Phage infection of the obligate intracellular bacterium, Chlamydia psittaci strain guinea pig inclusion conjunctivitis

The infectious cycle of phiCPG1, a bacteriophage that infects the obligate intracellular pathogen, Chlamydia psittaci strain Guinea Pig Inclusion Conjunctivitis, was observed using transmission electron microscopy of phage-hyperinfected, Chlamydia-infected HeLa cells. Phage attachment to extracellular, metabolically dormant, infectious elementary bodies and cointernalisation are demonstrated. Following entry, phage infection takes place as soon as elementary bodies differentiate into metabolically active reticulate bodies. Phage-infected bacteria follow an altered developmental path whereby cell division is inhibited, producing abnormally large reticulate bodies, termed maxi-reticulate bodies, which do not mature to elementary bodies. These forms eventually lyse late in the chlamydial developmental cycle, releasing abundant phage progeny in the inclusion and, upon lysis of the inclusion membrane, into the cytosol of the host cell. Structural integrity of the hyperinfected HeLa cell is markedly compromised at late stages. Released phage particles attach avidly to the outer leaflet of the outer membranes of lysed and unlysed Chlamydiae at different stages of development, suggesting the presence of specific phage receptors in the outer membrane uniformly during the chlamydial developmental cycle. A mechanism for phage infection is proposed, whereby phage gains access to replicating chlamydiae by attaching to the infectious elementary body, subsequently subverting the chlamydial developmental cycle to its own replicative needs. The implications of phage infection in the context of chlamydial infection and disease are discussed.     

Morphology of Halprowia (Chlamydia) isolated in Reiter's syndrome]

Morphology and ultrastructure of Halprowia arthritidis, strain SR-1 (HSR), isolated from the synovial fluid of a patient with Reiter's syndrome, was studied in the membranes of the yolk sacs of the developing chick embryos and the L-cell culture. In acridine orange staining for light and fluorescent microscopy there was revealed intracellular cytoplasmic inclusions containing HSR structures at various stages of its reproduction characteristic of halprowia (chlamydia). The direct immunofluorescent method demonstrated the presence of a characteristic HSR antigen not only in the developed inclusions, but also at the early stages of infection, when the morphological HSR structures could not be found by light microscopy. The ultrastructure of the HSR inclusions and forms in the cycle of development (of the initial and elementary bodies) of the SR-1 strain was typical of other halprowia. A peculiar structure of a complex of cell wall and cytoplasmic membrane of the elementary body was described. Taking into consideration the biological characteristics of HSR revealed earlier it can be considered to be a typical representative of Halprowiales s. Chlamydiales. The data obtained on other halprowia, pointing out the fact that criteria of compactness and diffuseness of inclusions, the presence of absence of glycogen in the inclusions could not serve as taxonomic signs in classification of halrpowia, were confirmed on a model of the SR-1 studied.     

Ultrastructure and Development of Oil Idioblasts in Annona muricata L.

The ultrastructure and development of oil idioblasts in theshoot apex and leaves in Annona muricata L. are described, andthree arbitrary developmental stages are distinguished: cellsin which no additional cell wall layers have been depositedagainst the initial primary cell wall, possessing an electron-translucentcytoplasm and distinct plastids which lack thylakoids (stage1); cells in which a suberized layer has been deposited againstthe primary wall (stage 2, the cytoplasm resembles that of thepreceding stage), and cells in which an additional inner walllayer has been deposited against the suberized layer, whichincreases in thickness with development (stage 3). In this stagean oil cavity is formed, surrounded by the plasmalemma, andattached to a bell-like protrusion of the inner wall layer,the cupule. A complex membranous structure occurs next to thecupule. Smooth tubular endoplasmic reticulum (ER), appearingas linearly arranged tubules, and groups of crystalline bodieswith an almost hexagonal outline are present. The final stagewas further subdivided into three subgroups (a, b, c) basedon the extent of the oil cavity, its contents, and the compositionof the cytoplasm, and increasing thickness of the inner walllayer. The oil is probably synthesized in the plastids, releasedinto the cytoplasm, and then passed through the plasmalemmasurrounding the oil cavity. Oil idioblasts, Annona muricata L., suberized layer, inner wall layer, oil cavity, cupule, smooth tubular ER, crystalline bodies     

