glutaraldehyde neither accelerates fixation nor enhances stabilization of cellular ultrastructure. For these reasons, addition of DMSO to fixatives is not recommended.     

The ultrastructure of Candida krusei

Various methods of chemical fixation and freeze-drying of Candida krusei were compared to determine the most appropriate method for the ultrastructural investigation of the thick walled organisms of this genus. Freeze-drying without chemical fixation was of little value because of insufficient variation in electron density. Potassium permanganate was able to penetrate the intact cell but failed to show cytoplasmic glycogen and lipid and some details of the cell wall. While normal glutaraldehyde, formaldehyde and osmium tetroxide treatment failed to permeate and preserve intracellular structures, several cycles of rapid freezing (–155°C) and thawing followed by glutaraldehyde fixation and osmium tetroxide post-fixation demonstrated the intracellular details of the majority of cells so treated.     

Effects of lead on hepatocyte ultrastructure in Carassius carassius (L.) var. auratus

Subcellular modifications in hepatocytes of Carassius carassius var. auratus subjected to 24 hr and 48 hr sublethal acute lead (5mg·1⁻¹) exposure were studied by electron microscopy. Cytological alterations were observed after 24 hr of treatment and became more evident after 48 hr. Lead induced an increase in nuclear heterochromatin and alterations in mitochondria, endoplasmic reticulum and Golgi complex ultrastructure. Glycogen granula decreased, and secondary lysosomes and lipid droplets increased. Furthermore, intracytoplasmic lumina with microvilli-bearing surfaces and numerous autophagic vacuola were observed after 48 hr of exposure.     

High enantiofacial selectivity in the OsO4 dihydroxylation of e-stilbene with a chiral biphenyl diamine catalyst

The chiral biphenyl-containing diamine 1 complexed with OsO₄ oxidized trans-stilbene at –80°C to (+)-(1R;2R)-1,2-diphenyl-1,2-ethanediol in high enantiomeric excess. On the other hand, (R)- or (S)-2,2'-bis(dimethylamino)-6,6'-dimethylbiphenyl proved to be ineffective in promoting oxidation. © 1992 Wiley-Liss, Inc.     

Effect of dimethylsulfoxide on two synthetic Asn-X-Thr containing substrates of the oligosaccharyltransferase

The enzymatic transfer of oligosaccharides from oligosaccharide lipids to synthetic Asn-X-Thr containing peptides of various length was studied in presence and absence of dimethylsulfoxide. Up to 13.5%, this solvent was found to specifically enhance the efficiency of a ribonuclease heptapeptide to be substrate of the thyroid oligosaccharyltransferase and in contrast, not that of a tripeptide. Circular dichroic analysis performed in various dimethylsulfoxidewater mixtures showed that only the heptapeptide underwent conformational modifications when increasing the concentration in dimethylsulfoxide. Some ordered structure in the immediate vicinity of the Asn-X-Thr signal sequence thus appears of importance in the N-glycosylation process.     

Cytotoxic effect of dimethylsulfoxide on the ultrastructure of cultured Rhesus kidney cells

Cultured Rhesus kidney cells were incubated in the 7.5 and 15% solutions at 4 and 25 ° C and for periods ranging from 10 to 60 min.The ultrastructural alterations, while varying from cell to cell, were evident even following the 10-min incubation interval. The most frequent and least severe structural changes included lipid accumulation, increased number of lysosomes, dilation and degranulation of rough surfaced endoplasmic reticulum, as well as the swelling of mitochondria and damage to mitochondrial membranes.The more severe instances of cellular lesions were represented by the extensive damage to cell membranes, karyorrhexis, total obliteration of the internal structure of mitochondria, and areas of complete loss of hyaloplasm.The extent and frequency of cellular lesions appeared to be determined primarily by the concentration of DMSO with the temperature and duration of incubation being important ancillary determinants.     

Golgi Method without Osmium Tetroxide for the Study of the Central Nervous System

A variant Golgi technique was developed that consisted of substituting osmium tetroxide with formaldehyde as the initial fixative in intracardiac perfusion, along with the addition of glacial acetic acid to the chromating fluid. This procedure avoids disposal of dangerous waste substances into the environment. Other advantages include 1) reduction of cost, danger to lab workers, and risk of disruption of the tissue slices during their handling by eliminating the osmium tetroxide, 2) clear tissue background, 3) greater quantity of impregnated neurons than in the classical procedure, with distinct morphological details easily identified even in gross sections and 4) reduction in processing time.     

