New natural noncannabinoid ligands for cannabinoid type-2 (CB2) receptors

Since the discovery that Delta 9-tetrahydrocannabinol and related cannabinoids from Cannabis sativa L. act on specific physiological receptors in the human body and the subsequent elucidation of the mammalian endogenous cannabinoid system, no other natural product class has been reported to mimic the effects of cannabinoids. We recently found that N-alkyl amides from purple coneflower (Echinacea spp.) constitute a new class of cannabinomimetics, which specifically engage and activate the cannabinoid type-2 (CB2) receptors. Cannabinoid type-1 (CB1) and CB2 receptors belong to the family of G protein-coupled receptors and are the primary targets of the endogenous cannabinoids N-arachidonoyl ethanolamine and 2-arachidonoyl glyerol. CB2 receptors are believed to play an important role in distinct pathophysiological processes, including metabolic dysregulation, inflammation, pain, and bone loss. CB2 receptors have, therefore, become of interest as new targets in drug discovery. This review focuses on N-alkyl amide secondary metabolites from plants and underscores that this group of compounds may provide novel lead structures for the development of CB2-directed drugs.

Differential effects of natural product microtubule stabilizers on microtubule assembly: single agent and combination studies with taxol, epothilone B, and discodermolide

A systematic comparison has been performed of the morphology and stability of microtubules (MTs) induced by the potent microtubule-stabilizing agents (MSAs) taxol, epothilone B (Epo B), and discodermolide (DDM) under GTP-free conditions. DDM-induced tubulin polymerization occurred significantly faster than that induced by taxol and Epo B. At the same time, tubulin polymers assembled from soluble tubulin by DDM were morphologically distinct (shorter and less ordered) from those induced by either taxol or Epo B, as demonstrated by electron microscopy. Exposure of MSA-induced tubulin polymers to ultrasound revealed the DDM-based polymers to be less stable to this type of physical stress than those formed with either Epo B or taxol. Interestingly, MT assembly in the presence of both DDM and taxol appeared to produce a distinct new type of MT polymer with a mixed morphology between those of DDM- and taxol-induced structures. The observed differences in MT morphology and stability might be related, at least partly, to differences in intramicrotubular tubulin isotype distribution, as DDM showed a different pattern of beta-tubulin isotype usage in the assembly process.

Detection of type III secretion genes as a general indicator of bacterial virulence

Type III secretion systems of Gram-negative bacteria are specific export machineries for virulence factors which allow their translocation to eukaryotic cells. Since they correlate with bacterial pathogenicity, their presence is used as a general indicator of bacterial virulence. By comparing the genetic relationship of the major type III secretion systems we found the family of genes encoding the inner-membrane channel proteins represented by the Yersinia enterocolitica lcrD (synonym yscV) and its homologous genes from other species an ideal component for establishing a general detection approach for type III secretion systems. Based on the genes of the lcrD family we developed gene probes for Gram-negative human, animal and plant pathogens. The probes comprise lcrD from Y. enterocolitica, sepA from enteropathogenic Escherichia coli, invA from Salmonella typhimurium, mxiA from Shigella sonnei, as well as hrcV from Erwinia amylovora. In addition we included as a control probe the flhA gene from E. coli K-12 to validate our approach. FlhA is part of the flagellar export apparatus which shows a high degree of similarity with type III secretions systems, but is not involved in pathogenicity. The probes were evaluated by screening a series of pathogenic as well as non-pathogenic bacteria. The probes detected type III secretion in pathogens where such systems were either known or were expected to be present, whereas no positive hybridization signals could be found in non-pathogenic Gram-negative bacteria. Gram-positive bacteria were devoid of known type III secretion systems. No interference due to the genetic similarity between the type III secretion system and the flagellar export apparatus was observed. However, potential type III secretion systems could be detected in bacteria where no such systems have been described yet. The presented approach provides therefore a useful tool for the assessment of the virulence potential of bacterial isolates of human, animal and plant origin. Moreover, it is a powerful means for a first safety assessment of poorly characterized strains intended to be used in biotechnological applications.

