Ultrastructural immunogold labelling of vimentin filaments on postembedding ultrathin sections of arachnoid villi and meningiomas

An immunoelectron microscopic technique for the labelling of vimentin intermediate filaments on postembedding ultrathin sections is reported. Arachnoid villi obtained at autopsy and meningiomas at surgery were fixed in 1% paraformaldehyde for 30 minutes, embedded without postfixation in Epon-Araldite mixture and polymerized at 37 degrees C for 3 weeks. Ultrathin sections were etched in 2% KOH for 3 minutes and incubated with anti-vimentin monoclonal antibodies which were subsequently labelled with goat anti-mouse IgG coupled to colloidal golds. All of these labelling procedures were consistently performed within 4 hours. In both arachnoidal and meningioma cells, immunogolds preferentially decorated the intermediate filaments in proportion to the concentration. Very few gold particles were seen over the nucleus, Golgi zone, mitochondria and the extracellular connective tissue fibres. The present technique may be applied to the immunogold labelling of intermediate filaments on postembedding ultrathin sections.     

Ultrastructure of Lymphocystis Virus

Lymphocystis virus obtained from bluegills (Lepomis macrochirus) was cultured in the permanent bluegill cell line BF-2 and examined by electron microscopy in ultrathin sections of cell cultures and in negative-contrast preparations from cells and from centrifuged culture medium. According to negative-contrast preparations, the icosahedral virions have an overall diameter close to but not exceeding 300 mμ. Delicate filaments seem to issue from the vertices. In collapsed virions, an ordered array of morphological units was seen. Positively contrasted virions in ultrathin sections show a shell with three dark (heavy metal-stained) layers alternating with and separated by two clear layers. The acquisition of an additional outer membrane during release from the cell, as found in African swine fever virus, was never seen. Morphologically, lymphocystis virus is considered to be closely related to Tipula iridescent virus.     

Prophage spontaneous activation promotes DNA release enhancing biofilm formation in Streptococcus pneumoniae

Streptococcus pneumoniae (pneumococcus) is able to form biofilms in vivo and previous studies propose that pneumococcal biofilms play a relevant role both in colonization and infection. Additionally, pneumococci recovered from human infections are characterized by a high prevalence of lysogenic bacteriophages (phages) residing quiescently in their host chromosome. We investigated a possible link between lysogeny and biofilm formation. Considering that extracellular DNA (eDNA) is a key factor in the biofilm matrix, we reasoned that prophage spontaneous activation with the consequent bacterial host lysis could provide a source of eDNA, enhancing pneumococcal biofilm development. Monitoring biofilm growth of lysogenic and non-lysogenic pneumococcal strains indicated that phage-infected bacteria are more proficient at forming biofilms, that is their biofilms are characterized by a higher biomass and cell viability. The presence of phage particles throughout the lysogenic strains biofilm development implicated prophage spontaneous induction in this effect. Analysis of lysogens deficient for phage lysin and the bacterial major autolysin revealed that the absence of either lytic activity impaired biofilm development and the addition of DNA restored the ability of mutant strains to form robust biofilms. These findings establish that limited phage-mediated host lysis of a fraction of the bacterial population, due to spontaneous phage induction, constitutes an important source of eDNA for the S. pneumoniae biofilm matrix and that this localized release of eDNA favors biofilm formation by the remaining bacterial population.     

