Regulation by ToxR-Like Proteins Converges on vttRB Expression To Control Type 3 Secretion System-Dependent Caco2-BBE Cytotoxicity in Vibrio cholerae.

UNLABELLED: Genes carried on the type 3 secretion system (T3SS) pathogenicity island of Vibrio cholerae non-O1/non-O139 serogroup strain AM-19226 must be precisely regulated in order for bacteria to cause disease. Previously reported results showed that both T3SS function and the presence of bile are required to cause Caco2-BBE cell cytotoxicity during coculture with strain AM-19226. We therefore investigated additional parameters affecting in vitro cell death, including bacterial load and the role of three transmembrane transcriptional regulatory proteins, VttRA, VttRB, and ToxR. VttRA and VttRB are encoded on the horizontally acquired T3SS genomic island, whereas ToxR is encoded on the ancestral chromosome. While strains carrying deletions in any one of the three transcriptional regulatory genes are unable to cause eukaryotic cell death, the results of complementation studies point to a hierarchy of regulatory control that converges on vttRB expression. The data suggest both that ToxR and VttRA act upstream of VttRB and that modifying the level of either vttRA or vttRB expression can strongly influence T3SS gene expression. We therefore propose a model whereby T3SS activity and, hence, in vitro cytotoxicity are ultimately regulated by vttRB expression. IMPORTANCE: In contrast to O1 and O139 serogroup V. cholerae strains that cause cholera using two main virulence factors (toxin-coregulated pilus [TCP] and cholera toxin [CT]), O39 serogroup strain AM-19226 uses a type 3 secretion system as its principal virulence mechanism. Although the regulatory network governing TCP and CT expression is well understood, the factors influencing T3SS-associated virulence are not. Using an in vitro mammalian cell model to investigate the role of three ToxR-like transmembrane transcriptional activators in causing T3SS-dependent cytotoxicity, we found that expression levels and a hierarchical organization were important for promoting T3SS gene expression. Furthermore, our results suggest that horizontally acquired, ToxR-like proteins act in concert with the ancestral ToxR protein to orchestrate T3SS-mediated pathogenicity.

Type 3 Secretion System Island Encoded Proteins Required for Colonization by Non-O1/non-O139 Serogroup Vibrio cholerae.

Vibrio cholerae is a genetically diverse species, and pathogenic strains can encode different virulence factors that mediate colonization and secretory diarrhea. Although the toxin co-regulated pilus (TCP) is the primary colonization factor in epidemic causing V. cholerae strains, other strains do not encode TCP and instead promote colonization via the activity of a type three secretion system (T3SS). Using the infant mouse model and T3SS-positive O39 serogroup strain AM-19226, we sought to determine which of 12 previously identified, T3SS translocated proteins (Vops) are important for host colonization. We constructed in frame deletions in each of the 12 loci in strain AM-19226, and identified five Vop deletion strains, including ΔVopM, which were severely attenuated for colonization. Interestingly, a subset of deletion strains was also incompetent for effector protein transport. Our collective data therefore suggest that several translocated proteins may also function as components of the structural apparatus or translocation machinery, and indicate that while VopM is critical for establishing an infection, the combined activities of other effectors may also contribute to the ability of T3SS-positive strains to colonize host epithelial cell surfaces.

Combined characterization of composite tabular silver halide microcrystals by cryo-EFTEM/EELS and cryo-STEM/EDX techniques.