Evaluation of a Novel Barrier—Type Teat Dip Product Applied to Ewes Post-Weaning

Teats of 36 ewes (72 udder halves or teats) were dipped with an experimental barrier - type teat dip product to evaluate product persistency post weaning. Persistency was evaluated one to two times/day and scored positive if the teat end orifice was covered and protected. Persistency or the percentage of teats covered/protected at 36, 54, 72, 96, 132, and 156 hours was 100%, 93%, 89%, 63%, 35%, and 24% respectively. Ewes will be dipped again pre-lambing and both persistency and bacteriology (mastitis prevention) will be evaluated compared to 36 control ewes.

Lions-type compactness and Rubik actions on the Heisenberg group

In this paper we prove a Lions-type compactness embedding result for symmetric unbounded domains of the Heisenberg group. The natural group action on the Heisenberg group TeX is provided by the unitary group U(n) × {1} and its appropriate subgroups, which will be used to construct subspaces with specific symmetry and compactness properties in the Folland-Stein’s horizontal Sobolev space TeX. As an application, we study the multiplicity of solutions for a singular subelliptic problem by exploiting a technique of solving the Rubik-cube applied to subgroups of U(n) × {1}. In our approach we employ concentration compactness, group-theoretical arguments, and variational methods.

Characterization of the Agrobacterium tumefaciens VirB2 pilin of the VirB/D4 Type IV Secretion System

The Agrobacterium tumefaciens VirB/D4 type IV secretion system (T4SS) delivers oncogenic T-DNA and effector proteins to susceptible plant cells. This leads to the formation of tumors termed Crown Galls. The VirB/D4 T4SS is comprised of 12 subunits (VirB1 to VirB11 and VirD4), which assemble to form two structures, a secretion channel spanning the cell envelope and a T-pilus extending from the cell surface. In A. tumefaciens, the VirB2 pilin subunit is required for assembly of the secretion channel and is the main subunit of the T-pilus. The focus of this thesis is to define key reactions associated with the T4SS biogenesis pathway involving the VirB2 pilin. Topology studies demonstrated that VirB2 integrates into the inner membrane with two transmembrane regions, a small cytoplasmic loop, and a long periplasmic loop comprised of covalently linked N and C termini. VirB2 was shown by the substituted cysteine accessibility method (SCAM) to adopt distinct structural states when integrated into the inner membrane and when assembled as a component of the secretion channel and the T-pilus. The VirB4 and VirB11 ATPases were shown by SCAM to modulate the structural state of membrane-integrated VirB2 pilin, and evidence was also obtained that VirB4 mediates extraction of pilin from the membrane. A model that VirB4 functions as a pilin dislocase by an energy-dependent mechanism was further supported by coimmunoprecipitation and osmotic shock studies. Mutational studies identified two regions of VirB10, an N-terminal transmembrane domain and an outer membrane-associated domain termed the antennae projection, that contribute selectively to T-pilus biogenesis. Lastly, characterization of a VirB10 mutant that confers a ‘leaky’ channel phenotype further highlighted the role of VirB10 in gating substrate translocation across the outer membrane as well as T-pilus biogenesis. Results of my studies support a working model in which the VirB4 ATPase catalyzes dislocation of membrane-integrated pilin, and distinct domains of VirB10 coordinate pilin incorporation into the secretion channel and the extracellular T-pilus.

Dose Reduction in Standard Head CT: First Results from a New Scanner Using Iterative Reconstruction and a New Detector Type in Comparison with Two Previous Generations of Multi-slice CT

PURPOSE Computed tomography (CT) accounts for more than half of the total radiation exposure from medical procedures, which makes dose reduction in CT an effective means of reducing radiation exposure. We analysed the dose reduction that can be achieved with a new CT scanner [Somatom Edge (E)] that incorporates new developments in hardware (detector) and software (iterative reconstruction). METHODS We compared weighted volume CT dose index (CTDIvol) and dose length product (DLP) values of 25 consecutive patients studied with non-enhanced standard brain CT with the new scanner and with two previous models each, a 64-slice 64-row multi-detector CT (MDCT) scanner with 64 rows (S64) and a 16-slice 16-row MDCT scanner with 16 rows (S16). We analysed signal-to-noise and contrast-to-noise ratios in images from the three scanners and performed a quality rating by three neuroradiologists to analyse whether dose reduction techniques still yield sufficient diagnostic quality. RESULTS CTDIVol of scanner E was 41.5 and 36.4 % less than the values of scanners S16 and S64, respectively; the DLP values were 40 and 38.3 % less. All differences were statistically significant (p < 0.0001). Signal-to-noise and contrast-to-noise ratios were best in S64; these differences also reached statistical significance. Image analysis, however, showed "non-inferiority" of scanner E regarding image quality. CONCLUSIONS The first experience with the new scanner shows that new dose reduction techniques allow for up to 40 % dose reduction while still maintaining image quality at a diagnostically usable level.

