Structure of a Burkholderia pseudomallei Trimeric Autotransporter Adhesin Head

Background

Pathogenic bacteria adhere to the host cell surface using a family of outer membrane proteins called Trimeric Autotransporter Adhesins (TAAs). Although TAAs are highly divergent in sequence and domain structure, they are all conceptually comprised of a C-terminal membrane anchoring domain and an N-terminal passenger domain. Passenger domains consist of a secretion sequence, a head region that facilitates binding to the host cell surface, and a stalk region.

Methodology/Principal Findings

Pathogenic species of Burkholderia contain an overabundance of TAAs, some of which have been shown to elicit an immune response in the host. To understand the structural basis for host cell adhesion, we solved a 1.35 Å resolution crystal structure of a BpaA TAA head domain from Burkholderia pseudomallei, the pathogen that causes melioidosis. The structure reveals a novel fold of an intricately intertwined trimer. The BpaA head is composed of structural elements that have been observed in other TAA head structures as well as several elements of previously unknown structure predicted from low sequence homology between TAAs. These elements are typically up to 40 amino acids long and are not domains, but rather modular structural elements that may be duplicated or omitted through evolution, creating molecular diversity among TAAs.

Conclusions/Significance

The modular nature of BpaA, as demonstrated by its head domain crystal structure, and of TAAs in general provides insights into evolution of pathogen-host adhesion and may provide an avenue for diagnostics.     

Headspace gas chromatography-mass spectrometry analysis of isoflurane enantiomers in blood samples after anesthesia with the racemic mixture

Several in vivo and in vitro studies on the stereoselective potency of isoflurane enantiomers suggest beneficial effects of the (+)-(S)-enantiomer. In order to detect possible differences in the pharmacokinetics of isoflurane enantiomers, a clinical study of 41 patients undergoing general anesthesia maintained with racemic isoflurane was performed. The isoflurane enantiomers were analyzed in blood samples drawn before induction, at cessation (day 0), and up to eight days after isoflurane anesthesia (day 1-8). A multipurpose sampler (Gerstel MPS) was used for the headspace gas chromatography-mass spectrometry (GC/MS) analysis, and it was combined with a cold injection system (Gerstel CIS 3) for coldtrapping, enrichment, and focusing of the analyte. The enantiomer separation was achieved by using a capillary column coated with octakis(3-O-butanoyl-2,6-di-O-pentyl)-gamma-cyclodextrin (Lipodex E) dissolved in the polysiloxane PS 255. Detection was done in the selected ion monitoring mode with ions m/z 117 and m/z 149. An enrichment of (+)-(S)-isoflurane in all blood samples drawn after anesthesia was found. The highest enantiomer bias, up to 52-54% (+)-(S)-isoflurane as compared to 50% for the racemate, was observed on day 2 for most of the patients. Furthermore, quantification of isoflurane in blood samples of five patients was done by enantiomer labeling, employing enantiomerically pure (+)-(S)-isoflurane as internal standard. The isoflurane concentration decreased rapidly from 383 nmol/ml to 0.6 nmol/ml (mean values) eight days after anesthesia. The present study shows differences in the pharmacokinetics of isoflurane enantiomers in man. However, it is not possible to distinguish between enantioselective distribution and enantioselective metabolism, if any.     

Association of Caldendrin splice isoforms with secretory vesicles in neurohypophyseal axons and the pituitary

Caldendrin is a neuronal calcium-binding protein, which is highly enriched in the postsynaptic density fraction and exhibits a prominent somato-dendritic distribution in brain. Two additional splice variants derive from the caldendrin gene, which have unrelated N-termini and were previously only detected in the retina. We now show that these isoforms are present in neurohypophyseal axons and on secretory granules of endocrine cells. In light of the described interaction of the Caldendrin C-terminus with Q-type Ca(v)2.1 calcium channels these data suggest that this interaction takes place in neurohypophyseal axons and pituitary cells indicating functions of the short splice variants in triggering Ca2+ transients to a vesicular target interaction.     