Electron Microscopic Observations on the Structure of the Envelopes of Mature Elementary Bodies and Developmental Reticulate Forms of Chlamydia psittaci

Purified suspensions of Chlamydia psittaci were prepared from L cells. Thin sections of intact elementary bodies and intact developmental reticulate bodies and of their purified envelopes were observed by electron microscopy. In both intact organisms and partially purified envelopes, two membranous structures, each appearing in electron micrographs as two darkly stained layers, were observed. In the elementary body sections, the outer membrane was round, apparently rigid, and was not soluble in 0.5% sodium dodecyl sulfate. The inner layer was irregular in shape and was completely removed by detergent treatment. We interpret these results to indicate that the outer rigid layer of the envelope is the cell wall and the inner layer is the cytoplasmic membrane. When the fragile reticulate body envelopes were similarly studied, the outer cell wall was clearly visible, and some evidence of an inner membrane was seen. After treatment with nucleases and detergent, all evidence of inner or cytoplasmic membrane was removed, but the outer cell wall remained. Thus, it appears that the cell wall of this organism is continuous throughout the growth cycle and that the fragility and lack of rigidity of the reticulate body cell is due to changes in chemical composition or structure of the cell wall.     

Ultrastructural analysis of the effects of erythromycin on the morphology and developmental cycle of Chlamydia trachomatis HAR-13

The effect of erythromycin (10 g/ml) on the morphology and developmental cycle of Chlamydia trachomatis HAR-13 was examined by electron microscopy. When the antibiotic was added later than 24 h post infection, the HAR-13 morphology or developmental cycle was not altered. Addition at 18 or 24 h post infection inhibited glycogen production, blocked the transformation of the reticulate body to elementary body, and produced ghost bodies and reticulate bodies twice the diameter of untreated reticulate bodies. When erythromycin was added within 12 h post infection, the conversion of the elementary body to reticulate body was inhibited. Erythromycin (10 g/ml) was bactericidal to strain HAR-13 throughout the developmental cycle.Abbreviations CXM cycloheximide media - IFU inclusion forming units - MEM minimal essential medium     

华北落叶松花粉发育过程中的钙动态分布

运用焦锑酸钾沉淀法研究了华北落叶松(Larix principis-rupprechtii Mayr)小孢子发育过程中不同阶段Ca2 的分布情况.减数分裂时期,小孢子囊壁表皮和中层细胞的细胞壁及细胞间隙Ca2 分布较多,绒毡层只有外切向面的细胞膜有Ca2 分布,小孢子母细胞的各部位则很少有Ca2 ;四分体时期,包围四分小孢子的胼胝质壁上有大量的Ca2 分布,在四分孢子壁上也有较多沉淀;游离小孢子时期,钙离子在小孢子壁的分布较四分体时期有所减少,而到花粉成熟时又逐渐增多;从四分体到花粉成熟,乌氏体周围的Ca2 有增多的趋势.对四分体外壁Ca2 的大量分布与花粉壁的形成及信号物质在花粉表面贮存的关系,以及小孢子囊的外壁、绒毡层和乌氏体在Ca2 向花粉运输中所起的作用进行了讨论.     

Early stages of nodulation in roots of Oxytropis arctobia (Leguminosae) induced by the arctic rhizobial strain N31

Early stages of nodulation of roots of the arctic legume Oxytropis arctobia by the arctic rhizobial strain N31 was characterized under controlled conditions. Root hair deformation occurred as a result of inoculation of seedlings. Infection threads were seen invading target cells in the root cortex of the newly formed nodule tissue. In emergent nodules, bacteria remained surrounded by a moderately electron-dense matrix, within the intercellular space. The target cells were rich in lipid bodies, proplastids, mitochondria and polyribosomes. Fibrillar material, microfilaments and small vesicles were present at the point of bacterial release, where the infection thread was devoid of its wall. Bacteria were found to be encircled by plasmalemma invaginations forming "symbiosomes". Lipid bodies were present near the membrane of the infection thread, close to the site of bacterial release, and in close association with plasmalemma.     

