Transmission electron microscopy studies of Moraxella (Branhamella) catarrhalis

A trypsin-sensitive 200-kDa protein has been reported to be exclusively associated with haemagglutinating isolates of Moraxella (Branhamella) catarrhalis. Transmission electron microscopy studies revealed that haemagglutination by M. catarrhalis to both human and rabbit erythrocytes was mediated by a trypsin-sensitive outer fibrillar coat. This fibrillar layer was absent on non-haemagglutinating isolates examined. Immuno-electron microscopy, using a polyclonal antiserum containing antibodies to the 200-kDa protein as a probe, showed that the 200-kDa protein is present on the outer fibrillar layer of the bacterium. These findings suggest that the haemagglutinin of M. catarrhalis is a 200-kDa protein present on the outer fibrillar coat.     

Moraxella (Branhamella) catarrhalis Adherence to Human Bronchial and Oropharyngeal Cells: The Role of Adherence in Lower Respiratory Tract Infections

To study the role of Moraxella (subgenus Branhamella) catarrhalis (B. catarrhalis) adherence to airway cells in lower respiratory tract infections, the in vitro attachments of B. catarrhalis to upper airway (oropharyngeal) and lower airway (bronchial) epithelial cells were compared. The adherence of 4 strains (1 nonfimbriated and 3 fimbriated) of B. catarrhalis to respiratory tract epithelial cells collected from 11 patients with chronic pulmonary disease (CPD) and 11 healthy individuals was evaluated. Both the fimbriated and nonfimbriated strains showed increased attachment to oropharyngeal cells in the CPD patients (mean ± SEM; 25.0 ± 3.2/cell; P < 0.01) when compared to the control subjects (12.1 ± 1.1/cell). On the average, the attachment to bronchial cells was 6.1 to 13.6 times greater per surface area (bacteria/μ²) than the attachment to oropharyngeal cells. The fimbriated strains tended to adhere in higher numbers to bronchial cells (19.0 ± 1.8/cell) than the nonfimbriated strain (8.7 ± 1.2/cell), although there was no difference between the CPD and control groups. In conclusion, the attachment of B. catarrhalis to oropharyngeal cells may be an enhancing factor for colonization in the upper respiratory tract in patients with CPD, and elevated adherence of the bacteria to bronchial cells may suggest pathogenic importance when mucociliary function is impaired.     

Complement resistance is a virulence factor of Branhamella (Moraxella) catarrhalis

Abstract The purpose of this study was to investigate complement resistance in Branhamella (Moraxella) catarrhalis isolated from healthy schoolchildren or sputum-producing adult patients. Two techniques were used: a serum bactericidal assay as the gold standard and an easier ‘culture and spot’ test. Children (age 4–13; n = 303) and patients ( n = 1047) showed high colonization/infection rates with B. catarrhalis (31% and 19%, respectively). Complement resistance or intermediate sensitivity occurred frequently in patient isolates (62% and 27%, respectively) and less often in children (33% and 8.5%, respectively; P ⪡ 0.0001). In young children (age 4–5 years), the proportion of complement-resistant strains was around 50%. Complement resistance in B. catarrhalis is associated with illness and may hence be considered a virulence factor.     

Moraxella (Branhamella) catarrhalis: Restriction enzyme analysis typing with HinfI, HaeIII and PstI

Abstract Restriction enzyme analysis typing with Hin fI, Hae III and Pst I was performed on Moraxella (Branhamella) catarrhalis strains consecutively collected from children suspected of respiratory tract infection and the type strain. Use of Hin fI gave the most distinct patterns. Great polymorphism was seen among strains.     

