Antigenic diversity of the S-layer proteins from pathogenic strains of Aeromonas hydrophila and Aeromonas veronii biotype sobria.

The antigenic relatedness of paracrystalline surface array proteins with subunit molecular weights of approximately 52,000 from isolates of Aeromonas hydrophila and Aeromonas veronii biotype sobria belonging to a single heat-stable serogroup was examined. Enzyme-linked immunosorbent assay and immunoblotting with two different polyclonal antisera against surface exposed and non-surface-exposed epitopes of the S-layer protein from A. hydrophila TF7 showed that the S-layer proteins of the mesophilic aeromonads were antigenically diverse. NH2-terminal amino acid sequence analysis of four antigenically different proteins showed that while the proteins were structurally related, they differed in primary sequence. Absorption experiments with heterologous live cells showed that cross-reactive epitopes were in non-surface-exposed regions of the S-layer proteins, while absorption with homologous live cells showed that the immunodominant epitopes of the S-layer protein of strain TF7 were strain specific and exposed on the surface of the native, tetragonal array produced by this strain. Proteolytic digestion of the TF7 S-layer protein with trypsin, chymotrypsin, or endoproteinase Glu-C produced an amino-terminal peptide of approximate Mr 38,000 which was refractile to further proteolytic cleavage under nondenaturing conditions. This peptide carried the immunodominant surface-exposed region of the protein, and chemical cleavage with cyanogen bromide further mapped the portion of these surface-exposed epitopes to a peptide of approximate Mr 26,000, part of which maps within the Mr 38,000 protease-resistant NH2-terminal peptide.     

Rapid Three-Dimensional Reconstruction at the Light Microscopic Level and a Technique for Re-Embedding the Same Semithin Sections for Electron Microscopic Examination

Rapid three-dimensional reconstruction of serial sections at the light microscopic and ultrastructural levels was accomplished using a two-step technique. Fixed specimens were embedded in Epon and 1 μm sections were cut and placed on glass slides. One of every four sections was drawn onto transparency film for rapid three-dimensional reconstruction. The semi-thin sections were re-embedded in Epon and sectioned at 90 nm for examination in the electron microscopy.     

Fluorescence and Electron Microscopic Localization of F-actin in the Ependymocytes

The organization of F-actin in the ventricular system has been reported to display pronounced regional differences with respect to shape, size, and development. However, the real roles played by F-actin in these cells cannot be understood unless the precise localization of F-actin is defined. In the present study, we used double-fluorescence labeling to further examine the localization of F-actin in the ependymocytes and its spatial relation to the other two cytoskeletal components, microtubules and intermediate filaments. Then we converted fluorescence signals for F-actin to peroxidase/DAB reaction products by use of a phalloidin-based FITC-anti-FITC system. This detection technique provided an overview of the distribution of F-actin in the ependymocytes at the ultrastructural level, and has been proven to be helpful in correlating light and electron microscopic investigations. (J Histochem Cytochem 57:741–751, 2009)     

Secretion of hemolysin of Aeromonas sobria as protoxin and contribution of the propeptide region removed from the protoxin to the proteolytic stability of the toxin

The sequence at the amino terminus region of the hemolysin ofAeromonas sobria is homologous with that of aerolysin of A. hydrophila. However, there is no homology between the two toxins in the sequence at the carboxy terminal region. It has been shown that aerolysin is secreted into culture supernatant as a protoxin. This proaerolysin is activated by the proteolytic removal of a carboxy terminal peptide. However, the role of the carboxy terminal region, which is removed in the activation process, has not been elucidated. In this study, we showed that hemolysin is also secreted as a protoxin into culture supernatant and that prohemolysin is cleaved by the protease of A. sobria between Ser-446 and Ala-447, resulting in the removal of a 42 amino acid peptide. The removal of the peptide converts the prohemolysin into active hemolysin. Subsequently, we mutated the hemolysin gene to delete the last several amino acid residues and expressed the genes in Escherichia coli, in order to examine the role of the carboxy terminal region of prohemolysin. The amounts of these mutant hemolysins accumulated in the periplasmic space of E. coli were very low compared with that of the wild-type. This observation indicated that the carboxy terminal region of prohemolysin contributes to the proteolytic stability of the toxin.     

Light and Electron Microscopic Observations on Encystment of Acanthamoeba palestinensis, Reich

SYNOPSIS. The ultrastructural changes occurring during encystment of Acanthamoeba palestinensis have been investigated. The cyst wall consists of endocyst and exocyst, both having the same fine structure. At irregular intervals in the cyst wall ostioles occupied by opercula are present. The nuclear membrane forms bulb-shaped projections and releases vesicles bounded by double membranes into the cytoplasm. Dense nucleolus-like bodies of different sizes and variable numbers are found in the nucleus of every cyst. The importance of the cyst structure as a taxonomic criterion is discussed.     

