Search

The partial 16S rRNA, rpoB, and cpn60 genes congruently allow this study to identify all the eight isolates as the species Campylobacter showae. To our knowledge, this is the first report to reveal the interspecies and intraspecies sequence variations present in the three genes of the C. showae isolates.

We set up a polarized cell culture model to study the pathogenicity of a common respiratory tract pathogen, Chlamydia pneumoniae . Immunofluorescence staining of ZO-1 (a tight junction protein) and Na + K + ATPase (a protein pump localized at the basolateral membrane in the polarized epithelial cells), as well as TER measurements, suggested that the filter-grown Calu-3 cells, but not the A549 cells, were polarized when grown on collagen-coated membranes. Both the flat and the filter-grown cultures were infected with C. pneumoniae . Infection in the polarized Calu-3 cultures produced more C. pneumoniae genome equivalents than infection in the flat cultures. However, this progeny was not as infective as that in the flat cultures. The maximum amount of C. pneumoniae was detected at 6 days postinfection in the filter-grown A549 cells, indicating a slower developmental cycle than that observed in the flat A549 cultures. The effect of cycloheximide on the growth of C. pneumoniae in the polarized cells was negligible. Furthermore, the infection in the polarized Calu-3 cells was resistant to doxycycline, and several cytokines were released mainly on the apical side of the polarized cells in response to C. pneumoniae infection. These findings indicate that the growth of chlamydiae was altered in the filter-grown epithelial culture system. The diminished production of infective progeny of C. pneumoniae , together with the resistance to doxycycline and polarized secretion of cytokines from the infected Calu-3 cells, suggests that this model is useful for examining epithelial cell responses to C. pneumoniae infection, and it might better resemble in vivo infection in respiratory epithelial cells.

The aim of the present work was to explore possibilities of photocatalytic TiO2 coating for reducing biofilms on non-living surfaces. The model organism, Deinococcus geothermalis, known to initiate growth of durable, colored biofilms on machine surfaces in the paper industry, was allowed to form biofilms on stainless steel, glass and TiO2 film coated glass or titanium. Field emission electron microscopy revealed that the cells in the biofilm formed at 45 degrees C under vigorous shaking were connected to the surface by means of numerous adhesion threads of 0.1-0.3 microm in length. Adjacent cells were connected to one another by threads of 0.5-1 microm in length. An ultrastructural analysis gave no indication for the involvement of amorphous extracellular materials (e.g., slime) in the biofilm. When biofilms on photocatalytic TiO2 surfaces, submerged in water, were exposed to 20 W h m(-2) of 360 nm light, both kinds of adhesion threads were completely destroyed and the D. geothermalis cells were extensively removed (from10(7) down to below 10(6) cells cm(-2)). TiO2 films prepared by the sol-gel technique were slightly more effective than those prepared by the ALD technique. Doping of the TiO2 with sulfur did not enhance its biofilm-destroying capacity. The results show that photocatalytic TiO2 surfaces have potential as a self-cleaning technology for warm water using industries.

Entacapone and tolcapone, novel catechol-O-methyl-transferase (COMT) inhibitors, have been developed for the treatment of Parkinson's disease in combination with levodopa. Three fatal cases of drug-induced hepatitis, one with hepatic necrosis and mitochondrial changes have been reported in clinical use of tolcapone. In vitro tolcapone has been shown to induce uncoupling of oxidative phosphorylation. Liver and skeletal muscle tissues from an oral rat toxicity study were used to investigate the influence of entacapone, tolcapone (300 and 500 mg/kg/day) or a known uncoupling agent, 2,4-dinitrophenol (DNP), (20 mg/kg/ day) on the cell morphology. Centrolobular hypertrophy was revealed in the histopathology of the liver in tolcapone-treated rats. Transmission electron microscopy (TEM) of the liver and skeletal muscle tissue, revealed mitochondrial swelling and reduced matrix density with deformation of cristae in the tolcapone and DNP groups. Intermyofibrillar edema was characteristic of the skeletal muscle tissue of DNP- and tolcapone-exposed animals. In the tolcapone group, also the sarcomeres were prominent. Treatment-related light microscopic or TEM findings were not observed either in entacapone-treated or control animals. The similarity of structural damages induced by both tolcapone- and DNP suggests that uncoupling of oxidative phosphorylation may contribute to the toxicity of tolcapone in the rat.

The radiation sensitivity of four strains of Bacillus cereus was investigated with attention to bacterial surface structure. All four strains were sensitive to radiation with gamma rays (D(10)=0.4 kGy). No crystalline surface protein layer could be detected on the cell surface. When cultured on solid media, an S-layer covered the cells of the two strains, and they were 2.6 times as resistant to radiation as the two reference strains without an S-layer. In SDS-PAGE, a major 97-kDa band from the resistant strains from plate cultures was replaced by a ca. 85-kDa protein band in samples from broth cultures. Electron microscopy, SDS-PAGE, Western blot and fluorescent antibody staining indicated that the higher resistance to radiation of the clinical strains from plate cultures was associated with the presence of the S-layer on the cell surface.

Nonopsonic phagocytosis of Bacillus cereus by human polymorphonuclear leukocytes (PMNs) with particular attention to bacterial surface properties and structure was studied. Two reference strains (ATCC 14579 T and ATCC 4342) and two clinical isolates (OH599 and OH600) from periodontal and endodontic infections were assessed for adherence to matrix proteins, such as type I collagen, fibronectin, laminin, and fibrinogen. One-day-old cultures of strains OH599 and OH600 were readily ingested by PMNs in the absence of opsonins, while cells from 6-day-old cultures were resistant. Both young and old cultures of the reference strains of B. cereus were resistant to PMN ingestion. Preincubation of PMNs with the phagocytosis-resistant strains of B. cereus did not affect the phagocytosis of the sensitive strain. Negatively stained cells of OH599 and OH600 studied by electron microscopy had a crystalline protein layer on the cell surface. In thin-sectioned cells of older cultures (3 to 6 days old), the S-layer was observed to peel off from the cells. No S-layer was detected on the reference strains. Extraction of cells with detergent followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a major 97-kDa protein from the strains OH599 and OH600 but only a weak 97-kDa band from the reference strain ATCC 4342. One-day-old cultures of the clinical strains (hydrophobicity, 5.9 to 6.0%) showed strong binding to type I collagen, laminin, and fibronectin. In contrast, reference strains (hydrophobicity, −1.0 to 4.2%) as well as 6-day-old cultures of clinical strains (hydrophobicity, 19.0 to 53.0%) bound in only low numbers to the proteins. Gold-labelled biotinylated fibronectin was localized on the S-layer on the cell surface as well as on fragments of S-layer peeling off the cells of a 6-day-old culture of B. cereus OH599. Lactose, fibronectin, laminin, and antibodies against the S-protein reduced binding to laminin but not to fibronectin. Heating the cells at 84°C totally abolished binding to both proteins. Benzamidine, a noncompetitive serine protease inhibitor, strongly inhibited binding to fibronectin whereas binding to laminin was increased. Overall, the results indicate that changes in the surface structure, evidently involving the S-layer, during growth of the clinical strains of B. cereus cause a shift from susceptibility to PMN ingestion and strong binding to matrix and basement membrane proteins. Furthermore, it seems that binding to laminin is mediated by the S-protein while binding to fibronectin is dependent on active protease evidently attached to the S-layer.