The combination of cryo-energy filtering transmission electron microscopy (EFTEM)/electron spectroscopic diffraction (ESD)/electron energy-loss spectroscopy (EELS) and cryo-energy-dispersive X-ray (EDX) analysis in the scanning transmission (STEM) and scanning (SEM) modes was applied for the characterization of composite tabular Ag(Br,I) microcrystals. A low-loss fine structure in EEL spectra between 4 and 26 eV was attributed to excitons and plasmons possibly superimposed with interband transitions and many-electron effects. The contrast tuning under the energy-filtering in the low-loss region was used to image the crystal morphology, defect structure (random dislocations and ¿111¿ stacking faults) and bend and edge contours as well as electron excitations in the microcrystals. Sharp extra reflections at commensurate positions in between the main Bragg reflections and diffuse honeycomb contours in ESD patterns of the microcrystals taken near the [111] zone were assigned to the number of defects in the shell region parallel to the grain edges and polyhedral clusters of interstitial silver cations, respectively. The imaginary part of the energy-loss function, Im (-1/epsilon), and the real and imaginary parts, epsilon1 and epsilon2, of the dielectric permittivity were determined by means of a Kramers-Kronig analysis. An assignment of exciton peaks based on calculations of electronic band structure of silver bromide is proposed. Inner-shell excitation bands of silver halide were detected in line with EDX-analyses. The energy-loss near-edge structure (ELNES) of the AgM4,5-edge governed by spin-orbital splitting between the 3d3/2- and 3d5/2-states has been evaluated. Combined silver and halide distributions were obtained by a three-window method (EFTEM) and by EDX/STEM including area mapping and line profiling of iodide.

Homotypic fusion between aggregated lysosomes triggered by elevated [Ca2+]i in fibroblasts.

Previous studies demonstrated that microinjection of antibodies to the cytoplasmic domain of the lysosomal glycoprotein lgp120 induces aggregation of lysosomes in NRK cells. Here we show that the antibody-clustered vesicles do not co-localize with MPR and ss-COP-containing organelles, confirming their lysosomal nature. Observations by transmission and high voltage electron microscopy indicated that, although tightly apposed to each other, aggregated lysosomes remained as separate vesicles, with an average diameter of 0.3-0.4 micron. However, when cells microinjected with antibody were exposed to the Ca2+ ionophore ionomycin, large vesicles were formed within the lysosome clusters, suggesting the occurrence of lysosome-lysosome fusion. Stereological measurements of lysosome diameters on confocal and transmission electron microscopy indicated that the large lgp120-positive vesicles could have originated from the fusion of 3 up to 15 individual lysosomes. To verify if agents that mobilize Ca2+ from intracellular stores had the same effect, anti-lgp120-microinjected cells were treated with thapsigargin, and with the receptor-mediated agonists bombesin and thrombin. Thapsigargin also induced the formation of large lgp120-containing vesicles, detected by both confocal and transmission electron microscopy. Analysis of antibody-clustered lysosomes in streptolysin O-permeabilized cells indicated that an intracellular free Ca2+ concentration of 1 microM was sufficient to trigger formation of large lysosomes.

Mitochondrial matrix granules: their behavior during changing metabolic situations and their relationship to contact sites between inner and outer mitochondrial membranes.

Since their discovery in the early fifties mitochondrial granules have been the subject of many researches. Some twenty years ago two hypotheses on their function were introduced. Peachey thought that the granules were a sink of cations and that they would eventually regulate the concentrations of these ions. Alternatively, Barnard thought that the granules were precursors of the mitochondrial inner membrane. There are only a few data on organic constituents of the granules. Phospholipids (e.g., cardiolipin) glycoprotein or lipids, calcium precipitable lipoprotein, cytochrome c oxidase seem to be present in the granules. There has been much debate on whether calcium is present or not. Reports are mostly based on X-ray microanalysis, the result of which depends on preparation techniques. In heart muscle in stimulating situations the NMG (native matrix granules) move towards the inner membrane and are incorporated in it. They appear to create contact sites between inner and outer mitochondrial membranes in which enzymes can function efficiently. It is hypothetized that the system, NMG-contact sites, forms the structural basis of a regulatory mechanism, by which cells can cope with a high and sudden energy demand.

The endothelium during cuff-induced neointima formation in the rabbit carotid artery.