Light and Electron Microscopic Observations on the Heterotrich Ciliate Climacostomum virens

SYNOPSIS. Alveolar membranes and an epiplasm exist under the cell membrane of the noncontractile heterotrich ciliate Climacostomum virens. Postciliary microtubular ribbons join at the right of each somatic kinety to form a Km fiber. Two transverse microtubular fibers occur per kinetosomal pair. A myonemal network interconnects the kinetosomal bases intrakinetally and interkinetally. Ultrastructural comparisons are made between the contractile and noncontractile heterotrichs.
The buccal cortex consists of an adoral zone of membranelles, a peristomal field, a buccal tube, the apical membranelles, and a haplokinety. The kineties of the peristomal field and buccal tube are rows of paired kinetosomes, with a postciliary ribbon of microtubules arising from the posterior kinetosome of each pair, and a transverse ribbon and an oblique ribbon from the anterior kinetosome. No Km fibers exist in this region. The haplokinety is a collar of paired kinetosomes surrounding the cytostome; a postciliary microtubular ribbon descends from each kinetosomal pair into the cytostomal region. Ultrastructural details of the buccal cortex of C. virens and other heterotrichs are compared. The nemadesmata which lie under the membranelles are implicated in the body bending of C. virens.
Algae endosymbiotic in the cytoplasm of C. virens are described.     

虎纹捕鸟蛛体表扫描电镜观察

对虎纹捕岛蛛(Ornithoctonus husoena)的眼、生殖球、颚叶、听毛、触毛、琴形器、跗节器、发音器、幼蛛及成蛛的纺绩器和螯肢等结构进行了扫描电镜观察。     

Scanning and Transmission Electron Microscopic Observations on Surface Structures of Three Turbellarian Species

The body surface of Microstomum lineare, Bothriomolus balticus and Archilopsis unipunctata was examined in scanning electron microscope. The threedimensional appearance of adhesive duo-gland organs, receptor cilia, globular bodies and pistil-like projections is described. The adhesive duo-gland papillae of Bothriomolus and Archilopsis appear as bouquetlike structures on either side of the tail as well as on the cilia covered body. In Microstomum the papillae occur as single projections among the ciliary body für. Supplementary TEM investigation revealed crystalline bodies in the cytoplasm of viscid gland cells.     

Electron Microscopic Observation of the Antibody-Induced Capsular Swelling Phenomenon in Klebsiella pneumoniae

The capsular swelling phenomenon of Klebsiella pneumoniae strain 277 was examined morphologically using the electron microscopy techniques of freeze-substitution. The capsules of strain 277 measured about 52 nm in thickness, and were composed of a number of fine fibers. After treating the bacteria with anti-capsular serum, the size of the capsules increased to about twice the normal size and they lost their electron density. The capsular fibers that are tightly packed in normal cells became loose and thus the identification of the individual capsular fibers was difficult in the swollen capsules. Capsule swelling was induced by washing the cells with phosphate-buffered saline. The removal of either divalent cations or some other materials might thus be important for maintaining the normal capsule structure. The mechanism of the swelling phenomenon was also discussed.     

Factors associated with the adherence and biofilm formation by Aeromonas caviae on glass surfaces

AIMS: Knowledge on factors of Aeromonas caviae promoting the formation of biofilms on surfaces. METHODS AND RESULTS: In nutrient broth under agitation, A. caviae LMG 13455 was able to form biofilms on the surfaces of glass flasks, but such biofilm formation was inconsistent. A derivative of this strain, called M12, promoted the rapid formation of reliable and strongly adherent biofilms with about half of the cells being adhered. In contrast with its parent, M12 was hydrophobic, displayed auto-aggregation in liquid medium, synthesized very little polysaccharides and was defective in swimming and swarming motilities, together with the appearance of a characteristic phenotype on motility soft agar. Further analyses demonstrated that most of these properties were related to a hyperpiliation of the cells through the presence of type IV pili, and suggested that a mechanism of genetic variation, by altering the nature of motility appendages, allows the variant bacteria to attach on inert surfaces. CONCLUSIONS: M12 is a stabilized variant of the parental strain, promoting strongly adherent biofilms through the type IV pili. SIGNIFICANCE AND IMPACT OF THE STUDY: The wild-type strain A. caviae LMG 13455 include subpopulations that are likely implicated in its adaptation to different environmental changes.     