Although S-layers are being increasingly identified on Bacteria and Archaea, it is enigmatic that in most cases S-layer function continues to elude us. In a few instances, S-layers have been shown to be virulence factors on pathogens (e.g. Campylobacter fetus ssp. fetus and Aeromonas salmonicida), protective against Bdellovibrio, a depository for surface-exposed enzymes (e.g. Bacillus stearothermophilus), shape-determining agents (e.g. Thermoproteus tenax) and nucleation factors for fine-grain mineral development (e.g. Synechococcus GL 24). Yet, for the vast majority of S-layered bacteria, the natural function of these crystalline arrays continues to be evasive. The following review up-dates the functional basis of S-layers and describes such diverse topics as the effect of S-layers on the Gram stain, bacteriophage adsorption in lactobacilli, phagocytosis by human polymorphonuclear leukocytes, the adhesion of a high-molecular-mass amylase, outer membrane porosity, and the secretion of extracellular enzymes of Thermoanaerobacterium. In addition, the functional aspect of calcium on the Caulobacter S-layer is explained.

The phagocytic ingestion of reference strains and clinical isolates of Fusobacterium nucleatum, Porphyromonas gingivalis, and Treponema denticola by polymorphonuclear leukocytes (PMNs) and the concomitant release of PMN granule proteinases were studied by specific functional and immunological assays. PMNs were incubated with the microorganisms anaerobically at 37 degrees C for indicated time periods. The suspensions and pellets were used for phagocytic ingestion assay and electron microscopic study, respectively. The supernatants were used for the measurements of the amounts and activities of the released PMN enzymes including PMN gelatinase (MMP-9), collagenase (MMP-8), serine proteases (elastase and cathepsin G), and lactate dehydrogenase (LDH). Both fluorescence microscopy and transmission electron microscopy showed that F. nucleatum, P. gingivalis and T. denticola were ingested by the PMNs in comparable numbers. However, measurements of the enzymes released from the triggered PMNs revealed major differences among the three species. High amount of elastase was released from the PMNs triggered by F. nucleatum, but not by P. gingivalis or T. denticola. The treatment of PMNs with P. gingivalis whole cells resulted in the release of gelatinase partly in the 82 kD active form, suggesting proteolytic activation of the degranulated 92 kD proMMP-9. The 82 kD active form of gelatinase was not detected upon triggering the PMNs with F. nucleatum and T. denticola. The PMN-bacteria interaction did not result in release of LDH from triggered PMNs indicating the proteinase release was not due to the PMN cell death. The results show that the susceptibilities of the 3 potentially periodontopathogenic microorganisms, F. nucleatum, P. gingivalis and T. denticola to phagocytic ingestion are not directly related to the amounts and activities of PMN enzymes released during the bacteria-PMN interactions. As PMN degranulation is considered as one of the major pathogenic mechanisms in periodontitis, the observed differences among the microorganisms may be important virulence characteristics of these species.

Background— Infectious agents, especially bacteria and their components originating from the oral cavity or respiratory tract, have been suggested to contribute to inflammation in the coronary plaque, leading to rupture and the subsequent development of coronary thrombus. We aimed to measure bacterial DNA in thrombus aspirates of patients with ST-segment–elevation myocardial infarction and to check for a possible association between bacteria findings and oral pathology in the same cohort. Methods and Results— Thrombus aspirates and arterial blood from patients with ST-segment–elevation myocardial infarction undergoing primary percutaneous coronary intervention (n=101; 76% male; mean age, 63.3 years) were analyzed with real-time quantitative polymerase chain reaction with specific primers and probes to detect bacterial DNA from several oral species and Chlamydia pneumoniae. The median value for the total amount of bacterial DNA in thrombi was 16 times higher than that found in their blood samples. Bacterial DNA typical for endodontic infection, mainly oral viridans streptococci, was measured in 78.2% of thrombi, and periodontal pathogens were measured in 34.7%. Bacteria-like structures were detected by transmission electron microscopy in all 9 thrombus samples analyzed; whole bacteria were detected in 3 of 9 cases. Monocyte/macrophage markers for bacteria recognition (CD14) and inflammation (CD68) were detected in thrombi (8 of 8) by immunohistochemistry. Among the subgroup of 30 patients with myocardial infarction examined by panoramic tomography, a significant association between the presence of periapical abscesses and oral viridans streptococci DNA–positive thrombi was found (odds ratio, 13.2; 95% confidence interval, 2.11–82.5; P =0.004). Conclusions— Dental infection and oral bacteria, especially viridans streptococci, may be associated with the development of acute coronary thrombosis.