Golgi Method without Osmium Tetroxide for the Study of the Central Nervous System

A variant Golgi technique was developed that consisted of substituting osmium tetroxide with formaldehyde as the initial fixative in intracardiac perfusion, along with the addition of glacial acetic acid to the chromating fluid. This procedure avoids disposal of dangerous waste substances into the environment. Other advantages include 1) reduction of cost, danger to lab workers, and risk of disruption of the tissue slices during their handling by eliminating the osmium tetroxide, 2) clear tissue background, 3) greater quantity of impregnated neurons than in the classical procedure, with distinct morphological details easily identified even in gross sections and 4) reduction in processing time.     

Metallic Lakes of the oxazines (Gallamin Blue,Gallocyanin and Coelestin Blue) as Nuclear Stain Substitutes for Hematoxylin

Becher's investigations upon the soluble metallic lakes of the oxazines have been re-investigated, extended and results described. Gallamin blue, gallocyanin and coelestin blue in combination with ferric ammonium sulfate gave the best results. The dyes are dissolved in a five per cent aqueous solution of ferric ammonium sulfate. The solution is boiled for 2–3 minutes, cooled, filtered and ready for immediate use. The iron lakes of these dyes stain nuclei excellently giving a deep blue or blue black in 3–5 minutes. No differentiation with acid is required. Coelestin blue gives the most stable solution and is recommended as a routine nuclear stain. The protoplasm remains practically colorless and counter-staining with acid dyes such as ethyl-eosin, orange G, or fuchsin gives pictures which cannot be distinguished from a good hematoxylin stain.

Counter-staining with van Gieson solution is also possible. Benda's modification of the van Gieson solution is recommended. Staining of fat with Sudan, scarlet red, etc., does not interfere with nuclear staining by these dyes.

As applied to the central nervous system these dyes are far superior to hematoxylin. Ganglion and glia cells are as excellently stained as with thionin.

The most widely used fixatives, namely formaldehyde, Mueller-formaldehyde, Zenker's and alcohol, give equally as good results. The nature of the staining process is briefly discussed and a prospectus offered.     

Na2CO3胁迫对星星草叶肉细胞超微结构的影响

利用透射电镜技术对Na₂CO₃胁迫下星星草叶肉细胞超微结构进行了观察。结果表明：未胁迫的叶肉细胞排列疏松,各种细胞器结构完整,叶绿体含少量淀粉粒和脂质球。轻度盐胁迫（2g/L,4g/L Na₂CO₃）对叶肉细胞超微结构影响较小。中度盐胁迫（6g/L,8g/L Na₂CO₃）引起叶肉细胞超微结构的变化,叶绿体类囊体肿胀,基粒紊乱,不含淀粉粒,脂质球数量增加,叶绿体由原来的梭形或椭球形变成圆球状;部分线粒体嵴消失,出现晶体结构;中央大液泡破裂;核逐渐降解。高度盐胁迫（10g/L,12g/L Na₂CO₃）下,叶绿体片层结构消失,脂质球数量增加,体积变大,被大量的膜片层所包围,叶绿体内、外膜消失,叶肉细胞中看不到叶绿体的存在;膜片层包围线粒体;叶肉细胞中可见大量的泡状结构和膜片层,叶肉细胞死亡。上述结果表明,细胞器特别是叶绿体膜结构的破坏与盐胁迫叶肉细胞最终死亡密切相关     

Novel free ceramides as components of the soldier defense gland of the Formosan subterranean termite (Coptotermes formosanus)

Of the lipid extracts of the defense secretion from the Formosan subterranean termite, Coptotermes formosanus Shiraki, on high-performance thin-layer chromatography analysis, no glycolipids or phospholipids were detected, but free fatty acids and three novel ceramides were found (termed TL-1, TL-2, and TL-3). Free fatty acids were confirmed to be lignoceric acid (C24:0) and hexacosanoic acid (C26:0), as described previously [Chen, J., G. Henderson, and R. A. Laine. 1999. Lignoceric acid and hexacosanoic acid: major components of soldier frontal gland secretions of the Formosan subterranean termite (Coptotermes formosanus). J. Chem. Ecol. 25: 817-824]. TL-1, TL-2, and TL-3 were characterized as ceramides differing in hydrophobicity based on results of matrix-assisted laser desorption-ionization time-of-flight mass spectrometry analysis, mild alkaline treatment, GC-MS analysis of fatty acid methylesters, and GC-MS analysis of sphingoid long-chain bases (LCBs) as trimethylsilyl derivatives. Fatty acids in TL-1 and TL-2 were C18:0, C20:0, and C22:0, and those in TL-3 were 2-hydroxy C18:0, C20:0, and C22:0. The most predominant LCB in TL-2 was a novel trihydroxy C(14)-sphingosine, 1,3,9-trihydroxy-2-amino-6-tetradecene. TL-3 contained C(18)-sphinganine and two kinds of novel sphingadienines, 1,3-dihydroxy-2-amino-7,10-hexadecadiene and 1,3-dihydroxy-2-amino-11,14-eicosadiene. Although examination of the biological activities of these novel ceramides was beyond the scope of these studies, because of the minuscule quantities available from termite secretions, it will be interesting in the future to synthesize these molecules for biological testing.     