Angiostrongylus cantonensis: Scanning Electron Microscopic Observations on the Cuticle of Moulting Larvae

Angiostrongylus cantonensis is a parasitic nematode that needs to develop in different hosts in different larval stages. Freshwater snails, such as Pomacea canaliculata, are the intermediate host, and rats are the definitive host. Periodic shedding of the cuticle (moulting) is an important biological process for the survival and development of the parasite in the intermediate and definitive hosts. However, there are few studies on the cuticle alterations between different stages of this parasite. In this study, we observed the ultrastructural appearance and changes of the cuticle of the 2nd/3rd stage larvae (L2/L3) and the 3rd/4th stage larvae (L3/L4) using a scanning electron microscope. We also first divided L2/L3 into late L2 and early L3. The late L2 lacked alae, but possessed a pull-chain-like fissure. Irregular alignment of spherical particles on the cuticle were noted compared to the L3. Alae appeared in the early L3. The old cuticle turned into a thin film-like structure which adhered to the new cuticle, and spherical particles were seen regularly arranged on the surface of this structure. Regular rectangular cavities were found on the surface of L3/L4. The caudal structure of L3/L4 was much larger than that of L3, but caudal inflation, such as seen in L4, was not observed. These results are the first to reveal the ultrastructural changes of the cuticle of A. cantonensis before and after moulting of L2/L3 and L3/L4.     

A Comparative Electron Microscopic Study of the Morphology of Toxoplasma gondii by Freeze-Etch Replication and Thin Sectioning Technic

SYNOPSIS. Freeze-etch preparations of Toxoplasma gondii reveal details of structure and organelles in 3-dimensional relationships. The subpellicular microtubules and their relationship to the polar ring, the tripartite pellicle, the pellicle constituents, and the spatial relationship of the rhoptries to the conoid and conoid canal are clearly demarcated.     

Electron Microscopic Reconstruction of Neural Circuitry in the Cochlea

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大连地区海生物中致病性弧菌及气单胞菌分布的研究

本文首次报道了大连地区海产品中致病性弧菌及气单胞菌生态分布的研究结果。从152份海产品中共检出88株致病性弧菌及气单胞菌,检出率为57.9％.在检查欧氏六线鱼、太平洋鲱鱼、蓝色马鲛;蝼蛄虾;紫贻贝、杂色蛤仔和毛蚶共7种海产品中,检出9种致病性弧菌和1种气单胞菌,即溶藻弧菌、副溶血性弧菌、麦氏弧菌、少女鱼弧菌、弗尼斯弧菌、拟态弧菌、河弧菌、非01群霍乱弧菌和创伤弧菌。鱼虾类中菌株的检出率明显高于贝类。检出菌株中溶藻弧菌所占比例最高(54.5％),其次为副溶血性弧菌(27.3％)。首次从海生物中分离出嗜水气单胞菌、少女鱼弧菌等致病原因菌株.     

Microscopic Observations on the Filopodia of Entamoeba histolytica*

Living Entamoeba histolytica trophozoites were examined by phase-contrast microscopy. Intact critical point dried trophozoites were examined by transmission electron microscopy at an accelerating voltage of 1000 kV (HVEM) and by scanning electron microscopy (SEM). Half and quarter m? thick sections of epoxy-embedded trophozoites were examined by HVEM. Many of the trophozoites of 2 strains examined had surface filopodia, 1 to over 100 pan in length. The cytoplasm of filopodia was continuous with the cytoplasm and bounded by surface plasmalemma bearing a glycocalyx. Structures called “surface-active lysosomes with trigger,”“dendritic plasmalemmal extensions,” and “extra-amebic vesicles” by previous investigators probably represent portions of filopodia demonstrated in the present study. Filopodia appear to be of frequent normal occurrence in E. histolytica and may function in: (a) endocytosis or pinocytosis; (b) exocytosis; (c) attachment to substratum; (d) penetration of tissue; (e) release of cytotoxic substances; or (f) contact cytolysis of host cells.     

Electron Microscopic Evidence in Support of α-Solenoid Models of Proteasomal Subunits Rpn1 and Rpn2

Rpn1 (109 kDa) and Rpn2 (104 kDa) are components of the 19S regulatory complex of the proteasome. The central portions of both proteins are predicted to have toroidal α-solenoid folds composed of 9-11 proteasome/cyclosome repeats, each ∼ 40 residues long and containing two α-helices and turns [A. V. Kajava, J. Biol. Chem. 277, 49791-49798, 2002]. To evaluate this prediction, we examined the full-length yeast proteins and truncated versions thereof consisting only of the repeat-containing regions by gel filtration, CD spectroscopy, and negative-staining electron microscopy (EM). All four proteins are monomeric in solution and highly α-helical, particularly the truncated ones. The EM data were analyzed by image classification and averaging techniques. The preponderant projections, in each case, show near-annular molecules 6-7 nm in diameter. Comparison of the full-length with the truncated proteins showed molecules similar in size and shape, indicating that their terminal regions are flexible and thus smeared to invisibility in the averaged images. We tested the toroidal model further by calculating resolution-limited projections and comparing them with the EM images. The results support the α-solenoid model, except that they indicate that the repeats are organized not as symmetrical circular toroids but in less regular horseshoe-like structures.     