Elastic fibres are an essential component of human placental stem villous stroma and an integrated part of the perivascular contractile sheath

The stroma of human placental stem villi is believed to consist only of reticular and collagen fibres. In the present study we were able to show for the first time by light (orcein staining) and electron microscopy large amounts of elastic fibres in the stem villous stroma. Electron microscopically, homogeneous elastin was found alone or in association with microfibrils. In addition, microfibrils were observed forming long bands. These three structures, generally known to form elastic connective tissue, were seen in close connection with placental extravascular smooth muscle cells, which belong to the perivascular contractile sheath (PVCS) of stem villi. Elastin was associated with these smooth muscle cells and connected to collagen fibres via microfibrils. Collagen fibres were additionally interconnected by spike-like structures. Extravascular smooth muscle cells revealed numerous adhesion plaques which occupied conspicuously long cytoplasmic faces of the plasma membrane. In cryostat sections, immunoreactivity of talin, an attachment protein of adhesion plaques linking intracellular α-actin filaments with extracellular fibronectin, was detected in extravascular and vascular (media) smooth muscle cells. The arrangement of placental extravascular smooth muscle cells, elastic and collagen fibres suggests a functional myofibroelastic unit within the PVCS, which surrounds the large foetal blood vessels possibly contributing to elasticity and supporting tensile and/or contracting forces within the stem villi. Received: 2 May 1995 / Accepted: 7 August 1995     

Identification of the nuclear matrix and chromosome scaffold in dinoflagellate Crypthecodinium cohnii~1

Dinoflagellate is one of the primitive eukaryotes,whosenucleus may represent one of the transition stages fromprokaryotic nucleoid to typical eukaryotic nucleus.Usingselective extraction together with embeddment-free sectionand whole mount electron microscopy,a delicate nuclearmatrix filament network was shown,for the first time,indinoflagellate Crypthecodinium cohnii nucleus.Chromosomeresidues are connected with nuclear matrix filaments to forma complete network spreading over the nucleus.Moreover,we demonstrated that the dinoflagellate chromosome retainsa protein scaffold after the depletion of DNA and solubleproteins.This scaffold preserves the characteristic mor-phology of the chromosome.Two dimensional elec-trophoreses indicated that the nuclear matrix and chromo- some scaffold are mainly composed of acidic proteins.Ourresults demonstrated that a framework similar to the nuclearmatrix and chromosome scaffold in mammalian cells appearsin this primitive eukaryote,suggesting that these structuresmay have been originated from the early stages of eukaryoteevolution.     

Filamentous structure of the external surface of abalone eggs revealed by quick-freeze deep-etch technique

The external surface of abalone eggs was examined by thin section and quick-freeze, deep-etch electron microscopy. In thin sections, networks of fine filaments were found interconnecting the adjacent microvilli on the surface of unfertilized eggs. Quick-freeze, deep-etch electron microscopy revealed the three-dimensional structure of these networks of filaments on the external surface of the egg. Mainly two networks of filaments were identified; one was composed of thicker (14–19 nm) filaments interconnecting with the neighboring microvilli nearly horizontally, and the other was composed of thinner (8–14 nm) branched filaments closely surrounding the microvilli surface as well as highly interconnecting neighboring microvilli in a polygonal pattern. The overall structure of the filamentous network on the egg surface showed no distinct alteration after fertilization. These networks of filaments observed on the egg surface may play a key role in sperm–egg interaction.     

La formation des canaux cuticulaires chez l'adulte de Tenebrio molitor L. étude ultrastructurale et remarques histochimiques

The sclerotized cuticle of adult Tenebrio shows (1) an exocuticle composed of rotating lamellate layers and of columns of cuticular material, the fibres of which run perpendicularly through the lamellae, (2) an endocuticle composed of layers with preferred orientation. In the exocuticle, the pore canals are numerous and run along the columns; they do not rotate with the lamellate layers. They show several filaments some of which leave the canals and form a dense intracuticular network. In the last layers of exocuticle, the pericolumnar canals fuse and form large endocuticular canals which rotate in phase with the cuticular fibres. The formation of columns and canals is in relation with cellular expansions which penetrate into the cuticle during cuticle deposition. Exocuticular columns seem characteristic of highly sclerotized cuticles and the intracuticular filaments may have a role in the transport of sclerotisation precursors.     