Targeting proteins to the cell wall of sporulating Bacillus anthracis

Dormant spores of Bacillus anthracis germinate during host infection and their vegetative growth and dissemination precipitate anthrax disease. Upon host death, bacilli engage a developmental programme to generate infectious spores within carcasses. Hallmark of sporulation in Bacillus spp. is the formation of an asymmetric division septum between mother cell and forespore compartments. We show here that sortase C (SrtC) cleaves the LPNTA sorting signal of BasH and BasI, thereby targeting both polypeptides to the cell wall of sporulating bacilli. Sortase substrates are initially produced in different cell compartments and at different developmental stages but penultimately decorate the envelope of the maturing spore. srtC mutants appear to display no defect during the initial stages of infection and precipitate lethal anthrax disease in guinea pigs at a similar rate as wild-type B. anthracis strain Ames. Unlike wild-type bacilli, srtC mutants do not readily form spores in guinea pig tissue or sheep blood unless their vegetative forms are exposed to air.     

An immunohistochemical analysis of rat hippocampal antigens in early postnatal ontogeny by using monoclonal antibodies]

In order to study the molecular mechanisms of neurogenesis, monoclonal antibodies (MAbs) were produced against antigens of the developing rat hippocampus. MAb 3G7-F8 was used for immunohistochemical localization of the corresponding antigen of paraffin sections of the rat brain at days 0, 5, 14, and 21 of the postnatal development. In the hippocampus of newborn and 5-day-old rats, positive immunostaining was observed in the cytoplasm and proximal segments of processes of neurons located in granular, polymorph, and pyramidal layers, as well as in entorhinal cortex. In granule cell bodies and neurons of entorhinal cortex specific staining decreased by day 14 and disappeared by day 21 after birth, whereas neurons of pyramidal and polymorph layers remained immunopositive. Diffuse specific staining in the cerebellum was observed beginning from day 5 after birth in the Purkinje cell layer. On days 14-21 positive reaction was observed in Purkinje cell bodies and in the layer containing dendrites of Purkinje cells and parallel fibers. External and internal granular layers remained immunonegative. No specific staining was observed in other regions of the brain, as well as in the control slices. These data suggest that the antigen detected by the 3G7-F8 antibody is involved in the formation of the neuronal connections.     

Human cytomegalovirus proteins PP65 and IEP72 are targeted to distinct compartments in nuclei and nuclear matrices of infected human embryo fibroblasts

The cellular distribution of the human cytomegalovirus (HCMV)-specific UL83 phosphoprotein (pp65) and UL123 immediate-early protein (IEp72) in lytically infected human embryo fibroblasts was studied by means of indirect immunofluorescence and confocal microscopy. Both proteins were found to have a nuclear localization, but they were concentrated in different compartments within the nuclei. The pp65 was located predominantly in the nucleoli; this was already evident with the parental viral protein, which was targeted to the above nuclear compartment very soon after infection. The nucleolar localization of pp65 was also observed at later stages of the HCMV infectious cycle. After chromatin extraction (in the so-called in situ nuclear matrices), a significant portion of the pp65 remained associated with nucleoli within the first hour after infection, then gradually redistributed in a perinucleolar area, as well as throughout the nucleus, with a granular pattern. A quite different distribution was observed for IEp72 at very early stages after infection of human embryo fibroblasts with HCMV; indeed, this viral protein was found in bright foci, clearly observable in both non-extracted nuclei and in nuclear matrices. At later stages of infection, IEp72 became almost homogeneously distributed within the whole nucleus, while the foci increased in size and were more evenly spread; in several infected cells some of them lay within nucleoli. This peculiar nuclear distribution of IEp72 was preserved in nuclear matrices as well. The entire set of data is discussed in terms of the necessity of integration for HCMV-specific products into the pre-existing nuclear architecture, with the possibility of subsequent adaptation of nuclear compartments to fit the needs of the HCMV replicative cycle.     