Annotating regulatory DNA based on man-mouse genomic comparison

Non-coding DNA segments that are conserved between the human and mouse genomic sequence are good indicators of possible regulatory sequences. Here we report on a systematic approach to delineate such conserved elements from upstream regions of orthologous gene pairs from man and mouse. We focus on orthologous genes in order to maximize our chances to find functionally similar regulatory elements. The identification of conserved elements is effected using the Waterman-Eggert local suboptimal alignment algorithm. We have modified an implementation of this algorithm such that it integrates the determination of statistical significance for the local suboptimal alignments. This has the effect of outputting a dynamically determined number of suboptimal alignments that are deemed statistically significant. Comparison with experimentally determined annotation shows a striking enrichement of regulatory sites among the conserved regions. Furthermore, the conserved regions tend to cover the promotor region described in the EPD database.     

The preservation of ultrastructure in saturated phosphatidyl cholines by tannic acid in model systems and type II pneumocytes

The preservation for electron microscopy of saturated phospholipids in general, and phosphatidyl choline (PC)in particular, remains and unsolved problem since OsO(4) and glutaraldehyde are incapable of interacting with PC directly. However, by introducing tannic acid preceding osmication, we were able to demonstrate highly ordered, preserved lamellar structures in model experiments with saturated PC, and in vivo experiments type II pneumocytes of lung tissue. The secretory bodies of the latter are known to contain a high proportion of these saturated phospholipids. In both cases, the repeating periodicity approximated 45 A. It was determined that tannic acid interacts with the choline component of PC to form a "complex," which then could be stabilized by treatment with OsO(4). In the absence of osmication, the PC-tannic acid complex acid did not survive conventional dehydration techniques, but osmication permitted conventional Epon embedment. Sphingomyelin (SPH), which contains choline, behaved similarly in model experiments. But there was no evidence of a comparable reaction with tannic acid using phosphatidyl ethanolamine (PEA), phosphatidyl serine (PS), or phosphstidy inositol (PI). Chemical studies indicted a high pH dependency for the formation of the PC- tannic acid complex. Also, experiments demonstrated its dissociation in various organic solvents. Sharp delineation and great contrast of the polar zones in the ordered lamellar structures was achieved by additional staining with lead citrate thus leading to the conclusion that tannic acid serves as a multivalent agent, capable of simultaneous interaction with saturated PC, OsO(4), and lead citrate stains.     

Cyclic amp-induced morphological transformation of cells infected by temperature-sensitive mouse sarcoma virus: Expression of transformation-associated markers

Normal rat kidney (NRK) cells infected with a temperature-sensitive (ts) mutant of mouse sarcoma virus (NRK [MSV-1b]) express the transformed phenotype when grown under permissive conditions, but acquire the normal phenotype when grown under restrictive conditions. Addition of 3', 5' cyclic adenosine monophosphate (cAMP) to NRK (MSV-1b) cells grown at the restrictive temperature results in morphological transformation. To determine whether other markers associated with the transformed phenotype were coordinately expressed after cAMP exposure, concanavalin A (Con A) agglutinability, hexose transport rate, and incorporation of radioactively labeled fucose into fucolipid III and fucolipid IV (FL III and FL IV ) of the cells were examined. NRK cells transformed by wild-type MSV or NRK(MSV- 1b) grown under permissive conditions were agglutinated by low concentrations of Con A and exhibited relatively high maximal agglutination levels which were specifically inhibited by α-methyl-D-mannoside. In contrast, NRK (MSV-1b) cells grown under restrictive conditions were weakly agglutinated by Con A and exhibited reduced maximal agglutination levels, similar to uninfected NRK cells. Treatment of NRK (MSV-1b) cells at the restrictive temperature with cAMP resulted in morphological transformation and a change in the pattern of incorporation of labeled fucose inot FL III and FL IV to one comparable to that of NRK (MSV-1b) cells at the permissive temperature or to NRK cells transformed by wild-type MSV. In contrast, cAMP treatment resulted in no increase in Con A agglutinability or 2 deoxy-D- [(3)H]glucose transport relative to mock treated cultures. The results demonstrate that cAMP-induced morphological transformation and altered fucolipid composition of NRK (MSV-1b) cells are not correlated with alterations in hexose transport rate or Con A agglutinability.     