Restriction Fragment Length Polymorphism (RFLP) of Genomic DNA of Moraxella (Branhamella) catarrhalis Isolates in a Hospital

Epidemiological typing, based on restriction fragment length polymorphism (RFLP) by pulsed-field gel electrophoresis (PFGE), was attempted for the 38 clinical isolates of Moraxella catarrhalis obtained at Shinshu University Hospital during the years 1987 and 1993. Digestion with SmaI or NotI generated well separable, 12 to 5 genomic DNA fragments ranging from 1,000 kb to 30 kb and the strains could be classified into 14 or 13 types, respectively. The electrophoretic profile differed with the strain in most of them and was hence useful to distinguish the each strain. Investigation for their RFLP have, however, suggested that majority of them, including the type strain ATCC25238, may have derived from a common ancestor.     

Construction of a Combined NotI/SmaI Physical and Genetic Map of Moraxella (Branhamella) catarrhalis Strain ATCC25238

Using pulsed field gel electrophoresis (PFGE) and Southern hybridization techniques, a physical map of Moraxella catarrhalis strain ATCC25238 was constructed to provide basic genetic knowledge of this bacterium that has attracted attention in recent years as a human pathogen. Restriction endonuclease NotI cut the genome into 10 fragments and SmaI into 9, and the molecular size of the genome was estimated to be 1,940 kilobases. Location of the 12 genes participating in the biosynthesis of purine, pyrimidine and nine kinds of amino acids were determined on the circular physical map of the strain.     

A Hammondia-like parasite from the European fox (Vulpes vulpes) forms biologically viable tissue cysts in cell culture

Tissue cysts of parasites of the genus Hammondia are rarely described in naturally or experimentally infected intermediate hosts. However, ultrastructural examinations on tissue cyst stages of Hammondia sp. are needed, e.g. to compare these stages with those of Neospora caninum and other related parasites. We describe a cell culture system employed to examine the in vitro development of tissue cysts of a Hammondia sp.-like parasite (isolate FOX 2000/1) which uses the European fox as a definitive host. Cells of a diploid finite cell line from embryonal bovine heart (KH-R; CCLV, RIE 090) were infected by inoculation of sporozoites und cultivated for up to 3 months. Transmission electron microscopic examination of 17 day old cell culture material revealed the presence of cyst walls. Infected cell cultures cultivated for 2 months were used to feed a fox. Six to 13 days post infection the fox shed large numbers (n=1.2 x 10(7)) of Hammondia-sp. like oocysts which could not be distinguished from those used to infect the cell culture as determined by DNA sequencing of the internal transcribed spacer 1 and the D2/D3 domain of the large subunit ribosomal DNA. To find out the proportion of parasitophorous vacuoles that had developed into tissue cysts, the expression of bradyzoite markers was examined by probing infected cell cultures with mouse polyclonal antibodies against Toxoplasma gondii bradyzoite antigen 1 (anti-BAG1) and rat monoclonal antibodies against a cyst wall protein (mAbCC2). Nineteen and 90 days post infection all parasitophorous vacuoles in the cell cultures were positive with anti-BAG1 and mAbCC2. This shows that biologically viable (i.e. infectious) tissue cysts of a fox-derived Hammondia sp. isolate (FOX 2000/1) can be efficiently produced in this cell culture system. Since in vitro cystogenesis of dog-derived Hammondia heydorni has not been observed yet, in vitro cyst formation might be one trait to separate fox-derived Hammondia sp. from H. heydorni on a species level.     

The sperm ultrastructure of Caurinus dectes Russell (Mecoptera: Boreidae) and its phylogenetic implications

The sperm structure of the enigmatic mecopteran species Caurinus dectes (Boreidae) is described for the first time. Diagnostic features are the bi-layered acrosome, a cylindric nucleus provided with two longitudinal opposite grooves, and a simple 9 + 2 axoneme which degenerates in the posterior tail end. The results are conform with the monophyly of Mecoptera including Boreidae. A possible autapomorphy of the order is the presence of the two longitudinal opposite grooves along the nucleus, and the presence of two electron-dense fibres beneath the axoneme. Some apparently plesiomorphic features are preserved in the sperm of Caurinus. Features characterizing the distal part of the flagellum, including the presence of an axial cylindrical structure and the distinctive type of axoneme degeneration, are potential synapomorphies of Caurinus and Boreus, i.e. autapomorphic traits of Boreidae.     

