Endothelial and virgultar cell formations in the mammalian lymph node sinus: endothelial differentiation morphotypes characterized by a special kind of junction (<Emphasis Type="Italic">complexus adhaerens</Emphasis>)

The lymph node sinus are channel structures of unquestionable importance in immunology and pathology, specifically in the filtering of the lymph, the transport and processing of antigens, the adhesion and migration of immune cells, and the spread of metastatic cancer cells. Our knowledge of the cell and molecular biology of the sinus-forming cells is still limited, and the origin and biological nature of these cells have long been a matter of debate. Here, we review the relevant literature and present our own experimental results, in particular concerning molecular markers of intercellular junctions and cell differentiation. We show that both the monolayer cells lining the sinus walls and the intraluminal virgultar cell meshwork are indeed different morphotypes of the same basic endothelial cell character, as demonstrated by the presence of a distinct spectrum of general and lymphatic endothelial markers, and we therefore refer to these cells as sinus endothelial/virgultar cells (SEVCs). These cells are connected by unique adhering junctions, termed complexus adhaerentes, characterized by the transmembrane glycoprotein VE-cadherin, combined with the desmosomal plaque protein desmoplakin, several adherens junction plaque proteins including α- and β-catenin and p120 catenin, and components of the tight junction ensemble, specifically claudin-5 and JAM-A, and the plaque protein ZO-1. We show that complexus adhaerentes are involved in the tight three-dimensional integration of the virgultar network of SEVC processes along extracellular guidance structures composed of paracrystalline collagen bundle “stays”. Overall, the SEVC system might be considered as a local and specific modification of the general lymphatic vasculature system. Finally, physiological and pathological alterations of the SEVC system will be presented, and the possible value of the molecular markers described in histological diagnoses of autochthonous lymph node tumors will be discussed. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.

Effects of benzoylphenylurea on chitin synthesis and orientation in the cuticle of the Drosophila larva

Chitin is an essential constituent of the insect exoskeleton, the cuticle, which is an extracellular matrix (ECM) covering the animal. It is produced by the glycosyltransferase chitin synthase at the apical plasma membrane of epidermal and tracheal cells. To fulfil its role in cuticle elasticity and stiffness it associates with proteins, thereby adopting a stereotypic arrangement of helicoidally stacked sheets, which run parallel to the surface of the animal. One approach to understand the mechanisms of chitin synthesis and organisation is to dissect these processes genetically. However, since only a few genes coding for factors involved in chitin synthesis and organisation have been identified to date using the model arthropod Drosophila melanogaster insight arising from mutant analysis is rather limited. To collect new data on the role of chitin during insect cuticle differentiation, we have analysed the effects of chitin synthesis inhibitors on Drosophila embryogenesis. For this purpose, we have chosen the benzoylphenylurea diflubenzuron and lufenuron that are widely used as insect growth regulators. Our data allow mainly two important conclusions. First, correct organisation of chitin seems to directly depend on the amount of chitin synthesised. Second, chitin synthesis and organisation are cell-autonomous processes as insecticide-treated larvae display a mosaic of cuticle defects. As benzoylphenylurea are used not only as insecticides but also as anti-diabetic drugs, the study of their impact on Drosophila cuticle differentiation may be fruitful for understanding their mode of action on a cellular pathway that is seemingly conserved between vertebrates and invertebrates.     

Classification of HER2/neu status in gastric cancer using a breast-cancer derived proteome classifier