Die Ultrastruktur des Conus papillaris im Auge der ErzschleicheChalcides chalcides (L.) (Lacertilia,Scincidae)

Zusammenfassung Die Strukturen des fingerförmigen Conus papillaris im Auge der ErzschleicheChalcides chalcides wurden licht- und elektronenmikroskopisch untersucht. Eine Abgrenzung zum Glaskörper hin fehlt. Das GefÄ\system gliedert sich in Arteriole, prÄkapillÄre GefÄ\e, Kapillaren und Venolen. Die Arteriole besitzt Endothelzellen mit kurzen FortsÄtzen und elektronendichten Cytosomen sowie eine locker angeordnete Muskelschicht. Die prÄkapillÄren GefÄ\abschnitte sind kurz und Ähneln im Aufbau der Arteriole. Die organellenreichen Endothelzellen der sehr langen Kapillaren weisen zürn GefÄ\lumen hin einen dichten Saum bis zu 1,2 m hoher, regelmÄ\ig angeordneter Mikrofalten auf, wÄhrend die basale ZelloberflÄche etwas weniger organisiert ist. In einer bindegewebigen GefÄ\scheide kommen PericytenauslÄufer vor. Die Venolen unterscheiden sich von der Arteriole durch eine grö\ere Zahl luminaler FortsÄtze und weniger Cytosomen im Endothel, sowie durch eine lockerer gebaute Muskelschicht. Im Interstitium finden sich Bindegewebszellen, Mastzellen und Pigmentzellen, deren Ultrastruktur beschrieben wird. Die zahlreichen marklosen vegetativen Nervenfasern enthalten auffallend wenige Schwannsche Zellen, deren Rolle formal z.T. von den Pigmentzellen eingenommen zu werden scheint. Die Nervenfasern dienen vermutlich teilweise der Innervation der zentralen GefÄ\e; andererseits kann erstmals in einem Conus papillaris auch eine Innervation von Pigmentzellen nachgewiesen werden.Die Befunde werden mit denen am Conus papillaris anderer Echsen und Pecten oculi von Vögeln im Hinblick auf strukturelle Gemeinsamkeiten zwischen beiden Organen verglichen. DerChalcidesconus entspricht in Form, GefÄ\architektur, Innervation und Mastzellgehalt dem typischen Echsenconus, wÄhrend die relative Kapillarvermehrung und die Gestaltung ihrer EndotheloberflÄche mit den VerhÄltnissen im Pecten oculi vergleichbar ist. Der Conus papillaris vonChalcides chalcides nimmt somit hinsichtlich seiner Ultrastruktur eine gewisse Mittelstellung zwischen Conus und Pecten ein. Vermutlich dient auch der Conus der ErnÄhrung der avaskulÄren Echsennetzhaut bzw. dem Austausch der intraokulÄren Flüssigkeit.

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Fine structure of the conus papillaris in the eye ofChalcides chalcides (L.) (Lacertilia, Scincidae)