Interferon-inducible Mx proteins belong to the family of large GTPases and are highly homologous with dynamins within their GTP-binding domain. Cytoplasmically localized human MxA protein mediates resistance to influenza and several other viruses, whereas human MxB protein has not been found to have any antiviral activity. Here we show that MxB protein is found both in the cytoplasm and in the nucleus, where it is localized in a granular pattern in the heterochromatin region beneath the nuclear envelope. Transfection experiments in COS cells of N-terminally deleted MxB constructs revealed a functional nuclear localization signal within the first 24 N-terminal amino acids. Nuclear 78-kDa and cytoplasmic 76-kDa forms of MxB protein were found in all of the cell lines studied and in human peripheral blood mononuclear cells. MxB protein proved to be a functional GTPase with activity comparable to that of MxA protein. N-terminally truncated (delta1-82) MxB protein lacking both the nuclear localization signal and a proline-rich domain had almost completely lost its GTPase activity. Analysis of peripheral blood mononuclear cells suggested that MxB protein expression is strictly regulated by interferon-alpha. This is the first documentation that human Mx protein resides in the nucleus. It also emphasizes that there are considerable differences in the localization and structure of functional domains within Mx proteins.

Titanium dioxide nanoparticles (TiO 2 NPs) have raised concern of environmental risks due to their widespread applications, but little is known about the potential toxicity of TiO 2 NPs to aquatic plants. The aim of this work was to study the effects of TiO 2 NPs on Lemna minor and to study the behavior of TiO 2 NPs under modified ISO 20079 test conditions. TiO 2 NPs had a tendency to aggregate in ISO (Steinberg) growth medium, but modification of the standard growth medium enabled the exposure of L. minor to TiO 2 NPs. By dilution of the growth medium (1:10), and exposure under semi-static conditions with medium renewal every second or third day, the size of TiO 2 particles remained rather stable throughout the test period. TiO 2 NPs showed n o adverse effect on the growth rate or chlorophyll a content of L. minor , even at a high exposure concentration of 5 mg L −1 and extended exposure time of 14 days. TiO 2 NPs attached onto L. minor cell walls, but no cellular uptake was observed. Although TiO 2 NPs were not toxic to L. minor , the potential transfer of TiO 2 NPs in aquatic food chains, e.g. attached to the plant leaves and other biological surfaces may be of importance, causing exposure of other organisms and contributing to the environmental fate of nanoparticles.

We have previously shown that strains of Eubacterium yurii are hydrophobic, as compared with human polymorphonuclear leukocytes (PMNs), possibly because of a crystalline surface layer (S-layer) covering the cell envelope of this potential endo-perio pathogen. The aim of the present study was to investigate the phagocytic ingestion by PMNs of the three E. yurii subspecies, with special attention to bacterial surface structures and hydrophobicity. Type strains of subspp. margaretiae, yurii, and schtitka, together with three clinical isolates from necrotic root canals, were studied. All strains were hydrophobic when tested by a two-phase partition method. E. yurii subspp. margaretiae strains ATCC43715T, ES4C, and ES14B-8E were resistant to PMN ingestion in the absence of opsonins, whereas strains of the two other subspecies were readily ingested. The presence of a resistant strain (subsp. margaretiae ATCC43715T) did not inhibit the ingestion of a sensitive strain (subsp. schtitka ATCC43716T). Ingestion of E. yurii subsp. margaretiae strains required opsonization by normal human serum or specific antibodies. Electron microscopy revealed an S-layer in all strains and fimbria-like structures in the subspp. margaretiae and yurii strains. The antiserum prepared against the S-protein of E. yurii subsp. margaretiae ATCC43715T showed only slight cross-reactivity with other E. yurii strains and indicated the presence of strain-specific rather than species- or subspecies-specific antigens in the S-protein of E. yurii subsp. margaretiae ATCC43715T. The results suggest that the mere presence of the S-layer or fimbria-like structures cannot explain the susceptibility to ingestion by the PMNs. It is possible that specific-receptor-mediated binding overrides the importance of hydrophobicity in the nonopsonic phagocytosis of E. yurii subspecies.

Excessive consumption of acid-containing drinks is associated with an increased risk of dental erosion. Milk has antierosive effects, and enrichment of acidic drinks with milk constitutes to reduce their erosive potential has been under increased interest. Our aim was to compare the erosive potential of conventional apple juice (85% juice), apple juice supersaturated with calcium phosphate (CaP), and apple juice enriched with calcium lactate gluconate (CaLG) in vitro. DeIonized water and citric acid (2%) were used as negative and positive controls. A total of 14-16 enamel specimens were incubated in each fluid for one hour. Erosion was examined by profilometry. One specimen per fluid was studied with a field emission scanning electron microscope for morphological changes. Citric acid caused erosion in all the specimens (100%), and conventional apple juice in 68.8% of the cases. One case in the CaP juice group showed visible erosion (6.7%), and the specimens incubated in the CaLG juice and water showed no visible erosion. Scanning electron microscopic data further confirmed the results. To conclude, addition of CaP and CaLG to apple juice reduced enamel erosion compared to conventional apple juice.

Resin salve prepared from Norway spruce (Picea abies) has been used for centuries in traditional medicine to treat skin diseases. The authors studied with transmission and scanning electron microscopy, and with electron physiology, changes in cell wall and cell membrane of Staphylococcus aureus after exposure of the bacterial cultures to resin. After exposure, cell wall thickening, cell aggregation, changed branching of fatty acids, and dissipation of membrane potential of the bacterial cells were observed. The authors conclude that spruce resin affects the cell viability via changes in the cell wall and membrane, and impairs, thereby, the synthesis of energy in the bacteria.

A motile Gram-positive bacterial strain (KL8) was isolated from indoor dust. It was identified by API-test50 CHB as a species of Bacillus. This Bacillus sp. strain KL8 was described using different electron microscopic techniques: negative staining, thin sectioning, metal shadowing and freeze-etching. An additional surface layer (S-layer) was the outermost layer of the cell wall of this flagellated bacterium. The hexagonally arranged protein lattice covering the cells had a lattice constant about 9–10 nm, which falls in the same range as that of Bacillus anthracis.

Chlamydia pneumoniae has been associated with cardiovascular diseases and has been shown by different methods to be present in atherosclerotic lesions. However, not all studies have found C. pneumoniae in atherosclerotic tissues. We compared polymerase chain reaction (PCR) methods, in situ hybridization (ISH), and measurement of chlamydial lipopolysaccharide (cLPS) by enzyme immunoassay (EIA) from homogenized atherosclerotic tissue in the detection of C. pneumoniae. In a study population of 110 patients with carotid artery disease, cLPS was found in 22.2%, and DNA by PCR was found in 34.3% and by ISH in 39.4% of the samples. Poor repeatability was shown to complicate PCR, and the technical problems inherent in ISH were not insignificant. In contrast, the cLPS EIA test was fast and easy to perform. If the sensitivity could be increased, for example, by testing multiple tissue pieces, cLPS EIA might provide a standardized commercial method for the detection of chlamydia in tissue samples, and it, thus, merits further characterization and validation in different patient populations.