HER2-testing in breast and gastric cancers is mandatory for the treatment with trastuzumab. We hypothesized that imaging mass spectrometry (IMS) of breast cancers may be useful for generating a classifier that may determine HER2-status in other cancer entities irrespective of primary tumor site. A total of 107 breast (n = 48) and gastric (n = 59) cryo tissue samples was analyzed by IMS (HER2 was present in 29 cases). The obtained proteomic profiles were used to create HER2 prediction models using different classification algorithms. A breast cancer proteome derived classifier, with HER2 present in 15 cases, correctly predicted HER2-status in gastric cancers with a sensitivity of 65% and a specificity of 92%. To create a universal classifier for HER2-status, breast and nonbreast cancer samples were combined, which increased sensitivity to 78%, and specificity was 88%. Our proof of principle study provides evidence that HER2-status can be identified on a proteomic level across different cancer types suggesting that HER2 overexpression may constitute a unique molecular event independent of the tumor site. Furthermore, these results indicate that IMS may be useful for the determination of potential drugable targets, as it offers a quicker, cheaper, and more objective analysis than the standard HER2-testing procedures immunohistochemistry and fluorescence in situ hybridization.     

Interaction of the sliding clamp beta-subunit and Hda, a DnaA-related protein

In Escherichia coli, interactions between the replication initiation protein DnaA, the beta subunit of DNA polymerase III (the sliding clamp protein), and Hda, the recently identified DnaA-related protein, are required to convert the active ATP-bound form of DnaA to an inactive ADP-bound form through the accelerated hydrolysis of ATP. This rapid hydrolysis of ATP is proposed to be the main mechanism that blocks multiple initiations during cell cycle and acts as a molecular switch from initiation to replication. However, the biochemical mechanism for this crucial step in DNA synthesis has not been resolved. Using purified Hda and beta proteins in a plate binding assay and Ni-nitrilotriacetic acid pulldown analysis, we show for the first time that Hda directly interacts with beta in vitro. A new beta-binding motif, a hexapeptide with the consensus sequence QL[SP]LPL, related to the previously identified beta-binding pentapeptide motif (QL[SD]LF) was found in the amino terminus of the Hda protein. Mutants of Hda with amino acid changes in the hexapeptide motif are severely defective in their ability to bind beta. A 10-amino-acid peptide containing the E. coli Hda beta-binding motif was shown to compete with Hda for binding to beta in an Hda-beta interaction assay. These results establish that the interaction of Hda with beta is mediated through the hexapeptide sequence. We propose that this interaction may be crucial to the events that lead to the inactivation of DnaA and the prevention of excess initiation of rounds of replication.     

Physicochemical properties of bacterial glycopolymers in relation to bioactivity

An overview is given on the physicochemical properties of bacterial glycopolymers, i.e., pure oligo- and polysaccharides as well as glycolipids. Data from analysis of the chemical and physicochemical properties of various sugar polymers are summarized. Furthermore, data are presented on the thorough characterization of the most important class of bacterial glycopolymers, the lipopolysaccharides (LPS). These data comprise the chemical characterization, the gel to liquid crystalline phase transition behaviour of their acyl chains, the ultrastructural studies of their morphology, and the investigation of the types of aggregate structures present above the critical micellar concentration (CMC). Furthermore, the relevance of these data with respect to an understanding of the various biological effects elicited by LPS is discussed.     

Characterization of prominin-2, a new member of the prominin family of pentaspan membrane glycoproteins

Prominin/CD133 is a 115/120-kDa integral membrane glycoprotein specifically associated with plasma membrane protrusions in epithelial and non-epithelial cells including neuroepithelial and hematopoietic stem cells. Here we report the identification as well as molecular and cell biological characterization of mouse, rat, and human prominin-2, a 112-kDa glycoprotein structurally related to prominin (referred to as prominin-1). Although the amino acid identity between prominin-2 and prominin-1 is low (<30%), their genomic organization is strikingly similar, suggesting an early gene duplication event. Like prominin-1, prominin-2 exhibits a characteristic membrane topology with five transmembrane segments and two large glycosylated extracellular loops. Upon its ectopic expression in Chinese hamster ovary cells as a green fluorescent protein fusion chimera, prominin-2 was also found to be associated with plasma membrane protrusions, as revealed by its co-localization with prominin-1, suggesting a related role. Consistent with this, prominin-2 shows a similar tissue distribution to prominin-1, being highly expressed in the adult kidney and detected all along the digestive tract as well as in various other epithelial tissues. However, in contrast to prominin-1, prominin-2 was not detected in the eye, which perhaps explains why a loss-of function mutation in the human prominin-1 gene causes retinal degeneration but no other obvious pathological signs. Finally, we present evidence for the existence of a family of pentaspan membrane proteins, the prominins, which are conserved in evolution.     