Intimal thickening in human arteries is considered as a site of predilection for atherosclerosis. The placement of a flexible, physically nonconstrictive, silicone cuff around the rabbit carotid artery induced a neointima composed of smooth muscle cells (SMCs) within 14 days. To investigate possible alterations of the endothelial cells (ECs) during neointima formation, their morphology was examined with scanning electron microscopy (SEM), transmission electron microscopy (TEM), and confocal microscopy. In the early postoperative period (6 hours), both cuffed and sham-operated arteries demonstrated small foci (5 to 200 microns) of denudation, presumably as a consequence of the manipulation. Within 24 hours the luminal surface of the cuffed and sham-operated arteries was completely covered with endothelium, which remained continuous throughout the study. However, after 1 week the ECs of the cuffed arteries contained a pronounced rough endoplasmic reticulum. From 6 hours until 3 days, polymorphonuclear leukocytes infiltrated the cuffed but not the sham-operated arteries from the lumen. Subendothelial SMC accumulation in the cuffed arteries began after this time period. At day 14 a full-blown neointima composed of longitudinally oriented SMCs had formed in the cuffed arteries. The sham-operated arteries did not develop a neointima. During neointima formation immunoreactivity for von Willebrand factor (vWf) increased in the ECs, and vWf was deposited in the extracellular spaces of the neointima. At day 14 the area of vWf deposits correlated positively with the area of the neointima (r = .73, P < .001). In subsequent weeks, the intimal area did not increase, and vWf deposits vanished from the neointimal matrix. The endothelium of the sham-operated arteries showed no change in vWf immunoreactivity compared with untreated arteries throughout the study. The altered ultrastructural morphology of the ECs and the concurrent vWf deposition in cuffed but not in sham-operated arteries point to alterations in EC function during the development of the neointima. The vWf secretion could possibly lead to increased adhesiveness of the extracellular matrix for the ECs as well as modulate neointima formation.

Catecholamines are present in a synaptic-like microvesicle-enriched fraction from bovine adrenal medulla.

"Synaptic-like microvesicles" are present in all neuroendocrine cells and cell lines. Despite their resemblance to small synaptic vesicles of the CNS, a thorough biochemical characterization is lacking. Moreover, the subcellular distribution of synaptophysin, the most abundant integral membrane protein of small synaptic vesicles, in adrenal medulla is still controversial. Using gradient centrifugation, we were able to compare the distribution of several markers for small synaptic vesicles and chromaffin granules. Synaptophysin was found at a high density (1.16 g/ml), purifying away from dopamine beta-hydroxylase and cytochrome b561. Both noradrenaline and adrenaline showed a parallel distribution with synaptophysin, suggesting their presence in synaptic-like microvesicles. Experiments in the presence of tetrabenazine did not influence the catecholamine content. Additionally, tetrabenazine binding showed a consistent shoulder in the region of synaptophysin. [3H]Noradrenaline uptake was blocked by tetrabenazine, but not by desipramine. Also chromogranin A parallels the distribution of synaptophysin; however, a localization in the Golgi cannot be ruled out. Synaptophysin was shown to undergo very fast phosphorylation, together with another triplet protein of approximately 18 kDa. In contrast, the latter showed a rather bimodal distribution coinciding with synaptophysin and dopamine beta-hydroxylase. Immunoelectron microscopy of synaptic-like microvesicle fractions showed an intense labeling for synaptophysin on 60-90-nm organelles. Whereas abundant gold labeling for cytochrome b561 was found over the entire surface of chromaffin granules, synaptophysin labeling was encountered mostly on vesicles adsorbed to granules. We conclude that catecholamines might be stored in synaptic-like microvesicles of the chromaffin cell.

Triphasic sequence of neointimal formation in the cuffed carotid artery of the rabbit.