Morphological and cytochemical studies of the effects of ecdysteroids in a lepidopteran cell line (IAL-PID2)

Summary The Indian meal-moth cell line, IAL-PID2, established from larval wing discs was examined from the 250th to the 300th passages. The cultured cells retain various structural and functional qualities of epidermal cells. Under hormone-free conditions PID2 cells grow as a monolayer of round or spindle-shaped cells. They appear as weakly active epidermal cells. The endoplasmic reticulum and Golgi apparatus are poorly developed and secretory activity is reduced. Culture conditions resulted in considerable cellular expansions, abundance of storage products (glycogen, lipids), and hypertrophy of the lysosomal system. The PID2 cell line retains the ability to respond to ecdysteroids; 20-hydroxyecdysone treatment (2×10^-6 M) triggered morphogenetic and secretory processes. Cells formed pseudoepithelial aggregates interconnected and linked by desmosome-like structures. The hormone-stimulated cells are involved in the biosynthesis of N-acetyl-D-glucosamine-rich glycoproteins. The glycosylation sites were located, by use of WGA-gold particles, on cellular expansions and all along the plasma membrane. The possible significance of these glycoproteins is discussed.     

Comparative morphology of spermatozoa and reproductive systems of zorapteran species from different world regions (Insecta,Zoraptera)

The male and female reproductive apparatus of Zorotypus magnicaudelli (Malaysia), Zorotypus huxleyi (Ecuador) and Zorotypus weidneri (Brazil) were examined and documented in detail. The genital apparatus and sperm of the three species show only minor differences. The testes are larger in Z. magnicaudelli. Z. huxleyi lacks the helical appendage in the accessory glands. A long cuticular flagellum is present in Z. magnicaudelli and in the previously studied Zorotypus caudelli like in several other species, whereas it is absent in Z. weidneri, Z. huxleyi, Zorotypus hubbardi, Zorotypus impolitus and Zorotypus guineensis. Characteristic features of the very similar sperm are the presence of: a) two dense arches above the axoneme; b) a 9 + 9+2 axoneme with detached subtubules A and B of doublets 1 and 6; c) the axonemal end degenerating with enlarging accessory tubules; d) accessory tubules with 17 protofilaments; e) three accessory bodies beneath the axoneme; and f) two mitochondrial derivatives of equal shape. The first characteristic (a) is unknown outside of Zoraptera and possibly autapomorphic. The sperm structure differs distinctly in Z. impolitus and Z. hubbardi, which produce giant sperm and possess a huge spermatheca. The presence of the same sperm type in species either provided with a sclerotized coiled flagellum in males or lacking this structure indicates that a different organization of the genital apparatus does not necessarily affect the sperm structure. The flagellum and its pouch has probably evolved within Zoraptera, but it cannot be excluded that it is a groundplan feature and was reduced several times. The fossil evidence and our findings suggest that distinct modifications in the genital apparatus occurred before the fragmentation of the Gondwanan landmass in the middle Cretaceous.     

The spermatozoon of Mengenilla moldrzyki (Strepsiptera,Mengenillidae): Ultrastructure and phylogenetic considerations