Distribution of insoluble polysaccharides, neutral lipids, and proteins in the developing anthers of Campsis radicans (L.) Seem. (Bignoniaceae)

In this study, distribution of polysaccharides, lipids, and proteins in the developing anthers of Campsis radicans (L.) Seem. was examined from sporogenous cell stage to mature pollen, using cytochemical methods. To detect the distribution and dynamic changes of insoluble polysaccharides, lipid bodies, and proteins in the anthers through progressive developmental stages, semi-thin sections of anthers at different developmental stages were stained with periodic-acid-Schiff (PAS) reagent, Sudan black B, and Coomassie brilliant blue, respectively, and examined under light microscope. Ultrastructural observations with TEM were also carried out to determine the storage form of starch in the connective tissue, and storage form of lipids in the tapetal cells. In sporogenous cell stage, anther wall contains numerous insoluble polysaccharides. However, from the sporogenous cell stage to the vacuolated microspore stage, the amount of insoluble polysaccharides in the anther wall decreases gradually. At bicellular pollen stage, tapetum degenerates completely and polysaccharides are not seen in the anther wall. Lipid bodies are observed in the cytoplasm of both middle layer and tapetal cells at tetrad stage, whereas they disappear in the vacuolated microspore stage. Compared with polysaccharides, proteins are limited in the anther wall at early stages of development. During pollen development, polysaccharides, proteins, and lipid bodies are scarce in the cytoplasm of sporogenous cells, but their amount increases at premeiotic stage. From tetrad stage to bicellular pollen stage, microspore cytoplasm contains variable amount of insoluble polysaccharide grains, lipid and protein bodies. At bicellular pollen stage, plentiful amount of starch granules are stored in the cytoplasm of the pollen grains. Proteins and lipid bodies are also present in the cytoplasm.     

Association of Experimental Chronic Arthritis with the Persistence of Group A Streptococcal Cell Walls in the Articular Tissue

A single injection of isolated fragments of group A streptococcal cell walls into the joints of rabbits stimulated an initial acute reaction which was followed by a prolonged inflammatory process. The chronic process was characterized by hyperplasia of the synovial cells, diffuse infiltration of the villi by macrophages, and focal collections of lymphocytes in the stroma of the villi. These histological changes were similar to those seen in the early stages of rheumatoid arthritis. Antibodies specific for the mucopeptide and group-specific C polysaccharide antigens of group A streptococcal cell walls were labeled with either fluorescein or (125)I, and were used to demonstrate antigen in the synovial tissues. The antigens persisted within macro-phages for at least 5 weeks. Their presence correlated with the evolution of the chronic inflammatory process.     

In vitro and in vivo functional activity of Chlamydia MurA,a UDP-N-acetylglucosamine enolpyruvyl transferase involved in peptidoglycan synthesis and fosfomycin resistance

Organisms of Chlamydia spp. are obligate intracellular, gram-negative bacteria with a dimorphic developmental cycle that takes place entirely within a membrane-bound vacuole termed an inclusion. The chlamydial anomaly refers to the fact that cell wall-active antibiotics inhibit Chlamydia growth and peptidoglycan (PG) synthesis genes are present in the genome, yet there is no biochemical evidence for synthesis of PG. In this work, we undertook a genetics-based approach to reevaluate the chlamydial anomaly by characterizing MurA, a UDP-N-acetylglucosamine enolpyruvyl transferase that catalyzes the first committed step of PG synthesis. The murA gene from Chlamydia trachomatis serovar L2 was cloned and placed under the control of the arabinose-inducible, glucose-repressible ara promoter and transformed into Escherichia coli. After transduction of a lethal DeltamurA mutation into the strain, viability of the E. coli strain became dependent upon expression of the C. trachomatis murA. DNA sequence analysis of murA from C. trachomatis predicted a cysteine-to-aspartate change in a key residue within the active site of MurA. In E. coli, the same mutation has previously been shown to cause resistance to fosfomycin, a potent antibiotic that specifically targets MurA. In vitro activity of the chlamydial MurA was resistant to high levels of fosfomycin. Growth of C. trachomatis was also resistant to fosfomycin. Moreover, fosfomycin resistance was imparted to the E. coli strain expressing the chlamydial murA. Conversion of C. trachomatis elementary bodies to reticulate bodies and cell division are correlated with expression of murA mRNA. mRNA from murB, the second enzymatic reaction in the PG pathway, was also detected during C. trachomatis infection. Our findings, as well as work from other groups, suggest that a functional PG pathway exists in Chlamydia spp. We propose that chlamydial PG is essential for progression through the developmental cycle as well as for cell division. Elucidating the existence of PG in Chlamydia spp. is of significance for the development of novel antibiotics targeting the chlamydial cell wall.     