The repair of corneal wounds requires both epithelial cell adhesion and migration. We have studied the early adhesion process of immortalized human corneal epithelial (HCE) cells and show by field emission scanning electron microscopy (FESEM) that the cells first adhere via foot-like process to the growth substratum and later present lamellar spreading. During early adhesion indirect immunofluorescence showed that the cells codeposited laminin (Lm) -332 and the large subunit of tenascin-C (Tn-C L ) as a demarcated plaque beneath the cells. Instead, unprocessed Lm-332 (α3′32) was found in a wider area in cells showing lamellar spreading and was also prominently expressed in the cytoplasm of the migrating marginal cells in the in vitro wounded HCE cultures. Confocal laser scanning microscopy (CLSM) showed that the Golgi apparatus was located to the vicinity of the Lm-332/Tn-C L -containing adhesion plaque and accordingly treatment of the cells with demecolcine, dispersing the Golgi apparatus, prevented the formation of plaques. This suggests that formation of the adhesion plaque depends on a direct vectorial secretion of Lm-332 and Tn-C L to the culture substratum. Instead, cytochalasin B treatment disrupted microfilaments and arborized the cells but did not affect the deposition of Tn-C L /Lm-332 as a plaque beneath the cells. The suggestion was supported by immunoprecipitation experiments which showed that Tn-C L and Lm α3′ chain were found in cell-free matrices on the culture substratum of spreading cells but not at all (Tn-C L ) or much less (Lm-332) in the culture medium. Quantitative cell adhesion experiments showed that HCE cells did not adhere to plain Tn-C coat and that integrin (Int) α 3 β 1 mediated the adhesion of HCE cells to purified Lm-332 and to Lm-332/Tn-C while Int β 4 did not mediate adhesion to these proteins. Taken together, our data suggest that Lm-332 and Tn-C L cooperate in early adhesion process of HCE cells. Furthermore, the results show that Lm-3′32 isoform functions in the spreading of the cells beyond the early adhesion stage and appears to emerge into HCE cells starting to migrate in experimental wounds.

Laminin alpha5-chain, a constituent of laminins-10 and -11, is expressed in endothelial basement membranes. In this study we evaluated the roles of alpha5 laminins and Lutheran blood group glycoproteins (Lu), recently identified receptors of the laminin alpha5-chain, in the adhesion of human dermal microvascular and pulmonary artery endothelial cells. Field emission scanning electron microscopy and immunohistochemistry showed that the endothelial cells spread on laminin-10 and formed fibronectin-positive fibrillar adhesion structures. Immunoprecipitation results suggested that the cells produced fibronectin, which they could use as adhesion substratum, during the adhesion process. When the protein synthesis during the adhesion was inhibited with cycloheximide, the formation of fibrillar adhesions on laminin-10 was abolished, suggesting that laminin-10 does not stimulate the formation of any adhesion structures. Northern and Western blot analyses showed that the cells expressed M(r) 78,000 and 85,000 isoforms of Lu. Quantitative cell adhesion assays showed that in the endothelial cell adhesion to laminin-10, Lu acted in concert with integrins beta(1) and alpha(v)beta(3), whereas in the adhesion to laminin-10/11, Lu and integrin beta(1) were involved. In the cells adhering to the alpha5 laminins, Lu and the integrins showed uniform cell surface distribution. These findings indicate that alpha5 laminins stimulate endothelial cell adhesion but not the formation of fibrillar or focal adhesions. Lu mediates the adhesion of human endothelial cells to alpha5 laminins in collaboration with integrins beta(1) and alpha(v)beta(3).Laminin alpha5-chain, a constituent of laminins-10 and -11, is expressed in endothelial basement membranes. In this study we evaluated the roles of alpha5 laminins and Lutheran blood group glycoproteins (Lu), recently identified receptors of the laminin alpha5-chain, in the adhesion of human dermal microvascular and pulmonary artery endothelial cells. Field emission scanning electron microscopy and immunohistochemistry showed that the endothelial cells spread on laminin-10 and formed fibronectin-positive fibrillar adhesion structures. Immunoprecipitation results suggested that the cells produced fibronectin, which they could use as adhesion substratum, during the adhesion process. When the protein synthesis during the adhesion was inhibited with cycloheximide, the formation of fibrillar adhesions on laminin-10 was abolished, suggesting that laminin-10 does not stimulate the formation of any adhesion structures. Northern and Western blot analyses showed that the cells expressed M(r) 78,000 and 85,000 isoforms of Lu. Quantitative cell adhesion assays showed that in the endothelial cell adhesion to laminin-10, Lu acted in concert with integrins beta(1) and alpha(v)beta(3), whereas in the adhesion to laminin-10/11, Lu and integrin beta(1) were involved. In the cells adhering to the alpha5 laminins, Lu and the integrins showed uniform cell surface distribution. These findings indicate that alpha5 laminins stimulate endothelial cell adhesion but not the formation of fibrillar or focal adhesions. Lu mediates the adhesion of human endothelial cells to alpha5 laminins in collaboration with integrins beta(1) and alpha(v)beta(3).

Attachment of bacteria to titanium may differ not only between bacterial species but also between strains within a species. The aim of the present in vitro study was to examine differences in bacterial attachment using 4 gram-negative anaerobic species of bacteria that are considered potential periodontal pathogens.The attachment of clinical and laboratory strains (n = 23) representing 2 Fusobacterium nucleatum subspecies, Porphyromonas gingivalis, and Prevotella intermedia to smooth, commercially pure titanium was examined using scanning electron microscopy.All bacterial strains were attached to the smooth titanium surface by their outer membrane. F nucleatum cells were poorly attached to the titanium, unlike P gingivalis or P intermedia cells, but only slight differences were observed in the quantity of attached cells between the strains within each bacterial group.In favorable conditions, some anaerobes can attach directly to an inert titanium surface. Microbial adhesion and subsequent colonization on the dental implant surface can lead to infection of the peri-implant tissue.The results indicated that the avidity of bacterial attachment to a smooth titanium surface varies between species of oral gram-negative anaerobes but not between strains.