Evidence for VirB4-mediated dislocation of membrane-integrated VirB2 pilin during biogenesis of the Agrobacterium VirB/VirD4 type IV secretion system.

Agrobacterium VirB2 pilin is required for assembly of the VirB/VirD4 type IV secretion system (T4SS). The propilin is processed by signal sequence cleavage and covalent linkage of the N and C termini, and the cyclized pilin integrates into the inner membrane (IM) as a pool for assembly of the secretion channel and T pilus. Here, by use of the substituted cysteine accessibility method (SCAM), we defined the VirB2 IM topology and then identified distinct contributions of the T4SS ATPase subunits to the pilin structural organization. Labeling patterns of Cys-substituted pilins exposed to the membrane-impermeative, thiol-reactive reagent 3-(N-maleimidopropionyl)biocytin (MPB) supported a topology model in which two hydrophobic stretches comprise transmembrane domains, an intervening hydrophilic loop (residues 90 to 94) is cytoplasmic, and the hydrophilic N and C termini joined at residues 48 and 121 form a periplasmic loop. Interestingly, the VirB4 ATPase, but not a Walker A nucleoside triphosphate (NTP) binding motif mutant, induced (i) MPB labeling of Cys94, a residue that in the absence of the ATPase is located in the cytoplasmic loop, and (ii) release of pilin from the IM upon osmotic shock. These findings, coupled with evidence for VirB2-VirB4 complex formation by coimmunoprecipitation, support a model in which VirB4 functions as a dislocation motor to extract pilins from the IM during T4SS biogenesis. The VirB11 ATPase functioned together with VirB4 to induce a structural change in the pilin that was detectable by MPB labeling, suggestive of a role for VirB11 as a modulator of VirB4 dislocase activity.

Identification and characterization of genes encoding phospholipid biosynthetic enzymes of {\it Saccharomyces cerevisiae\/}

Phospholipids are the major component of cellular membranes. In addition to its structural role, phospholipids play an active and diverse role in cellular processes. The goal of this study is to identify the genes involved in phospholipid biosynthesis in a model eukaryotic system, Saccharomyces cerevisiae. We have focused on the biosynthetic steps localized in the inner mitochondrial membrane; hence, the identification of the genes encoding phosphatidylserine decarboxylase (PSD1), cardiolipin synthase (CLS1), and phosphatidylglycerophosphate synthase (PGS1).^ The PSD1 gene encoding a phosphatidylserine decarboxylase was cloned by complementation of a conditional lethal mutation in the homologous gene in Escherichia coli strain EH150. Overexpression of the PSD1 gene in wild type yeast resulted in 20-fold amplification of phosphatidylserine decarboxylase activity. Disruption of the PSD1 gene resulted in 20-fold reduction of decarboxylase activity, but the PSD1 null mutant exhibited essentially normal phenotype. These results suggest that yeast has a second phosphatidylserine decarboxylation activity.^ Cardiolipin is the major anionic phospholipid of the inner mitochondrial membrane. It is thought to be an essential component of many biochemical functions. In eukaryotic cells, cardiolipin synthase catalyzes the final step in the synthesis of cardiolipin from phosphatidylglycerol and CDP-diacylglycerol. We have cloned the gene CLS1. Overexpression of the CLS1 gene product resulted in significantly elevated cardiolipin synthase activity, and disruption of the CLS1 gene, confirmed by PCR and Southern blot analysis, resulted in a null mutant that was viable and showed no petite phenotype. However, phospholipid analysis showed undetectable cardiolipin level and an accumulation of phosphatidylglycerol. These results support the conclusion that CLS1 encodes the cardiolipin synthase of yeast and that normal levels of cardiolipin are not absolutely essential for survival of the cell.^ Phosphatidylglycerophosphate (PGP) synthase catalyzes the synthesis of PGP from CDP-diacylglycerol and glycerol-3-phosphate and functions as the committal and rate limiting step in the biosynthesis of cardiolipin. We have identified the PGS1 gene as encoding the PGP synthase. Overexpression of the PGS1 gene product resulted in over 15-fold increase in in vitro PGP synthase activity. Disruption of the PGS1 gene in a haploid strain of yeast, confirmed by Southern blot analysis, resulted in a null mutant strain that was viable but had significantly altered phenotypes, i.e. inability to grow on glycerol and at $37\sp\circ$C. These cells showed over a 10-fold decrease in PGP synthase activity and a decrease in both phosphatidylglycerol and cardiolipin levels. These results support the conclusion that PGS1 encodes the PGP synthase of yeast and that neither phosphatidylglycerol nor cardiolipin are absolutely essential for survival of the cell. ^