Pyocyanin Promotes Extracellular DNA Release in Pseudomonas aeruginosa

Bacterial adhesion and biofilm formation are both dependent on the production of extracellular polymeric substances (EPS) mainly composed of polysaccharides, proteins, lipids, and extracellular DNA (eDNA). eDNA promotes biofilm establishment in a wide range of bacterial species. In Pseudomonas aeruginosa eDNA is major component of biofilms and is essential for biofilm formation and stability. In this study we report that production of pyocyanin in P. aeruginosa PAO1 and PA14 batch cultures is responsible for promotion of eDNA release. A phzSH mutant of P. aeruginosa PAO1 that overproduces pyocyanin displayed enhanced hydrogen peroxide (H₂O₂) generation, cell lysis, and eDNA release in comparison to its wildtype strain. A ΔphzA-G mutant of P. aeruginosa PA14 deficient in pyocyanin production generated negligible amounts of H₂O₂ and released less eDNA in comparison to its wildtype counterpart. Exogenous addition of pyocyanin or incubation with H₂O₂ was also shown to promote eDNA release in low pyocyanin producing (PAO1) and pyocynain deficient (PA14) strains. Based on these data and recent findings in the biofilm literature, we propose that the impact of pyocyanin on biofilm formation in P. aeruginosa occurs via eDNA release through H₂O₂ mediated cell lysis.     

Intermediate-sized filaments present in Sertoli cells are of the vimentin type.

The cytoplasmic structure of Sertoli cells of rat testes has been studied by electron microscopy of ultrathin sections. Sertoli cells contain numerous intermediate-sized (7-11 nm) filaments which form a meshwork extending throughout the whole cytoplasm. Often the frequency of such filaments appears especially high in juxtanuclear and cortical regions, including the apical recesses containing the spermatids. Examination of frozen sections of testes by indirect immunofluorescence microscopy using guinea pig antibodies to prekeratin and vimentin has shown the absence of intermediate-sized filaments of the cytokeratin type in all cells of the testes but the presence of filaments of the vimentin type in Sertoli cells as well as in cells of the interstitial space. These results show that the intermediate-sized filaments, abundant in Sertoli cells, are of the vimentin type. In addition we conclude that the "germ epithelium" differs from others true epithelia by the absence of cytokeratin filaments and typical desmosomes and, in Sertoli cells, the presence of vimentin filaments, suggestive of a mesenchymal character or derivation.     

Different patterns of collagen-proteoglycan interaction: a scanning electron microscopy and atomic force microscopy study

The extracellular matrix of unfixed, unstained rat corneal stroma, visualized with high-resolution scanning electron microscopy and atomic force microscopy after minimal preliminary treatment, appears composed of straight, parallel, uniform collagen fibrils regularly spaced by a three-dimensional, irregular network of thin, delicate proteoglycan filaments. Rat tail tendon, observed under identical conditions, appears instead made of heterogeneous, closely packed fibrils interwoven with orthogonal proteoglycan filaments. Pre-treatment with cupromeronic blue just thickens the filaments without affecting their spatial layout. Digestion with chondroitinase ABC rids the tendon matrix of all its interconnecting filaments while the corneal stroma architecture remains virtually unaffected, its fibrils always being separated by an evident interfibrillar spacing which is never observed in tendon. Our observations indicate that matrix proteoglycans are responsible for both the highly regular interfibrillar spacing which is distinctive of corneal stroma, and the strong interfibrillar binding observed in tendon. These opposite interaction patterns appear to be distinctive of different proteoglycan species. The molecular details of proteoglycan interactions are still incompletely understood and are the subject of ongoing research.     