We thank the authors of the letters for their interest in our publication.1 Their comments focused on Chlamydia pneumoniae –negative finding, role of infection burden and coexistence of multiple pathogens, clinical usefulness of findings, and how the chronic oral infection is involved in acute coronary events. For the last 2 decades, C pneumoniae has been the strongest candidate bacteria behind atherosclerotic inflammation. According to the recent review by Joshi et al,2 there are a total of 1652 published articles on this subject, and various techniques (immunocytochemistry, polymerase chain reaction [PCR], in situ hybridization, electron microscopy, and culturing) have been used to determine the presence of C pneumoniae in vascular tissues. The range of C pneumoniae –positive patients varies greatly across studies from 0% to 100%. Apfalter et al3 reported that 89% of all patients studied had C pneumoniae –specific antibodies, but the pathogen was not detected in a single carotid atheroma by real-time PCR and cell culture in carotid endarterectomy samples. So …

The microflora isolated from food-packaging board is dominated by paenibacilli; a number of these micro-organisms have been characterized using a polyphasic approach. The highest 16S rRNA gene similarity was found between these isolates and Paenibacillus azotofixans ATCC 35681(T) (97.7 %). The main fatty acid of the paperboard isolates was C(16 : 0) (34-45 %); straight-chain fatty acids made up 41-60 % of the total cellular fatty acids, thus distinguishing these strains from other Paenibacillus species. The paperboard isolates produced cyclodextrins from starch. The spore surface had a characteristic ribbed ornamentation. Spores and vegetative cells frequently had pilus-like appendages. Based on phylogenetic data and phenotypic and chemotaxonomic characteristics, it is proposed that the isolates represent a novel species, Paenibacillus stellifer sp. nov., with IS 1(T) (=DSM 14472(T)=CCUG 45566(T)) as the type strain.

Hard metal lung diseases (HML) are rare, and complex to diagnose. We describe the case of a patient with allergic alveolitis accompanied by rheumatoid arthritis. A sharpener of hard metal by trade, our patient was a 45-year-old, nonsmoking Caucasian female who experienced symptoms of cough and phlegm, and dyspnea on exertion. Preliminary lung findings were inspiratory rales in both basal areas, decreased diffusion capacity and a radiological picture resembling sarcoidosis. A high-resolution computed tomography scan indicated patchy alveolitis as well. An open lung biopsy revealed non-necrotizing granulomas consisting of epitheloid cells and surrounded by lymphocytes, plasma cells and a few eosinophils. These cells also occupied the thickened alveolar interstitium. Macrophages in the alveolar spaces, some of them multinuclear, contained dust particles. Hard metal alveolitis is clinically well known and, in this patient, has been described histologically. After the patient had quit working with hard metal and following corticosteroid therapy, pulmonary symptoms and signs were relieved. During this recovery period, however, she contracted rheumatoid arthritis.

Bacillus cereus is a causative agent in both gastrointestinal and in nongastrointestinal infections. Enterotoxins, emetic toxin (cereulide), hemolysins, and phoshpolipase C as well as many enzymes such as beta-lactamases, proteases and collagenases are known as potential virulence factors of B. cereus. A special surface structure of B. cereus cells, the S-layer, has a significant role in the adhesion to host cells, in phagocytosis and in increased radiation resistance. Interest in B. cereus has been growing lately because it seems that B. cereus-related diseases, in particular food poisonings, are growing in number.

The cbsA gene of Lactobacillus crispatus strain JCM 5810, encoding a protein that mediates adhesiveness to collagens, was characterized and expressed in Escherichia coli . The cbsA open reading frame encoded a signal sequence of 30 amino acids and a mature polypeptide of 410 amino acids with typical features of a bacterial S-layer protein. The cbsA gene product was expressed as a His tag fusion protein, purified by affinity chromatography, and shown to bind solubilized as well as immobilized type I and IV collagens. Three other Lactobacillus S-layer proteins, SlpA, CbsB, and SlpnB, bound collagens only weakly, and sequence comparisons of CbsA with these S-layer proteins were used to select sites in cbsA where deletions and mutations were introduced. In addition, hybrid S-layer proteins that contained the N or the C terminus from CbsA, SlpA, or SlpnB as well as N- and C-terminally truncated peptides from CbsA were constructed by gene fusion. Analysis of these molecules revealed the major collagen-binding region within the N-terminal 287 residues and a weaker type I collagen-binding region in the C terminus of the CbsA molecule. The mutated or hybrid CbsA molecules and peptides that failed to polymerize into a periodic S-layer did not bind collagens, suggesting that the crystal structure with a regular array is optimal for expression of collagen binding by CbsA. Strain JCM 5810 was found to contain another S-layer gene termed cbsB that was 44% identical in sequence to cbsA . RNA analysis showed that cbsA , but not cbsB , was transcribed under laboratory conditions. S-layer-protein-expressing cells of strain JCM 5810 adhered to collagen-containing regions in the chicken colon, suggesting that CbsA-mediated collagen binding represents a true tissue adherence property of L. crispatus .

Polyethyleneimine (PEI), a polycationic polymer substance used in various bioprocesses as a flocculating agent and to immobilize enzymes, was recently shown to make Gram-negative bacteria permeable to hydrophobic antibiotics and to detergents. Because this suggests impairment of the protective function of the outer membrane (OM), the effect of PEI on the ultrastructure of Salmonella typhimurium was investigated. Massive alterations in the OM of PEI-treated and thin-sectioned bacteria were observed by electron microscopy. Vesicular structures were seen on the surface of the OM, but no liberation of the membrane or its fragments was evident. Since a potential mechanism for the action of PEI could be its binding to anionic LPSs on the OM surface, the interaction of PEI with isolated LPSs was assayed in vitro. The solubility of smooth-type LPSs of Salmonella, regardless of the sugar composition of their O-specific chains, was not affected by PEI, nor was that of Ra-LPS (lacking O-specific chains but having a complete core oligosaccharide). PEI strongly decreased the solubility of rough-type LPSs of the chemotypes Rb2 and Re, whereas it had only a weak effect on the abnormally cationic Rb2-type pmrA mutant LPS, suggesting that the negative charge to mass ratio of LPS plays a critical role in the interaction.