Summary The conal process (conus papillaris) within the eye ofChalcides chalcides was studied by light and electron microscopy. The finger-like organ is not sharply bordered against the vitreous body. The vascular system consists of a central arteriole, precapillary vessels, capillaries and venules. The endothelial cells of the arteriole contain some luminal processes and many electron dense cytosomes. The arteriole is enveloped by a single layer of loosely arranged muscle cells. The precapillary vessels are short and in general resemble the arteriolar structure. The capillaries are forming long loops. Their endothelial cells are richly studded with cellular organelles, especially mitochondria and micropinocytotic vesicles. Their luminal surface is organized into numerous regularly arranged about 1.2 m high microfolds, whereas the basal area is developed in a similar but slightly decreased way. The capillaries are enveloped by a connective tissue vessel sheath and some pericyte processes. The endothelium of the venules differs from that of the arteriole in a higher number of luminal processes and lesser dense cytosomes. The muscle cells are loosely connected to each other forming up to 3 layers. The intervascular space of the conus is occupied by connective tissue cells, mast cells, and pigment cells. Connective tissue cells are represented by small rounded elements. Pigmented cells contain many mitochondria, some filaments, dense areas beneath the inner leaflet of the unit membrane, and they are covered in part by basement membrane-like material. Mast cells are richly supplied by specific granules, on which exocytotic processes could not be observed. The conus is innervated by numerous unmyelinated vegetative nerve fibers. Schwann cells are rare and in part seem to be replaced by pigment cells. The nerve fibers probably innervate the central vessels. In addition, for the first time an innervation of conal pigment cells is found.Our findings are compared to those of the conal process of other lizards and those of the pecten oculi within the bird's eye. Form. vascular architecture, innervation and content of mast cells resemble the situation in typical lizards' conal processes, whereas lengthening of capillary vessels and organization of the luminal surface of their endothelial cells correspond to those found in birds' pecten oculi. Therefore, the conal process ofChalcides chalcides based on its ultrastructure, seems to represent a position in the middle between the typical conus papillaris of lizards and the pecten oculi of birds. It is assumed that like the pecten the conal process serves to the nutrition of the avascular retina and/or the exchange of intraocular fluids.

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Wir danken Frau I. Schroeder und Frl. S. Seidel (Münster) sowie Herrn D. Müller (Münster) und Herrn Ch. Fiebiger (Marburg) sehr herzlich für ihre technische Mitarbeit.     

In situ hybridization at the electron microscope level: hybrid detection by autoradiography and colloidal gold

In situ hybridization has become a standard method for localizing DNA or RNA sequences in cytological preparations. We developed two methods to extend this technique to the transmission electron microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope in situ hybridization. Radioactively labeled complementary RNA (cRNA) is hybridized to metaphase chromosomes deposited on electron microscope grids and fixed in 70 percent ethanol vapor; hybridixation site are detected by autoradiography. Specific and intense labeling of chromosomal centromeric regions is observed even after relatively short exposure times. Inerphase nuclei present in some of the metaphase chromosome preparations also show defined paatterms of satellite DNA labeling which suggests that satellite-containing regions are associate with each other during interphase. The sensitivity of this method is estimated to at least as good as that at the light microscope level while the resolution is improved at least threefold. The second method, which circumvents the use of autoradiogrphic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction is improved at least threefold. The second method, which circumvents the use of autoradiographic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction with an antibody against biotin and secondary antibody adsorbed to the surface of over centromeric heterochromatin and along the associated peripheral fibers. Labeling is on average ten times that of background binding. This method is rapid and possesses the potential to allow precise ultrastructual localization of DNA sequences in chromosomes and chromatin.     

IkappaB kinase is a critical regulator of chemokine expression and lung inflammation in respiratory syncytial virus infection

Respiratory syncytial virus (RSV) is the major etiologic agent of severe epidemic lower respiratory tract infections in infancy. Airway mucosal inflammation plays a critical role in the pathogenesis of RSV disease in both natural and experimental infections. RSV is among the most potent biological stimuli that induce the expression of inflammatory genes, including those encoding chemokines, but the mechanism(s) that controls virus-mediated airway inflammation in vivo has not been fully elucidated. Herein we show that the inoculation of BALB/c mice with RSV results in rapid activation of the multisubunit IkappaB kinase (IKK) in lung tissue. IKK transduces upstream activating signals into the rate-limiting phosphorylation (and proteolytic degradation) of IkappaBalpha, the inhibitory subunit that under normal conditions binds to the nuclear factor (NF)-kappaB complex and keeps it in an inactive cytoplasmic form. Mice treated intranasally with interleukin-10 or with a specific cell-permeable peptide that blocks the association of the catalytic subunit IKKbeta with the regulatory protein NEMO showed a striking reduction of lung NF-kappaB DNA binding activity, chemokine gene expression, and airway inflammation in response to RSV infection. These findings suggest that IKKbeta may be a potential target for the treatment of acute or chronic inflammatory diseases of the lung.