Even though the spermatozoa of several strepsipteran species were described earlier, no data were available for the basal family Mengenillidae. Well-fixed material of the recently described Tunisian species Mengenilla moldrzyki was used for a detailed examination of the sperm ultrastructure. The total length is c. 30 μm. The head region contains a conical acrosome vesicle (0.3-0.35 μm) and an elongated nucleus (7.3 μm) with dense chromatin. Some granular material along with a uniformely dense centriole adjunct and two mitochondrial derivatives are visible at the posterior end of the nucleus. The material of the centriole adjunct does not extend along the flagellum and accessory bodies are absent. The mitochondrial derivatives are elongated structures crossed by a longitudinal crista but lacking parallel transverse cristae and paracrystalline material in the dense matrix. The mitochondrial derivatives gradually reduce their size and end at the most posterior tail region. The flagellar axoneme has a 9 + 9 + 2 pattern and originates beneath the nucleus. In the terminal tail region the axoneme gradually disintegrates. Despite the extreme specialization of the endoparasitc group, strepsipteran spermatozoa are mostly characterized by plesiomorphies. The pattern within the order is largely uniform, but Mengenilla displays several apomorphic features compared to the presumptive strepsipteran groundplan (e.g., absence of crystallizations and cristae in the mitochondrial derivatives). The subdivision of the intertubular material into two compartments with a dense beak-like structure adhering to the tubular wall supports a clade Coleopterida (=Strepsiptera + Coleoptera) + Neuropterida.     

Crystalloid inclusions in the Sertoli cell of the koala,Phascolarctos cinereus (Marsupialia)

Summary Crystalloid inclusions are a common feature in the basal region of Sertoli cells in the koala, Phascolarctos cinereus. Generally located near the nucleus, they are non membrane-bounded, slender rectangular structures composed of tubules which are orientated at right angles to the long axis of the crystalloid and regularly arranged in rows parallel to this long axis. The tubules in adjacent rows are offset from one another at definite angles and extensively interconnected by filaments. Neither the composition nor function of the crystalloids has been determined, but their association with tonofilaments and the presence of ribosomes in the vicinity suggests that they are most likely proteinaceous.The authors would like to thank Mr. D. Harbrow, Mr. S. Brown and Ms. B. Canty for technical assistance; the Queensland National Parks and Wildlife Service and the Victorian Ministry of Conservation (Fisheries and Wildlife Division) for providing permits to work on this protected species; the staff of the Lone Pine Koala Sanctuary, Brisbane, for their assistance in obtaining animals. This work was supported by a grant from the Australian Research Grants Committee, Number DI-77/15525     

An improved method for detecting cytostatic toxin (emetic toxin) of Bacillus cereus and its application to food samples

Abstract We developed an improved HEp-2 cell assay method for the detection of Bacillus cereus toxin, which affects the proliferation of HEp-2 cells. The cytostatic toxin was stable upon exposure to heat, pH 2, pH 11 and trypsin, which suggests it is an emetic. Using the HEp-2 cell assay, we examined the distribution and contamination of B. cereus strains that produced an emetic toxin in various foods. Although there were 228 enterotoxin producers among 310 B. cereus strains obtained from foods, 16 of them produced the cytostatic type (emetic toxin). All of the strains that produced the cytostatic toxin were of the H.1 serotype.     

Ultrastructural details of Sertoli cell junctional complexes in vivo and their modifications in tissue culture

Summary This study was carried out using thin sections, lanthanum tracing, and freeze-cleaving in order to investigate the Sertoli cell junctions of rat testis in vivo and their maintenance in culture. The presence of a new type of membrane specialization has been revealed. This consists of a close association between tight junctions and nexuses. The Sertoli cell contact specializations show a progressive disorganization in vitro correlated with the duration of the period in culture. The relationship between the morphological changes in Sertoli cell junctions and the lack of spermatogenesis in culture is discussed.Research performed under the C.N.R. project Biology of Reproduction     

Morphology of the male reproductive system and sperm ultrastructure of the egg parasitoid Gryon pennsylvanicum (Ashmead) (Hymenoptera,Platygastridae)