Objectives. We sought to study the possible presence of Chlamydia pneumoniaein aortic valve stenosis (AVS). Background. Inflammation and immune mechanisms are considered important for the pathogenesis of nonrheumatic AVS. All chlamydial species are able to cause heart infections, and seroepidemiologic studies have indicated an association between chronic C. pneumoniaeinfection and coronary artery disease. Furthermore, the organism has been demonstrated in atherosclerotic lesions. Methods. Aortic valve specimens with varying degrees of macroscopic disease were obtained from 35 subjects—17 consecutive patients undergoing aortic valve replacement for treatment of nonrheumatic AVS and 18 age-matched subjects at autopsy. The possible presence of C. pneumoniaein aortic valves was studied by immunohistochemical analysis, polymerase chain reaction or transmission electron microscopy, or a combination of these. Results. Positive immunohistochemical staining with C. pneumoniaespecific antibody was found in 9 (53%) of 17 patients with advanced aortic valve disease requiring surgical treatment (group A), 8 (80%) of 10 cadavers with clearly macroscopic aortic valve pathology (group B) and 1 (12%) of 8 grossly normal cadaver control subjects (group C). Statistical significance with regard to the presence of C. pneumoniaewas found when combined diseased subjects (groups A and B: total 17 of 27 subjects) were compared with group C (p = 0.018). However, when group A was compared with group C, there was only marginal statistical significance (p = 0.088). Finally, there was a strong statistical significance (p = 0.015) when groups B and C were compared. Chlamydia pneumoniaeDNA was also found in three stenotic valves, and in two of the three tested valve specimens chlamydia-like particles were seen by electron microscopy. Conclusions. Chlamydia pneumoniaeis frequently present in nonrheumatic AVS. Similarly, the high number of C. pneumoniaeinfections detected in the early lesions of “degenerative” AVS suggest that this pathogen may play an etiologic role in the development of this disease. The validity of this relation requires additional study. (J Am Coll Cardiol 1997;29:1054–9) © 1997 by the American College of Cardiology

Tissue destruction during periodontitis is believed to be primarily brought about by leukocyte proteinases. We postulate that oral spirochetes cause discharge of polymorphonuclear leukocyte (PMN) lysosomal enzymes. Effects of Treponema denticola 53-kDa outer membrane protein, lipopolysaccharide (LPS), and peptidoglycan on degranulation of matrix metalloproteinases (MMP)-8 (collagenase) and -9 (gelatinase), cathepsin G, and elastase by human peripheral blood PMNs were studied by specific enzyme assays and Western blot analysis. T. denticola 53-kDa outer membrane protein was found to be a particularly efficient inducer of MMP-8 release. The induction was comparable with that of phorbol myristate acetate, a known inducer of PMN specific granule discharge. All of the treponemal substances, most notably the 53-kDa protein and LPS, induced release of MMP-9, a component of C-type granules. Both collagenase and gelatinase released from PMNs were mostly in active forms. Release of cathepsin G and elastase was also observed with the 53-kDa protein treatment. The other T. denticola substances did not induce release of these serine proteinases. Lactate dehydrogenase was not released from PMNs by the treatments, indicating that the degranulation was specific and not caused by toxic effects of the substances. This was confirmed by transmission electron microscopy of PMNs treated with the 53-kDa protein that showed rapid vacuole formation and cell shape changes but no disintegration of the cells. Thus, T. denticola may participate in the PMN-dependent extracellular matrix degradation during the course of periodontal inflammation by triggering the secretion and activation of matrix metalloproteinases.

SUMMARY The surface ultrastructure of three anaerobic Gram-positive cocci frequently encountered in oral infections, Peptostreptococcus micros, P. magnus and P. anaerobius, was studied. The type strains of P. micros (DSM 20468) and P. anaerobius (ATCC 27337), several clinical isolates of both species and the type strain of P. magnus (DSM 20470) were included. Thin-sectioned cells studied by electron microscopy revealed a homogeneous layer outside the peptidoglycan layer in P. anaerobius. In P. micros and P. magnus a more amorphous layer was present. No periodic structures were seen in negatively stained whole cells of these three species. However, in freeze-etched cells of P. anaerobius a crystalline surface protein layer (S-layer) was detected. No periodicity was seen in any of the P. micros strains or the magnus type strain by the methods used, but a periodic pattern was observed in negatively stained specimens of cell wall fragments of sonicated P. anaerobius cells. No capsular material was visible outside the S-layer in P. anaerobius. The cells of the Peptostreptococcus spp. were extracted for 30 min with detergents and urea. One er cent SDS and 6 M urea both extracted a major 78 kDa protein from all strains of P. anaerobius. Extraction of P. micros and P. magnus cells did not reveal any major protein bands comparable to that of P. anaerobius. Surface biotinylation of cells followed by Western blotting and detection by alkaline-phosphatase-conjugated extravidin showed strong staining of the 78 kDa band in P. anaerobius, further indicating that this molecule is located on the surface of the cell and is the S-protein. Another protein, of 127 kDa, was also detected by surface biotinylation in all P. anaerobius strains. No differences were detected in colony morphology or cell surface structures between the four strains. Isoelectric focusing of the proteins of P. anaerobius revealed that the S-proteins of individual strains have different pl values, ranging from 5.3 to 6.9.

The cell envelopes of bacteria from clinical infections consist of the cytoplasmic membrane and the cell wall. The cytoplasmic membrane is the site of many important biosynthetic activities of the cell, while the cell wall is mainly responsible for the maintenance of the cell shape. In gram-positive bacteria peptidoglygan surrounds the cytoplasmic membrane, in gram-negative cells a thin peptidoglycan layer is further surrounded by the outer membrane. In both gram-positive and gram-negative species amorphous polymeric material (e.g., capsular polysaccharides) may be present on the cell surface.