Effects of cryopreservation on the developmental competence, ultrastructure and cytoskeletal structure of porcine oocytes

The purpose of this study was to determine ultrastructural and cytoskeletal changes that result from vitrification of porcine germinal vesicle- (GV-) and meiosis II- (MII-) stage oocytes. To investigate the effects of vitrification on developmental competence, oocytes were divided into three groups: fresh GV-oocytes (control), vitrified GV-oocytes, and vitrified MII-oocytes. In both GV- and MII-oocytes, vitrification resulted in a high proportion with normal morphology (92.4 vs. 94.2%, P > 0.05), while vitrified GV-oocytes yielded a higher survival rate than did vitrified MII-oocytes (56.8 vs. 41.9%, P < 0.05). In vitrified GV-oocytes, 12 of 154 oocytes underwent cleavage after fertilization in vitro, and 6 of these developed to the 8-cell stage, 3 developed to the 16-cell stage, and 3 developed into morulae. No cleavage was obtained from vitrified MII-oocytes. For ultrastructural analysis of oocytes, fresh and vitrified-warmed GV- and MII-oocytes were randomly selected for transmission electron microscopy (TEM). Results showed that vitrification caused various degrees of cryodamage in GV-oocytes. Cumulus cells of some oocytes were separated from the cumulus-oocyte complex (COC), and the zona pellucida adjacent to cumulus cells was fractured. The gap junctions between cumulus cells were ruptured, and many microvilli were disrupted or disappeared. Only homogeneous lipid droplets were observed. After vitrification, cortical granules still lined the oolemma of MII-oocytes. Only morphologically irregular, nonhomogeneous lipid droplets surrounding large vacuoles were found. To examine cytoskeletal structures, fresh and vitrified-warmed MII-oocytes were analyzed by laser-scanning confocal microscopy (LSCM); vitrified-warmed GV-oocytes were cultured for 42-44 hr before LSCM. Of 58 control oocytes, 79.5% displayed normal spindles with chromosomes aligned along the equatorial plate. In vitrified oocytes the percentage with normal spindle organization was decreased significantly in both vitrified GV-oocytes and MII-oocytes (10.1 and 12.9%, respectively, P < 0.05). The proportion of oocytes with normal distribution of F-actin was lower for vitrified GV- and MII-oocytes than for controls (16.9 and 37.2% vs. 72.3%). Results of this experiment suggest that irreversible damage to the cytoskeleton of porcine GV- and MII-oocytes after vitrification could be an important factor affecting developmental competence.     

Pb、Cd污染胁迫对大羽藓超微结构的影响

大羽藓在Ph、Cd溶液中培养7d后,观察其超微结构的变化发现：Pb、Cd胁迫使得细胞的超微结构遭到破坏,如叶绿体的外膜破裂,不连续,甚至完全消失;类囊体片层膨胀,或高浓度培养中叶绿体完全解体;线粒体外膜断裂或消失;细胞核被膜破坏,染色质凝聚,核质解体。仅在Cd100mg／L的培养中观察到内质网断裂呈片段状。同时在Pb的培养中发现了大量的黑色颗粒,而在Cd的培养中却未发现。     

Effect of mitotic inhibitors on the ultrastructure of root meristem cells

After 5 h in 10^-3M vinblastine the most obvious effects upon Vicia faba L. cells are seen in the spindle apparatus. These include the microtubules themselves as indicated by c-type metaphases and the pole regions of otherwise intact spindles, leading to multipolar anaphases and to telophases with more than two daughter nuclei. Vesicle transport may be undisturbed and new cell walls can be formed, although cases of disturbed cell plate and cell wall formation were noted occasionally, accompanied by myelinizations in phragmosomes. After 24 h in the same concentration of vinblastine, divisions are no longer observed and the plasma membranes are severely affected. They show extensive myelinizations, accumulations of lipids and dehiscence from the cell walls which are frequently thickened and irregularly formed. Of the other cellular membranes, the nuclear envelope and, more frequently, the tonoplast may be affected. Electron-dense deposits appear in the vacuoles. Comparable, though less severe, changes including multipolar anaphases and myelinizations result from treatment with lumicolchicine, but not with colchicine. Vinblastine leads to the appearance of filament bundles both in cytoplasm and karyoplasm, lumicolchicine to morphologically identical filaments in the cytoplasm alone. The nature of these filaments is unknown.Abbreviation VLB vincaleukoblastine     