Electron Microscopic Studies on the Intra-flagellar Structures of Trypanosomes*

SYNOPSIS. The intraflagellar structure (IFS) of the flagella of Trypanosoma brucei was examined on the basis of ultrathin sections in various planes. The IFS is composed of filaments approximately 50 A thick. These filaments seem to be identical with the protofilaments found earlier to be the basic elements of the contractile flagellar fibrils. The fibrillar system is firmly connected with the IFS and the latter is attached to the flagellar membrane by filaments. The lattice-like appearance of the IFS is caused by longitudinal and oblique filaments running in different planes. The structure of this network is discussed in detail. The IFS may serve as an abutment for the contractile flagellar fibrils.     

Hydrogen peroxide-dependent DNA release and transfer of antibiotic resistance genes in Streptococcus gordonii

Certain oral streptococci produce H(2)O(2) under aerobic growth conditions to inhibit competing species like Streptococcus mutans. Additionally, H(2)O(2) production causes the release of extracellular DNA (eDNA). eDNA can participate in several important functions: biofilm formation and cell-cell aggregation are supported by eDNA, while eDNA can serve as a nutrient and as an antimicrobial agent by chelating essential cations. eDNA contains DNA fragments of a size that has the potential to transfer genomic information. By using Streptococcus gordonii as a model organism for streptococcal H(2)O(2) production, H(2)O(2)-dependent eDNA release was further investigated. Under defined growth conditions, the eDNA release process was shown to be entirely dependent on H(2)O(2). Chromosomal DNA damage seems to be the intrinsic signal for the release, although only actively growing cells were proficient eDNA donors. Interestingly, the process of eDNA production was found to be coupled with the induction of the S. gordonii natural competence system. Consequently, the production of H(2)O(2) triggered the transfer of antibiotic resistance genes. These results suggest that H(2)O(2) is potentially much more than a simple toxic metabolic by-product; rather, its production could serve as an important environmental signal that facilitates species evolution by transfer of genetic information and an increase in the mutation rate.     

Changes in three-dimensional architecture of microfilaments in cultured vascular smooth muscle cells during phenotypic modulation

To investigate changes in the three-dimensional microfilament architecture of vascular smooth muscle cells (SMC) during the process of phenotypic modulation, rabbit aortic SMCs cultured under different conditions and at different time points were either labelled with fluorescein-conjugated probes to cytoskeletal and contractile proteins for observation by confocal laser scanning microscopy, or extracted with Triton X-100 for scanning electron microscopy. Densely seeded SMCs in primary culture, which maintain a contractile phenotype, display prominent linear myofilament bundles (stress fibres) that are present throughout the cytoplasm with alpha-actin filaments predominant in the central part and beta-actin filaments in the periphery of the cell. Intermediate filaments form a meshed network interconnecting the stress fibres and linking directly to the nucleus. Moderately and sparsely seeded SMCs, which modulate toward the synthetic phenotype during the first 5 days of culture, undergo a gradual redistribution of intermediate filaments from the perinuclear region toward the peripheral cytoplasm and a partial disassembly of stress fibres in the central part of the upper cortex of the cytoplasm, with an obvious decrease in alpha-actin and myosin staining. These changes are reversed in moderately seeded SMCs by day 8 of culture when they have reached confluence. The results reveal two changes in microfilament architecture in SMCs as they undergo a change in phenotype: the redistribution of intermediate filaments probably due to an increase in synthetic organelles in the perinuclear area, and the partial disassembly of stress fibres which may reflect a degradation of contractile components.     

Evidence of compositional differences between the extracellular and intracellular DNA of a granular sludge biofilm

Aims: Extracellular polymeric substances (EPS) are an important component of microbial biofilms, and it is becoming increasingly apparent that extracellular DNA (eDNA) has a functional role in EPS. This study characterizes the eDNA extracted from the novel activated sludge biofilm process of aerobic granules. Methods and Results: Exposing the sludge to cation exchange resin (CER) was used for the extraction of eDNA and intracellular DNA (iDNA) from aerobic granules. This was optimized for eDNA yield while causing minimal cell lysis. We then compared the DNA composition of these extractions using randomly amplified polymorphic DNA (RAPD) fingerprinting and PCR‐based denaturing gradient‐gel electrophoresis (DGGE). Upon the analysis of the genomic DNA and the 16S rRNA genes, differences were detected between the sludge biofilm eDNA and iDNA. Conclusions: Different bacteria within the biofilm disproportionally release DNA into the EPS matrix of the biofilm. Significance and Impact of the Study: The findings further the idea that eDNA has a functional role in the biofilm state, which is an important conceptual information for industrial application of biofilms.     