An ultrastructural comparison of the cell envelopes of selected strains of Nocardia asteroides and Nocardia brasiliensis

Growth curves were determined for three strains each ofNocardia asteroides andNocardia brasiliensis. Two strains ofN. brasiliensis and one strain ofN. asteroides had longer lag periods of growth than the remaining three strains. All strains had generation times of approximately 5.5 hours.The ultrastructure of the cell envelope of eachNocardia strain in early stationary phase growth was also examined. All the strains had typical trilaminar cell walls and cell membranes. The thickness of the cell wall layers, especially the inner peptidoglycan layer, varied from strain to strain. The inner layer of two strains ofN. brasiliensis and one strain ofN. asteroides was 12 nm or more in thickness, while that of the remaining three strains was 7 nm thick. These observed differences in growth patterns and/or thickness of the cell wall layers could be correlated to the varying degress of virulence as well as the divergent pathologies exhibited by these organisms.     

草鱼呼肠孤病毒在CIK细胞中复制及形态发生的研究

以草鱼呼肠孤病毒（ＧＣＲＶ）感染的草鱼肾细胞系（ＣＩＫ）为模型,进行了草鱼呼肠孤病毒在细胞内的形态发生的研究。当病毒以感染复数为５￣１０ＰＦＵ／ＣＥＬＬ感染ＣＩＫ细胞时,在病毒感染细胞４ｈ以内的切片中,可观察到脱去部分外层衣壳的不完整病毒颗粒。感染细胞８ｈ,可观察到浆胞内病毒发生基质,其内含有大量的直径约５０ｎｍ的亚病毒颗粒,无外层蛋白结构。感染１２￣１６ｈ后,这些亚病毒颗粒装配上外层蛋白结构,形     

A comparison between osmiophilic reagents and the Gomori lead method for the electron cytochemical demonstration of two lysosomal enzymes in oral epithelium

Synopsis Osmiophilic reagents were used to study the histochemical localization of acid phosphatase and non-specific esterase in the keratinized oral mucosa of rat. The reaction product from both enzymes was found in the epithelium and in cells of the corium as discrete granules, suggestive of a lysosomal localization. Treatment with E-600 before incubation for non-specific esterase did not change this localization. The osmium black end-product, due to acid phosphatase activity, was examined with the electron microscope and compared with the localization obtained by the Gomori lead phosphate technique. Both methods produced a reaction product in membrane-bounded bodies resembling lysosomes, as described in other tissues. These organelles were present in the basal prickle and granular cell layers of the epithelium. In the keratinized layer the reaction product was localized between the cell membranes of the deeper cells and no deposits were present in the cells. It is suggested that the osmiophilic reagents provide a good alternative to the Gomori method for the localization of lysosomal acid phosphatase at both the light and electron microscope levels.     

Characteristics of the cytodifferentiation of omega-particle symbiotic bacteria from the micronucleus of Paramecium caudata clone M1-48]

The structure of the omega-particle-bacteria, growing in the micronucleus of Paramecium caudatum (Ciliata, Protozoa), was studied by electrom microscopy in the course of their life cycle. The cytoplasm of the spindle-shaped vegetative cells contains a large number of dense particles and transparent regions comprising the fibrillar material. Such cells, via several intermediate stages, are transformed into elongated twisted cells that are regarded as spores. The spore consists of two parts: homogeneous, and that containing the membrane system and rounded light bodies. The membranes are often double and connected with the fibrils. The cell wall is constructed, during all stages, of the outer membrane layer and the inner electron-dense layer.     