Biophysical characterization of triacyl monosaccharide lipid a partial structures in relation to bioactivity

Synthetic triacyl glucosamine monosaccharide lipid A part structures corresponding to the non-reducing moiety of enterobacterial lipid A with an acyloxyacyl chain linked to position 3 of the glucosamine and an unbranched chain linked to position 2 (group 1) and vice versa (group 2) were analyzed biophysically: Fourier-transform infrared spectroscopy was performed to characterize the gel-to-liquid crystalline phase transition, the phosphate band contour, and the orientation of the glucosamine with respect to the membrane surface. Small-angle x-ray diffraction was applied for the elucidation of the supramolecular aggregate structure and, with that, of the molecular shape. With fluorescence resonance energy transfer the lipopolysaccharide-binding protein (LBP)-mediated intercalation of the lipid A partial structures into phospholipid liposomes was monitored. The physical data clearly exhibit a classification of the synthetic compounds into two groups: group 1 compounds have sharp phase transitions, indicating dense acyl chain packing and an inclination of the glucosamine backbone with respect to the membrane surface of 30 degrees with the phosphate buried in the membrane. Group 2 compounds have a very broad phase transition, indicating poorly packed acyl chains, and an inclination of -30 degrees with the phosphate group sticking outward. For the first group unilamellar phases are observed superimposed by a non-lamellar structure, and for the second one only multilamellar aggregate structures. The cytokine-inducing capacity in human mononuclear cells is relatively high for the first group and low or absent for the second group. Based on these data a model of the intra and intermolecular conformations is proposed which also extends the concept of "endotoxic conformation."     

Effects of morphine on the phenotypic expression of cell surface markers on murine lymphocytes.

There is a growing literature indicating that opioid abuse by human addicts and opioid administration to animals have profound effects on the immune system. In the present study, implantation of morphine pellets in mice was associated with reduced phenotypic expression of the cell surface antigens specific to T-lymphocytes and to helper and cytotoxic/suppressor T-lymphocyte subtypes. The effect of morphine, as measured by flow cytometry using monoclonal antibodies specific for antigens expressed by these cells, was dose-dependent. The decrease in expression of antigens was apparent as early as 24 h after morphine pellet implantation and continued for 3 days. In addition, a time-dependent increase in the expression of these antigens was observed in placebo- and morphine-treated mice, suggesting that the pellets had a small antigenic effect. However, at all times studied, morphine-treated mice had fewer cells expressing the antigens than placebo-treated mice. Our results provide additional evidence that the use of opioids by IV drug abusers compromises their immune function.     

Assessment of differences in ascomycete communities in the rhizosphere of field-grown wheat and potato

To assess effects of plant crop species on rhizosphere ascomycete communities in the field, we compared a wheat monoculture and an alternating crop rotation of wheat and potato. Rhizosphere soil samples were taken at different time points during the growing season in four consecutive years (1999-2002). An ascomycete-specific primer pair (ITS5-ITS4A) was used to amplify internal transcribed spacer (ITS) sequences from total DNA extracts from rhizosphere soil. Amplified DNA was analyzed by denaturing gradient gel electrophoresis (DGGE). Individual bands from DGGE gels were sequenced and compared with known sequences from public databases. DGGE gels representing the ascomycete communities of the continuous wheat and the rotation site were compared and related to ascomycetes identified from the field. The effect of crop rotation exceeded that of the spatial heterogeneity in the field, which was evident after the first year. Significant differences between the ascomycete communities from the rhizospheres of wheat in monoculture and one year after a potato crop were found, indicating a long-term effect of potato. Sequencing of bands excised from the DGGE gels revealed the presence of ascomycetes that are common in agricultural soils.