A nonocclusive silicone cuff placed around the rabbit carotid artery results in a diffuse intimal thickening. The early stages of this phenomenon were studied by light microscopy, immunohistochemistry, and electron microscopy. Neointimal formation appeared to be triphasic. The first phase started 2 hours after cuff placement, with vascular infiltration by polymorphonuclear leukocytes (PMNs). In the second phase, starting within 12 hours, 1.90 +/- 0.36% of the medial smooth muscle cells (SMCs) were replicating, as demonstrated by their immunoreactivity for proliferating cell nuclear antigen (PCNA). The third phase was characterized by the appearance, from day 3 onward, of subendothelial SMCs that were immunoreactive for alpha-SMC actin and vimentin. A few cells showed immunoreactivity for PCNA. During this phase all the PMNs disappeared, but SMC replication in the media was still present, as indicated by the presence of mitoses and the persisting immunoreactivity for PCNA (0.76 +/- 0.22% at day 7). In the third phase the number of subendothelial cells increased (104 +/- 15 SMC nuclei per section at day 7, of which 8.89 +/- 2.26% were PCNA-positive) and was associated with deposition of collagen type IV and fibronectin. At 14 days a complete, circular neointima was present and contained 2.13 +/- 0.28% replicating SMCs. The media showed 0.44 +/- 0.08% cell-cycling SMCs, which was still four times higher than normal. During the first week there was also a significantly higher PCNA activity in the media of sham-operated carotid arteries (no cuff present) than in nonsurgical ones. However, this did not lead to the formation of a neointima. We conclude that in the cuff system SMC replication in the media precedes the neointimal formation. The system can be used to study SMC replication, migration, and neointimal formation with minimal medial SMC damage.

Ultrastructurally normal and motile spermatozoa in a fertile man with Kartagener's syndrome.

The findings in a 40-year-old man with Kartagener's triad (sinusitis, bronchiectasis, and situs inversus) and corrected transposition of the great vessels are presented. Electron microscopy revealed normal ultrastructure of the axoneme in both respiratory cilia and sperm tails. Light microscopic evaluation of the spermatozoa showed 50 percent motility, suggesting normal fertility. This assumption is confirmed, as the patient has two children. We suggest that an abnormal, uncoordinated motility pattern of the ultrastructurally normal respiratory cilia results in improper mucociliary clearance. This coordination is not needed in swimming spermatozoa, which could explain the apparent paradox between bronchopulmonary symptoms and normal fertility in our patient.

[Detection of mitochondrial contact sites and creatine kinase activity in Ca(2+)-stimulated rat hearts]. / Detekcia kontaktných miest mitochondrií a kreatínkinázovej aktivity v Ca(2+)-stimulovaných srdciach potkanov.

Contact sites are created by fusion of the inner and outer mitochondrial membrane. They represent a dynamic microcompartment for creatine kinase activity. In this microenvironment the active sites of creatine kinase, oxidative phosphorylation, and ADP/ATP transport interact during basal and stimulated metabolism. Electron microscopic studies showed that the occurrence rate of contact sites was changing according to the energy state of the mitochondrion (Biermans et al., 1989). The aim of this study was to stimulate the metabolism of the rat heart by extracellular calcium and to investigate the formation of contact sites and creatine kinase activity. Isolated rat hearts were perfused with Krebs-Henseleit solution containing 1.6 mmol Ca. After 15 min stabilization the hearts were subjected to 0.6, 0.9, 1.8, 2.2, 2.6, or 3.6 mmol Ca for a period of 15 min. Stimulation of the isolated rat hearts with increasing calcium concentrations was physiologically manifested by enhanced dp/dt and LVP values. A higher frequency rate of mitochondrial contact sites was observed and these were associated with higher creatine kinase activity. We suppose that induction of contact site formation is related to the functional activity of the Ca stimulated rat heart and thus efficient production and transport of energy is provided. (Tab. 1, Fig. 6, Ref. 18.).

Cytotoxic activity of tumor necrosis factor is mediated by early damage of mitochondrial functions. Evidence for the involvement of mitochondrial radical generation.