– The hydrophobicities of human polymorphonuclear leukocytes (PMNLs) and Bacteroides buccae, B. oris, B. oralis, B.veroralis, B. buccalis, B. heparinolyticus, B. intermedius, B. denticola, B. loescheii, B. melaninogenicus, Porphyromonas gingivalis, P. endodontalis, Wolinella recta, and Eubacterium yurii were studied by the hexadecane method. The majority of the strains were equally or less hydrophobic than the PMNLs. Only in the case of E. yurii and the only strain of B. buccalis were all strains more hydrophobic than the PMNLs. However, some strains of B. intermedius, B. oris, B. denticola, and P. gingivalis were also more hydrophobic than the PMNLs. With the exception of B. intermedius and species with a crystalline surface protein layer (S-layer), the strains of all other species with a thick capsule were more hydrophilic than the strains with little or no extracellular polymeric material. All strains of the S-layer species were either quite hydrophilic or hydrophobic depending on the species, totally irrespective of the presence of the capsule. The results suggest that the S-layers of oral anaerobic bacteria may be important determinants of cell surface hydrophobicity.

The phagocytic ingestion of clinical isolates and reference strains of Bacteroides buccae, Bacteroides oris, Porphyromonas gingivalis and Fusobacterium nucleatum by polymorphonuclear leukocytes (PMNs) was studied. Special attention was focused on the hydrophobicity of the strains. B. buccae strains, less or equally hydrophobic than PMNs, were poorly ingested without opsonization. Hydro-phobic, but not hydrophilic, strains of B. oris and both hydrophilic and hydro-phobic P. gingival's and F. nucleatum strains were readily ingested without opsonization. Hydrophobicity thus contributes to the adherence of bacteria to PMNs in some, but not all, species tested. Normal human serum enhanced the ingestion of B. buccae, but failed to do so after heat-inactivation. Heat-inactivation of the immune serum to B. buccae strain ES57 did not reduce opsonic activity suggesting that specific antibodies enhanced the ingestion of B. buccae.

– Transferring actively growing bacteria or Streptococcus mutans ATCC 27351 into a xylitol-containing reaction mixture caused distinct alterations in bacterial ultrastructure without notable effect on the total viability of the strain. Incubations in media containing 50 mg/ml of glucose, fructose, sucrose, lactose, sorbitol or mannitol as the primary carbon source did not affect bacterial ultrastructure. These fermentations were reflected biochemically in the amounts of insoluble glucans, as expected. A negative correlation was found between the cell mass and the lipoteichoic acid formation. But these aspects could not be visualized in the electron microscope. In the xylitol series, however, degrading cells and autolysis, intracellular vacuoles and lamellated formations in the cytoplasmic membrane were frequently seen independent of the concentration of xylitol in the reaction mixtures. In freeze-fracturing replicas, however, the membrane intercalated particles of the cytoplasmic membranes seemed to be unaffected and like those in the controls. Minor ultrastructural changes in the fracture-faces were detected. Despite the alterations in ultrastructure of the xylitol-incubated bacteria, there was no difference in their viability when compared to the controls.

The structures of crystalline layers from six Bacteroides strains were studied by electron microscopy. Two different hexagonal crystalline surface layers were found, one with a unit cell spacing of 21.5 nm and another with a spacing of 7.7 nm. A three-dimensional structure of the 21.5-nm layer and a two-dimensional projection of the 7.7-nm layer were determined to 3.0- and 3.8-nm resolution, respectively, by computerized image processing of electron micrographs. Both of these two crystalline layers were found in all six strains studied: B. pentosaceus NP333T and WPH61, B. capillus ATCC 33690T and ATCC 33691, and B. buccae ATCC 33574T and ES57. This further supports the identity of B. pentosaceus, B. capillus, and B. buccae as suggested by M. Haapasalo, K. Lounatmaa, H. Ranta, H. Shah, and K. Ranta (Int. J. Syst. Bacteriol. 35:65-72, 1985). The surface layer with 21.5-nm spacing is an intricate network with two classes of pores through the layer.

Synechocystis CB3, isolated from the Gulf of Finland, was covered by innumerable flexible pili (fimbriae) with an approximate diameter of 6 nm. The Synechocystis CB3 pili had a tendency to attach side by side thus forming characteristic bundles consisting of several dozens of individual pilus filaments. The Synechocystis CB3 pili were isolated and purified using deoxycholate and urea, and found to be very similar to other bacterial pili in their subunit Mr (21 kDa) and amino acid composition (46% hydrophobic amino acids). The amino acid analysis revealed also small amounts of glucosamine in the pilus preparation.

Type 1 pili from Salmonella typhimurium LT2 were purified and characterized. The pilus filaments were 6 nm in diameter and over 1 microns long. Estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the molecular weight of the pilin was 21,000. The isoelectric point of the filament was 4.1. Hydrophobic amino acids comprised 40.3% of the total amino acids of the pilin, which contained more proline, serine, and lysine than reported for the type 1 pilin of Escherichia coli. Purified pili agglutinated both horse and chicken erythrocytes and yeast cells but not bovine, sheep, or human erythrocytes. Horse erythrocyte agglutination was inhibited at lower concentrations by alpha-methyl-D-mannoside than by yeast mannane and D-fructose. Agglutination was not affected by D-galactose or sucrose. Results of the present study confirm the role of type 1 pili as Salmonella hemagglutinins and show chemical differences between the type 1 pili of S. typhimurium and E. coli.

– Bacteroides sp. was isolated in human periapical osteitis and shown to be biochemically closely related to B. ruminicola ssp. brevis and B. capillus. Electron microscopic examination revealed an external cell-wall layer (S-layer). The fractionation of cells by various methods gave partially purified sheets corresponding to the observed layer with a hexagonal molecular arrangement.

The outer membrane (OM) structure of Nitrosospira sp. X101 was studied by different electron microscopic techniques and SDS-PAGE. A crystalline outer membrane protein was visible in freeze-etched cells, occasionally seen also in the thin sectioned cells, but was difficult to see in a negatively-stained preparation. The lattice probably consists of large globular protein subunits with a hexagonal arrangement. The molecular weights of the major proteins in the cell envelope are 35 kDa, 40 kDa and 42 kDa.