MORPHOLOGICAL AND CHEMICAL STUDIES OF COLLAGEN FORMATION : I. The Fine Structure of Guinea Pig Granulomata

This paper describes electron microscopic studies of developing connective tissue in granulomata induced by the subcutaneous injection of carrageenin into guinea pigs. Seven days after injection the granulomata contained many fibroblasts and exhibited rapid production of collagen. The fibroblasts were characterised by an extensively developed endoplasmic reticulum and showed numbers of fine, unstriated filaments in the outer regions of the cytoplasm. The filaments, about 50 A in diameter, tended to lie parallel to and closely adjacent to the cell boundary. The cytoplasmic membrane was frequently ill defined or disrupted, particularly bordering regions in which filaments occurred. In longitudinal sections of extended cell processes, filaments were abundant and, in some instances, the cytoplasmic membrane was barely detectable. In the extracellular space striated collagen fibrils were usually accompanied by filaments, 50 to 100 A in diameter, and these often exhibited the characteristic periodicity of collagen, particularly after intense electron bombardment. Much cellular debris was present in the extracellular space. These observations have led to the suggestion that connective tissue precursors are released from fibroblasts by the disintegration or dissolution of the cytoplasmic membrane and the shedding of cytoplasmic material, as in the apocrine gland cells. In some instances this release may take the form of the elongation from the cell of extended processes; disintegration of the cytoplasmic membrane surrounding these processes then leaves the contents in the extracellular phase.     

Ultrastructural and cytochemical investigations on the functional role of proteinaceous nuclear inclusions (PNIs) in chlorenchyma plant cells

Summary— Detailed investigations on the fine ultrastructural organization of different forms of proteinaceous nuclear inclusions (PNls) in chlorenchyma plant cells suggest they consist of the same elementary subunits. Previous high magnification of TEM micro-graphs had shown that the amorphous type of inclusion (A) was mainly composed of elementary fibrils measuring 3.0–3.5 nm in diameter, with no orderly spatial arrangement. New computer image treatments of electron micrographs allowed us to establish that the 8.0–13.0 nm thick filaments — forming the fibrillar (F), crystalline (C) and lamellar (L) inclusions — consist of two elementary fibrils which are coiled in a helix with variable pitch, depending on the type of inclusion. A further secondary coiling of two filaments, about 8.0–9.0 nm in diameter, gives the 20.0–25.0 nm thick tubules which form the characteristic tubular inclusion (T). Correlating the distributive data of PNIs with observations on their ultrastructural morphology and with micrographs of partial aggregation or disgregation patterns of the inclusions, led to the hypothesis that the different forms are not different classes of proteins, but simply different stages of structural complexity of the same protein. To determine whether the intranuclear inclusion protein is nucleolar or nucleolus-associated, cytochemical and immunocytochemical tests were performed on ultrathin sections or leaf lamina tissue in block. These techniques proved that PNIs do not belong to the class of argyrophilic proteins (AgNOR-proteins), and particularly not nucleolin and fibrillarin, two of the major nucleolar proteins. Structural similarities to other plant inclusions, especially P-proteins, and to animal and plant intermediate cytoskeletal filaments (IFs) are discussed with regard to the functional role of PNIs.     