Factors Associated with Bone Level Alterations at Implants with Inner-Cone Connection and Platform Switching

Purpose.This retrospective cohort study evaluated factors for peri-implant bone level changes (ΔIBL) associated with an implant type with inner-cone implant-abutment connection, rough neck surface, and platform switching (AT). Materials and Methods. All AT placed at the Department of Prosthodontics of the University of Bern between January 2004 and December 2005 were included in this study. All implants were examined by single radiographs using the parallel technique taken at surgery (T0) and obtained at least 6 months after surgery (T1). Possible influencing factors were analysed first using t-test (normal distribution) or the nonparametric Wilcoxon test (not normal distribution), and then a mixed model q variance analysis was performed. Results. 43 patients were treated with 109 implants. Five implants in 2 patients failed (survival rate: 95.4%).Mean ΔIBL in group 1 (T1: 6–12 months after surgery) was −0.65 ± 0.82mm and −0.69 ± 0.82mm in group 2 (T1: >12 months after surgery) (

The pre- and post-somatic segments of the human type I spiral ganglion neurons--structural and functional considerations related to cochlear implantation.

Human auditory nerve afferents consist of two separate systems; one is represented by the large type I cells innervating the inner hair cells and the other one by the small type II cells innervating the outer hair cells. Type I spiral ganglion neurons (SGNs) constitute 96% of the afferent nerve population and, in contrast to other mammals, their soma and pre- and post-somatic segments are unmyelinated. Type II nerve soma and fibers are unmyelinated. Histopathology and clinical experience imply that human SGNs can persist electrically excitable without dendrites, thus lacking connection to the organ of Corti. The biological background to this phenomenon remains elusive. We analyzed the pre- and post-somatic segments of the type I human SGNs using immunohistochemistry and transmission electron microscopy (TEM) in normal and pathological conditions. These segments were found surrounded by non-myelinated Schwann cells (NMSCs) showing strong intracellular expression of laminin-β2/collagen IV. These cells also bordered the perikaryal entry zone and disclosed surface rugosities outlined by a folded basement membrane (BM) expressing laminin-β2 and collagen IV. It is presumed that human large SGNs are demarcated by three cell categories: (a) myelinated Schwann cells, (b) NMSCs and (c) satellite glial cells (SGCs). Their BMs express laminin-β2/collagen IV and reaches the BM of the sensory epithelium at the habenula perforata. We speculate that the NMSCs protect SGNs from further degeneration following dendrite loss. It may give further explanation why SGNs can persist as electrically excitable monopolar cells even after long-time deafness, a blessing for the deaf treated with cochlear implantation.

Narrow dust jets in a diffuse gas coma: a natural product of small active regions on comets

Comets often display narrow dust jets but more diffuse gas comae when their eccentric orbits bring them into the inner solar system and sunlight sublimates the ice on the nucleus. Comets are also understood to have one or more active areas covering only a fraction of the total surface active with sublimating volatile ices. Calculations of the gas and dust distribution from a small active area on a comet’s nucleus show that as the gas moves out radially into the vacuum of space it expands tangentially, filling much of the hemisphere centered on the active region. The dust dragged by the gas remains more concentrated over the active area. This explains some puzzling appearances of comets having collimated dust jets but more diffuse gaseous atmospheres. Our test case is 67P/Churyumov–Gerasimenko, the Rosetta mission target comet, whose activity is dominated by a single area covering only 4% of its surface.