We describe the comparative ultrastructures of bile-sensitive Bacteroides species which were isolated from oral cavities and ferment xylose and arabinose. Reference strains Bacteroides buccae ATCC 33574T(T = type strain), Bacteroides capillus ATCC 33690Tand ATCC 33691, and Bacteroides pentosaceus NP333Tand WPH61 and Bacteroides sp. strains ES42 and ES57 all had an extra surface layer (S-layer) outside the outer membrane. No S-layer was detected in Bacteroides oris ATCC 33573Tand ATCC 27518, Bacteroides oralis, Bacteroides veroralis, or Bacteroides sp. strains ES2759 and ES2834. The deoxyribonucleic acid guanine-plus-cytosine contents and both the malate dehydrogenase and glutamate dehydrogenase mobilities of the strains with an S-layer were identical. We suggest that the oral pentose-fermenting Bacteroides isolates with an S-layer may belong to the same species.

Pectinatus cerevisiophilus strains were subjected to mild hydrochloric acid extraction. Electron microscopic examination revealed that the bacterial cells still retained their original form. The extract contained vesicles, derived mostly from the outer membranes of the bacteria. The extracted material from ten isolates was studied by SDS polyacrylamide gel electrophoresis and a number of different protein patterns were found. These allowed two main groups and four subgroups to be distinguished among the isolates. The molecular weights of the major proteins varied between 13 and 62 kilodaltons. The mild hydrochloric acid extractable protein patterns provide a useful tool for typing different strains.

Mercaptoethanol (EtSH) was shown to enhance the solubility of the 39.5 kDa outer membrane (OM) protein of elementary bodies of chlamydia trachomatis in detergents. In the presence of mild detergents and EtSH almost selective solubilization of the 39.5 kDa protein and chlamydial LPS was obtained. This solution was further applied to gel filtration in the presence of SDS to purify the 39.5 kDa protein free of LPS.

A motile Gram-positive bacterial strain (KL1) was isolated from degrading wood of birch (Betula sp.). It was identified as a species of Bacillus. The Bacillus sp. strain KL1 was characterised with different electron microscopic techniques; negative staining, thin sectioning and freeze-etching. The cell wall structure of this flagellated bacterium was exceptional, with a periodic layer (S-layer), on both sides of the unperiodic cell wall layer. The tetragonally arranged protein lattice, also found around the cells in the medium, had a lattice constant of about 16 nm.

Summary The three-dimensional structures of the crystalline surface layers of two species of Eubacteria have been determined by electron microscopy and computerized image processing. The S-layer of Eubacterium sp. ES4C has tetragonal symmetry, with a unit cell spacing of 10.6 nm and a thickness of 9.5 nm, while that of Eubacterium sp. AHN 990 has hexagonal symmetry a = b = 15.7 nm and a thickness of 13 nm. The resolutions in the reconstructions were 2.5 nm and 1.8 nm, respectively. The reconstruction of the S-layer of strain ES4C reveals a distinct domain structure: a major tetramer, arms connecting adjacent unit cells, and a minor tetramer. The S-layer of strain AHN 990, on the other hand, has a rather complex arrangement, centred around the six-fold axis.

Cholesterol and intramembrane particle distribution on autophagic vacuole membranes was studied in Ehrlich ascites cells using filipin labelling and freeze-fracture electron microscopy. Unsaturated fatty acids were stained using imidazole-buffered osmium tetroxide. Autophagocytosis was induced with vinblastine, and early autophagic vacuoles were accumulated by lowering the ATP level in the cells with iodoacetate. Filipin labelling was observed in the limiting membranes of later, apparently hydrolase-containing autophagic vacuoles, whereas the most newly-formed, doublemembrane limited vacuoles were not labelled. The limiting membranes of late, residual body-type vacuoles either showed patchy filipin-induced deformation or were completely smooth. Imidazolebuffered osmium tetroxide stained the membranes of newly-formed or developing autophagic vacuoles partly or entirely. The membranes of older vacuoles stained more weakly. Intramembrane particle density on the P-face of the outer limiting membranes of newly-formed autophagic vacuoles was similar to that on endoplasmic reticulum, and the density seemed to increase slightly later on. The size of the P-face particles increased when the vacuoles became older. The limiting membranes of late, residual body-type vacuoles were almost smooth. The inner limiting membranes and the membranes inside the autophagic vacuoles were always almost particle-free. In conclusion, the amount of cholesterol, unsaturated fatty acids and protein in autophagic vacuole membranes changes during vacuole maturation.

Background: Seroepidemiologic studies have indicated an association between chronic Chlamydia pneumoniae infection and coronary heart disease. The organism, which is a common respiratory pathogen, has been demonstrated in atherosclerotic lesions of the aorta and coronary arteries. Abdominal aortic aneurysms are frequently associated with atherosclerosis, and inflammation may actually be an important factor in aneurysmal dilatation. Hence it could be assumed that C. pneumoniae may play a role in maintaining an inflammation and triggering the development of aortic aneurysms. Methods and Results: Specimens from abdominal aortic aneurysm were examined for the presence of C. pneumoniae by immunohistochemical analysis, the polymerase chain reaction amplifying omp1 gene, transmission electron microscopy, and culture methods with histologically atherosclerosis-negative human aortic tissues used as a control group. Chlamydial lipopolysaccharide and C. pneumoniae specific antigens were found by immunohistochemistry in 12 and 8 of 12 aneurysm specimens, respectively, and C. pneumoniae DNA could be demonstrated in 6 of 6 aneurysm specimens studied. Furthermore electron microscopy revealed the presence of Chlamydia -like elementary bodies in three of four aneurysm specimens tested. None of the control samples gave positive reaction in the polymerase chain reaction, and C. pneumoniae antigens were not detected in any of them. Conclusions: C. pneumoniae is frequently found in the vessel wall of abdominal aortic aneurysm. The potential etiopathogenetic role of C. pneumoniae in the development of these aneurysms remains to be studied. (J Vasc Surg 1997;25:499-505.)

On the basis of phenetic and chemotaxonomic criteria a new nonmotile anaerobic gram-negative rod-shaped bacterium from dental root canals is described. The new species has characteristics in common with species of the genus Bacteroides, but there are also many differences. According to a detailed biochemical analysis the new species is closer to the genus Mitsuokella than to Bacteroides. We propose a new species, Mitsuokella dentalis. The type strain of M. dentalis is strain DSM 3688.