Structural mitochondrial damage accompanies the cytotoxic effects of several drugs including tumor necrosis factor (TNF). Using various inhibitors of mitochondrial electron transport we have investigated the mechanism of TNF-mediated cytotoxicity in L929 and WEHI 164 clone 13 mouse fibrosarcoma cells. Inhibitors with different sites of action modulated TNF cytotoxicity, however, with contrasting effects on final cell viability. Inhibition of mitochondrial electron transport at complex III (cytochrome c reductase) by antimycin A resulted in a marked potentiation of TNF-mediated injury. In contrast, when the electron flow to ubiquinone was blocked, either at complex I (NADH-ubiquinone oxidoreductase) with amytal or at complex II (succinate-ubiquinone reductase) with thenoyltrifluoroacetone, cells were markedly protected against TNF cytotoxicity. Neither uncouplers nor inhibitors of oxidative phosphorylation nor complex IV (cytochrome c oxidase) inhibitors significantly interfered with TNF-mediated effects, ruling out the involvement of energy-coupled phenomena. In addition, the toxic effects of TNF were counteracted by the addition of antioxidants and iron chelators. Furthermore, we analyzed the direct effect of TNF on mitochondrial morphology and functions. Treatment of L929 cells with TNF led to an early degeneration of the mitochondrial ultrastructure without any pronounced damage of other cellular organelles. Analysis of the mitochondrial electron flow revealed that TNF treatment led to a rapid inhibition of the mitochondria to oxidize succinate and NADH-linked substrates. The inhibition of electron transport was dose-dependent and became readily detectable 60 min after the start of TNF treatment, thus preceding the onset of cell death by at least 3-6 h. In contrast, only minor effects were observed on complex IV activity. The different effects observed with the mitochondrial respiratory chain inhibitors provide suggestive evidence that mitochondrial production of oxygen radicals mainly generated at the ubisemiquinone site is a causal mechanism of TNF cytotoxicity. This conclusion is further supported by the protective effect of antioxidants as well as the selective pattern of damage of mitochondrial chain components and characteristic alterations of the mitochondrial ultrastructure.

Phosphorus, calcium and lead distribution in collagen in lead induced soft tissue calcification. An ultrastructural and X-ray microanalytical study.

Repeated intraperitoneal injections of lead acetate in rats caused a calcification of the skin of the abdomen near the site of the injections. In the lead-induced calcifications, electron dense collagen bundles could be observed. On the surface of the collagen fibrils, needle-like crystals were visible. With energy-dispersive X-ray analysis, phosphorus, calcium and lead were detected in the electron dense collagen bundles. X-ray maps of the P-K alpha, Ca-K alpha, and Pb-L alpha plus Pb-L beta lines showed an equivalent distribution along the collagen fibrils for phosphorus and calcium. The occurrence of the most electron dense areas in the STEM-image was comparable to the lead distribution. A good correlation existed between the structural and the elemental images of the same area. Although the medicinal use of preparations containing lead is no longer recommended, some are still prescribed. From our results we can conclude that they should not be applied to injured or inflamed skin.

Influence of fixation procedures on the microanalysis of lead-induced intranuclear inclusions in rat kidney.

Using Laser Microprobe Mass Analysis (LAMMA), we studied the chemical composition of lead-induced intranuclear inclusions in rat kidney tissue prepared by three different wet chemical fixation procedures for transmission electron microscopy. Fixation with glutaraldehyde-Na2S gave the same results as fixation with glutaraldehyde only: a high lead concentration could be detected. Therefore, for lead strongly bound to proteins, precipitation procedures are not essential. Post-fixation with osmium tetroxide drastically changed the composition of the inclusions: the lead concentration decreased substantially, while sodium, calcium, and barium were introduced. The osmium tetroxide fixative was found to be the source of the contamination. It also contained aluminum, and we suggest that other proteins (e.g., in neurofibrillary tangles) might be able to take up Al out of solution and that care must be exercised in interpreting the microanalytical results of osmium-fixed material. For the microanalysis of the lead inclusions, fixation with glutaraldehyde only provides a good compromise between preservation of the ultrastructure and maintenance of the element distribution.