Evaluation of Electron Microscopic Sample Preparation Methods and Imaging Techniques for Characterization of Cell-Mineral Aggregates

Scanning Electron Microscopy (SEM) is used to image geomicrobiological samples, typically containing interfaces between “hard and soft materials” such as minerals and cells, which represent challenges for artifact-free preparation for high-resolution imaging. We used cell-mineral aggregates produced during microbial Fe(II) oxidation and Fe(III) reduction to evaluate different sample preparation and imaging techniques. Both rapid freezing and standard critical point drying (CPD) preserve structures of geomicrobiological samples, at least the ones obtained for Fe(II)-oxidizing and Fe(III)-reducing bacteria, without artifacts. We recommend a SEM sample preparation scheme for geomicrobiological specimens and discuss critical parameters like fixation, dehydration, coating, and acceleration voltages.     

Scanning Electron Microscopic Observation of Differentiation from the Reproductive Short Form to the Infectious Long Form of Holospora obtusa

To identify the surface features of Holospora obtusa during its differentiation from the reproductive short form to the infectious long form, bacteria of four different buoyant densities were isolated by Percoll density gradient centrifugation of homogenates of host cells or isolated macronuclei, and examined with a scanning electron microscope. Bacteria of buoyant density 1.09 g/ml were reproductive short forms as well as cells at various stages in the elongation process including fully elongated ones. Bacteria of buoyant densities 1.11 g/ml and 1.13 g/ml were premature long forms and those of 1.16 g/ml were mature infectious long forms. Bacteria of buoyant density 1.09 g/ml had an entirely rough surface while those of buoyant densities 1.11 g/ml and 1.13 g/ml were smooth and had wale-like stripes on their surface. A small tapered tip was observed at one end of the bacteria of buoyant density 1.13 g/ml. Bacteria of buoyant density 1.16 g/ml had an entirely smooth surface, but one end always showed a rough surface; this locally differentiated surface of the special tip of the infectious long form may be responsible for both the nuclear and species specificities of the infectivity of H. obtusa. These observations indicate that the surface of H. obtusa changes during differentiation and the special tip develops in bacteria of buoyant density 1.13 g/ml.     

Classification and Reconstruction of a Heterogeneous Set of Electron Microscopic Images: A Case Study of GroEL–Substrate Complexes

Image analysis methods were used to separate images of a large macromolecular complex, the chaperonin GroEL, in a preparation in which it is partially liganded to a nonnative protein substrate, glutamine synthetase. The relatively small difference (6%) in size between the chaperonin in its free and complexed forms, and the absence of gross changes in overall conformation, made separation of the two types of particles challenging. Different approaches were evaluated and used for alignment and classification of images, both in two common projections and in three dimensions, yielding 2D averages and a 3D reconstruction. The results of 3D analysis describe the conformational changes effected by binding of this particular protein substrate and demonstrate the utility of 2D analysis as an indicator of structural change in this system.     

中海拔地区慢性暴露大鼠心肌及肝脏的光镜及电镜观察

目的 探讨中度海拔高度地区慢性低氧大鼠心肌、肝的组织学及超微结构变化。方法 本实验用Wistar大鼠20只,雌雄各半,六日内从海拔5米运至海拔3418米饲养,8周后断头处死大鼠,留取心肝组织作光电镜观察,同时高原暴露前后测定血RBC数及Hb含量。结果 心肝组织学改变主要为细胞水肿,即心肌颗粒变性,肝细胞疏松化,其次为心肌、肝细胞嗜酸性变。心肌组织有少量小灶状坏死,肝组织中未见坏死。超微结构主要有肌浆网扩张,线粒体肿胀,糖元颗粒减少,未见不可逆性损伤如线粒体出现杆状嵴、三膜嵴及核染色质边聚现象。毛细血管内皮细胞多有突起伸向管腔,胞质空泡变性,微饮泡较少。另外,高原暴露后RBC数及Hb含量明显升高。结论 该海拔地区慢性低氧大鼠心肌、肝组织及毛细血管的病变是可逆性的; 左右心室病变程度无显著性差异; 肝组织的病变程度明显轻于心肌组织。