The determination of the location of the antigens of the causative agent of melioidosis at the ultrastructural level by using monoclonal antibodies]

The indirect immunoperoxidase method with the use of monoclonal antibodies has been employed to determine the localization of antigens of melioidosis agents. The formed reaction products were then detected by the electron microscopic analysis. The most important in melioidosis pathogenesis antigens 6 and 8 (AG 6 and AG 8) were determined to localize on the cell wall and in the capsule-like mucous layer, respectively. Monoclonal antibodies to AG 8 due to their marked cross-linking properties promote stabilization, preservation and detection of labile melioidosis agent. At the same time the mucous layer prevents monoclonal antibodies to AG 6 to access to the corresponding antigen, localized on the cell wall and makes its detection difficult.     

Structural Changes Associated with Resistance of Soybean to Heterodera glycines

Subcellular responses to infection by Race 3 of Heterodera glycines in susceptible (''Lee'') and resistant (''Forrest'' and ''Bedford'') soybean cultivars were compared. Syncytial formation, initiated in susceptible as well as resistant soybean cultivars, was characterized by wall perforations, dense cytoplasm, and increased endoplasmic reticulum, In susceptible plants, syncytia developed continuously until nematode maturity. This included hypertrophy of nuclei, increase of rough endoplasmic reticulum in early stages of infection, and formation of wall ingrowths at a late stage of infection. In the resistant reaction in Forrest, a necrotic layer surrounded syncytium component cells demarcating them from surrounding normal cells and leading to syncytial necrosis. Wall appositions were prominently formed near the necrotic layer, and the cytoplasm of the syncytium component cells was extremely condensed. The whole syncytium became necrotic at a late stage of infection. Bedford had nuclear degeneration prior to cytoplasmic degradation. Chromatin was often scattered throughout the syncytial cytoplasm. Finally the whole syncytium became degenerated with plasmalemma completely detached from the syncytial cell walls. The differences in resistant responses reflect a difference in genetic composition of the soybean cultivars tested.     

ULTRASTRUCTURE OF CARPOSPOROGENESIS IN THE PARASITIC RED ALGA FAUCHEOCOLAX ATTENUATA SETCH. (RHODYMENIALES,RHODYMENIACEAE)

Carpospore differentiation in Faucheocolax attenuata Setch. can be separated into three developmental stages. Immediately after cleaving from the multinucleate gonimoblast cell, young carpospores are embedded within confluent mucilage produced by gonimoblast cells. These carpospores contain a large nucleus, few starch grains, concentric lamellae, as well as proplastids with a peripheral thylakoid and occasionally some internal (photosynthetic) thylakoids. Proplastids also contain concentric lamellar bodies. Mucilage with a reticulate fibrous substructure is formed within cytoplasmic concentric membranes, thus giving rise to mucilage sacs. Subsequently, these mucilage sacs release their contents, forming an initial reticulate deposition of carpospore wall material. Dictyosome vesicles with large, single dark-staining granules also contribute to wall formation and may create a separating layer between the mucilage and carpospore wall. During the latter stages of young carpospores, starch is polymerized in the perinuclear cytoplasmic area and is in close contact with endoplasmic reticulum. Intermediate-aged carpospores continue their starch polymerization. Dictyosomes deposit more wall material, in addition to forming fibrous vacuoles. Proplastids form thylakoids from concentric lamellar bodies. Mature carpospores are surrounded by a two-layered carpospore wall. Cytoplasmic constituents include large floridean starch granules, peripheral fibrous vacuoles, mature chloroplasts and curved dictyosomes that produce cored vesicles which in turn are transformed into adhesive vesicles. Pit connections remain intact between carpospores but begin to degenerate. This degeneration appears to be mediated by microtubules.