Ultrastructural localization of aluminium in liver of aluminium maltol-treated rabbits by laser microprobe mass analysis.

By means of laser microprobe mass analysis (LAMMA), we have studied the ultrastructural localization of aluminium in livers of aluminium maltol-treated rabbits. This animal model was developed to study long-term aluminium toxicity using systemic (intravenous) administration of aluminium. We could only detect aluminium in electron-dense inclusion bodies found in large, sometimes multinucleated cells. These results prove that the actual observation of aluminium deposits in liver with LAMMA gives more information than bulk analysis and can be very useful to explore mechanisms of toxicity.

Histological study of the thin replacement membrane of human tympanic membrane perforations.

A histological study was done on the thin, nearly transparent replacement membrane of tympanic membrane perforations. Human tympanic membranes that were rejected for transplantation, were studied by light and electron microscopy. The abrupt reduction in thickness at the margin of the covered perforation, is entirely due to the reduction of the lamina propria. Even in the thinnest parts of the replacement membrane, a lamina propria is present, separated by continuous basement membranes from the epithelium and mucosa, and measuring no more than some 2-3 microns in thickness. This lamina propria consists of fibrils and interfibrillar matrix, but fibroblasts appear to be lacking. The epithelial layer does not contain basal cells, confirming the thesis that the upper layers are not generated by in situ proliferation, but that they have migrated from the periphery.

Freeze-fracture study of heart mitochondria in the condensed or orthodox state.

Rat heart mitochondria were isolated and forced in a well-defined metabolic state. After freeze-fracturing, the intramembrane particle dimension and density on both fracture faces of the inner mitochondrial membrane were measured. No significant differences could be calculated between the diameter of the membrane particles in the five different states. However, the particle density on the fracture faces of the inner mitochondrial membrane in the condensed configuration is significantly smaller than in the orthodox configuration on the 99.5% level of confidence. These results are compared with the literature, where conflicting data have been published about these particle densities.

A simplified 'sandwich' technique for in situ embedding and perpendicular sectioning of monolayer cultures of human skin fibroblasts.

In the processing of cell cultures, grown as a monolayer in tissue culture dishes for electron microscopy, the sectioning of the monolayer is an essential step. The monolayer can be sectioned either parallel or perpendicular to the plane of growth. Several methods for the perpendicular way of sectioning have already been described. We propose a simplified method in which the monolayer is sandwiched between two layers of resin, one of which is a prepolymerized block, the other being a layer of resin, applied at a second stage. Sectioning of this 'flat embedded' specimen yields thin sections perpendicular to the plane of growth of the monolayer without elaborate orientating procedures. The advantage of this procedure is that it can be done using only routine embedding techniques, avoiding special materials or complex manipulations. This sandwich technique provides an excellent mechanical fixation of the monolayer and protects it against external damage.

Examination of sexually abused children.

The approach to the examination of sexually abused children differs from that of normal for sick children and also from that of adults who have been the victims of rape. While the physical and psychological welfare of the patient must be the prime concern, medico-legal aspects are equally important. In the interests of the patient the initial examination and subsequent management and follow-up should be done by those with specific skills and with the support of members of disciplines essential to the total management of the complex and not uncommon problem of sexual abuse in children.

Immunocytochemical investigation of native matrix granules of the rat heart mitochondrion.

We have examined the ultrastructure and the protein content of native matrix granules (NMG) in rat heart mitochondria, by postembedding immunocytochemistry. Cytochrome c oxidase was found to be present in these granules. It is believed that these granules contain incomplete inner mitochondrial membrane fractions, which can be incorporated in the membrane after stimulation of the metabolism.