冬凌草甲素对金黄色葡萄球菌生物膜的抑制机制

【背景】金黄色葡萄球菌是一种常见的食源性致病菌,易在食品及加工器具表面形成生物膜,引起食品腐败和疾病的传播,威胁食品安全。【目的】研究冬凌草甲素抑制金黄色葡萄球菌生物膜形成的作用机制。【方法】使用结晶紫染色法和扫描电镜观察冬凌草甲素对金黄色葡萄球菌生物膜形成的抑制作用,刚果红平板法定性检测冬凌草甲素对细胞间多糖黏附素(polysaccharideintercellular adhesion,PIA)合成的影响,分光光度法测定冬凌草甲素对供试菌株胞外DNA (eDNA)释放量的影响,RT-PCR技术检测冬凌草甲素对供试菌株ica A、cid A、agr A和sar A基因表达量的影响。【结果】冬凌草甲素对金黄色葡萄球菌生物膜形成有较强的抑制作用;冬凌草甲素能显著抑制PIA的合成,且呈浓度剂量依赖;冬凌草甲素能抑制供试菌株e DNA的释放量,其中1/4最小抑菌浓度(minimum inhibitory concentration,MIC)的冬凌草甲素作用金黄色葡萄球菌16 h后,与对照组相比,e DNA的释放量降低了48.62%;冬凌草甲素可显著抑制金黄色葡萄球菌生物膜形成相关基因的表达,其中1/2MIC的冬凌草甲素作用金黄色葡萄球菌16 h后,ica A、cid A、agr A和sar A基因的表达量分别比对照降低了91.6%、94.7%、77.6%和70.4%。【结论】冬凌草甲素通过抑制ica A和cid A基因的表达,影响PIA的合成和eDNA的释放,进而干预生物膜的形成。     

Paracrystalline inclusions of nutritive cells of galls induced by Diplolepis rosae L. on Rosa canina L

Numerous crystalline inclusions are found in the nutritive cells lining the larval cavities of the galls induced by Diplolepis rosae L. on Rosa canina L. Electron microscopic investigations show that these intracytoplasmic inclusions consist of staggered, closely parallel, and slightly wavy filaments. In each filament three fibrils can be distinguished inside a less electron-dense matrix. The different aspects that the filaments may present according to the section plans are studied with an electron microscope equipped with a goniometric stage. Enzymatic digestion of ultrathin sections show that the paracrystals are essentially composed of proteins. This conclusion is sustained by the results of an ultrastructural autoradiographic study using tritiated aminoacids. The physiological significance of these paracrystals is discussed: their presence is probably related to a larval action which stimulates proteosynthesis in the cells surrounding the consumed ones.     

A dual role of extracellular DNA during biofilm formation of Neisseria meningitidis

Major pathogenic clonal complexes (cc) of Neisseria meningitidis differ substantially in their point prevalence among healthy carriers. We show that frequently carried pathogenic cc (e.g. sequence type ST‐41/44 cc and ST‐32 cc) depend on extracellular DNA (eDNA) to initiate in vitro biofilm formation, whereas biofilm formation of cc with low point prevalence (ST‐8 cc and ST‐11 cc) was eDNA‐independent. For initial biofilm formation, a ST‐32 cc type strain, but not a ST‐11 type strain, utilized eDNA. The release of eDNA was mediated by lytic transglycosylase and cytoplasmic N‐acetylmuramyl‐l ‐alanine amidase genes. In late biofilms, outer membrane phospholipase A‐dependent autolysis, which was observed in most cc, but not in ST‐8 and ST‐11 strains, was required for shear force resistance of microcolonies. Taken together, N. meningitidis evolved two different biofilm formation strategies, an eDNA‐dependent one yielding shear force resistant microcolonies, and an eDNA‐independent one. Based on the experimental findings and previous epidemiological observations, we hypothesize that most meningococcal cc display a settler phenotype, which is eDNA‐dependent and results in a stable interaction with the host. On the contrary, spreaders (ST‐11 and ST‐8 cc) are unable to use eDNA for biofilm formation and might compensate for poor colonization properties by high transmission rates.