低温逆境对不同核桃品种抗氧化系统及超微结构的影响

为揭示核桃抗寒机理,确定核桃抗寒性鉴定适宜的生化指标,以展叶期抗寒性不同的哈特雷、晋龙1号和晋龙2号3个品种1年生枝条的叶片为材料,测定了1℃低温下抗氧化酶活性及超氧阴离子(O2(-))含量的变化,并采用透射电子显微镜观察低温逆境对抗寒性差异大的哈特雷和晋龙2号叶肉细胞超微结构的影响.结果表明:低温胁迫前后抗寒性强的哈特雷叶片中超氧化物歧化酶(SOD)和过氧化物酶(POD)的活性最高,超氧阴离子含量最低,叶肉细胞超微结构较稳定,叶片没有明显冷害症状.抗寒性差的晋龙2号随着低温胁迫时间的延长,3种抗氧化酶活性的下降幅度最大,O2(-)含量始终处于高水平;胁迫72 h时细胞叶绿体普遍膨胀,基粒片层变薄,数目减少,部分叶绿体被膜及质膜清晰度下降,部分顶端小叶叶缘呈水浸状,表现出冷害症状.可见,低温逆境下核桃叶肉细胞超微结构的稳定性与其品种的抗寒性密切相关.SOD、POD活性以及O2(-)含量可作为展叶期核桃抗寒性鉴定的生化指标;低温胁迫下核桃叶片细胞内膜系统的损伤与活性氧积累之间可能存在一定的相互关系.     

ting类旋壁超微构造的研究

电子显微镜下观察表明,除史塔夫ting类外,ting类旋壁有8种类型,各分层的超微构造是由开头不同、粒径不一及排列方式各异的方解石晶粒组成。旋壁基本结构包括致密层、透明层、内疏松层、原蜂巢层（?原始层）、蜂巢层及后透明层。ting类标本受后期次生作用,可改变组成旋壁的方解石晶粒原有的形状,晶粒被重结晶,但其旋壁的分层结构仍保留原生的特征。     

The effect of zinc on endothelium-dependent relaxation of blood-vessels and on the ultrastructure of endothelial cells under immobilization stress

We investigated the effects of zinc on the function and ultrastructure of endothelial cells in the case of a 48-day immobilization stress provoked in Chinchilla male rabbits (n=18) by placing them in metal hutches. Half of those rabbits (n=9) received an daily oral supplement of zinc at a dose of 0.3 mg/kg body weight (in the form of zinc acetate). The control rabbits had no intervention and received no supplement of zinc. The relaxation of smooth muscles from thoracic aorta as mediated by acetylcholine at concentrations from 10(-8) mol/L to 10(-4) mol/L was determined in isometric regime. Responses were expressed as the percentage of relaxation to prostaglandin F2alpha (2.10(-5) mol/L)-induced precontraction. The ultrastructure of endothelial cells was evaluated by electron microscopy. The level of total cholesterol and zinc in the blood serum was determined by an enzymatic method and by atomic absorption spectrometry, respectively. In rabbits receiving no zinc supplement, the relaxation of smooth muscles under the influence of acetylcholine concentrations from 10(-8) mol/L to 10(-4) mol/L was significantly (P < 0.05-0.01) lower than in rabbits receiving a supplement of zinc and lower than in control rabbits. Also, in the rabbits not receiving the zinc supplement, the level of total blood serum cholesterol was increased, but the concentration of zinc decreased. In rabbits receiving the zinc supplement, the contractility of the smooth muscles effected by acetylcholine did not change as compared with control rabbits, and we found a normal structure of endothelial cells and a normal level of total cholesterol and zinc in their blood serum. Thus, zinc played an important role in the maintenance of the normal ultrastructure and function of the endothelial cells in the rabbits receiving zinc under immobilization stress.     

Effect of the lipopeptide antibiotic, iturin A, on morphology and membrane ultrastructure of yeast cells

Abstract The effects of iturin A, at fungicidal concentrations, on yeast cells were studied by scanning electron microscopy and by transmission electron microscopy. A depression, observed in each iturin A-treated cell, was the consequence of the release of electrolytes and other cytoplasmic components. Iturin A passes through the cell wall and disrupts the plasma membrane with the formation of small vesicles and the aggregation of intramembranous particles. Moreover, iturin A passes through the plasma membrane and interacts with the nuclear membrane and probably with membranes of other cytoplasmic organelles.