Gryon pennsylvanicum is a platygastrid hymenopteran that has lately received increasing attention in Europe due to its possible use in biological control of the conifer seed bug pest Leptoglossus occidentalis. Here the male reproductive system and the spermatogenesis of this species, along with those of Gryon muscaeformis, are examined ultrastructurally for the first time. The male genital system is formed by a pair of testes, each containing only one follicle, a pair of accessory glands and deferent ducts connected to a single ejaculatory duct. All the stages of spermatogenesis are described in detail. Characteristic features of the Gryon spp. sperm, which are 100 μm long, are the presence of a polygonal nucleus, only one mitochondrial derivative, the occurrence of the centriole adjunct and a typical insect 9 + 9 + 2 flagellar axoneme. The single derivative, however, results from a process in which one of the two mitochondria is lost during spermiogenesis. Unlike in other insects, two centrioles occur in spermatids as a consequence of the ameiotic parthenogenesis. These characteristics stand as a valuable tool for phylogenetic inferences. Furthermore this study suggests a useful strategy for laboratory mass rearing.     

A comparative fine structural and phylogenetic analysis of resting cysts in oligotrich and hypotrich Spirotrichea (Ciliophora)

So far, neither morphology nor gene sequences have provided a reliable classification of halteriid and hypotrichid spirotrichs. Thus, we performed a comparative study on the fine structure of the resting cysts in some representative species, viz., the oligotrichs Halteria grandinella and Pelagostrombidium fallax and the oxytrichid hypotrichs Laurentiella strenua, Steinia sphagnicola, and Oxytricha granulifera. Main results include: (i) there are three different, very likely non-homologous cyst surface ornamentations, viz., spines (generated by the ectocyst), thorns (generated by the mesocyst), and lepidosomes (produced in the cytoplasm); (ii) Halteria has a perilemma; (iii) Halteria, Meseres and Pelagostrombidium have fibrous lepidosomes, while those of Oxytricha are tubular; (iv) the cyst wall structure of Pelagostrombidium and Strombidium is distinctly different from that of halteriids and oxytrichids, which are rather similar in this respect; (v) cyst ornamentation does not provide a reliable phylogenetic signal in oxytrichid hypotrichs because ectocyst spines occur in both flexible and rigid genera. The new observations and literature data were used to investigate the phylogeny of the core Spirotrichea. The Hennigian argumentation scheme and computer algorithms showed that the spirotrichs are bound together by the macronuclear reorganization band, the subepiplasmic microtubule basket, and the apokinetal stomatogenesis. The Hypotrichida and Oligotrichida are united by a very strong synapomorphy, viz., the perilemma, not found in any other member of the phylum. Halteriid and oligotrichid spirotrichs form a sister group supported by as many as 13 apomorphies. Thus, the molecular data, which classify the halteriids within the core hypotrichs, need to be reconsidered.     

Micromorphology of Scirpus (Cyperaceae) and related genera in south-west Europe

Glume surface, fruit surface, perianth bristles and pollen morphology in Scirpus L. and related genera from south-west Europe have been investigated by light and scanning electron microscopy. The results of this analysis confirm the heterogeneity of the group and provide further support to the current recognition of the genera Scirpus L., Bolboschoenus (Rchb.) Palla , Scirpoides Ség., Schoenoplectus (Rchb.) Palla, Isolepis R.Br., Trichophorum Pers. and Blysmus Panz. as well as to a re-appraisal of subg. Actaeogeton (Rchb.) Oteng-Yeb. of Schoenoplectus .  © 2004 The Linnean Society of London, Botanical Journal of the Linnean Society , 2004, 145 , 45–58.     

白豆杉细胞培养

紫杉醇是从短叶红豆杉〔Ｔａｘｕｓｂｒｅｖｉｆｏｌｉａ(Ｃｈｅｎｇ)Ｃｈｅｎｇ〕树皮中分离出来的二萜类生物碱[1],现已成为知名抗癌药物。紫杉醇主要来源于红豆杉属植物。白豆杉〔Ｐｓｅｕｄｏｔａｘｕｓｃｈｉｅｎｉｉ(Ｃｈｅｎｇ)Ｃｈｅｎｇ〕属红豆杉科白豆杉属,为...