Mechanism of anti-tumor activity of adenovirus type 5 E1A in ovarian cancer

The adenovirus type 5 E1A gene was originally developed as a gene therapy to inhibit tumorigenicity of HER-2-overexpressing cells by transcriptional downregulation of HER-2. Our goal is to improve the overall efficacy of E1A gene therapy. To achieve this goal, we have conducted two preclinical experiments. ^ First, we hypothesized that Bcl-2 overexpressing ovarian cancer is resistant to E1A gene therapy. This hypothesis is based on that the 19 kDa protein product of the adenoviral E1B gene which is homologous to Bcl-2 inhibits E1A-induced apoptosis. Treating high Bcl-2-xpressing cells with E1A in combination with an antisense oligonucleotide to Bcl-2 (Bcl-2-ASO) resulted in a significant decrease in cell viability due to an increased rate of apoptosis relative to cells treated with E1A alone. In an ovarian cancer xenograft model, mice implanted with low HER-2, high Bcl-2 cells, treated with E1A plus Bcl-2-ASO led to prolonged survival. Bcl-2 thus may serve as a predictive molecular marker enabling us to select patients with ovarian cancer who will benefit significantly from E1A gene therapy. ^ Second, we elucidated the molecular mechanism governing the anti-tumor effect of E1A in ovarian cancer to identify a more potent tumor suppressor gene. We identified PEA-15 (phospho-protein enriched in astrocytes) upregulated in E1A transfected low HER-2-expressing OVCAR-3 ovarian cancer cell, which showed decreased cell proliferation. PEA-15 moved ERK from the nucleus to the cytoplasm and inhibited ERK-dependent transcription and proliferation. Using small interfering RNA to knock down PEA-15 expression in OVCAR-3 cells made to constitutively express E1A resulted in accumulation of phosphoERK in the nucleus, an increase in Elk-1 activity, DNA synthesis, and anchorage-independent growth. PEA-15 also independently suppressed colony formation in some breast and ovarian cancer cell lines in which E1A is known to have anti-tumor activity. We conclude that the anti-tumor activity of E1A depends on PEA-15. ^ In summary, (1) Bcl-2 may serve as a predictive molecular marker of E1A gene therapy, allowing us to select patients and improve efficacy of E1A gene therapy. (2) PEA-15 was identified as a component of the molecular mechanism governing the anti-tumor activity of E1A in ovarian cancer, (3) PEA-15 may be developed as a novel therapeutic gene. ^

Heavy metal and petroleum product concentrations in waters at the Obskaya 1 drill site

During recent years, the basins of the Kara Sea (Kamennomysskaya, Obskaya, and Chugor'yakhinskaya structures) in the Russian Federation have been considered as promising regions for oil and gas exploration and, simultaneously, as possible paths of relatively cheap pipeline and tanker transportation of hydrocarbons projected for recovery. On the other hand, exploration operations, recovery, and transportation of gas pose a considerable risk of accidents and environmental pollution, which causes a justified concern about the future state of the ecological system of the Gulf of Ob and the adjoining parts of the Kara Sea. Therefore, regular combined environmental investigations (monitoring) are the most important factor for estimating the current state and forecasting the dynamics of the development of estuary systems. The program of investigations (schedule, station network, and measured parameters) is standardized in accordance with the international practice of such work and accounts for the experience of monitoring studies of Russian and foreign researchers. Two measurement sessions were performed during ecological investigations in the region of exploration drilling: at the beginning at final stage of drilling operations and borehole testing; in addition, natural parameters were determined in various parts of the Ob estuary before the beginning of investigations. Hydrophysical and hydrochemical characteristics of the water medium were determined and bottom sediments and water were analyzed for various pollutants (petroleum products, heavy metals, and radionuclides). The forms of heavy-metal occurrence in river and sea waters were determined by the method of continuous multistep filtration, which is based on water component fractionation on membrane filters of various pore sizes. These investigations revealed environmental pollution by chemical substances during the initial stage of drilling operations, when remains of fuels, oils, and solutions could be spilled, and part of the chemical pollutants could enter the environment. Owing to horizontal and vertical turbulent diffusion, wave mixing, and the effect of the general direction of currents in the Ob estuary from south to north, areas are formed with elevated concentrations of the analyzed elements and compounds. However, the concentration levels of chemical pollutants are practically no higher than the maximum admissible concentrations, and their substantial dissipation to the average regional background contents can be expected in the near future. Our investigations allowed us to determine in detail the parameters of anthropogenic pollution in the regions affected by hydrocarbon exploration drilling in the Obskii and Kamennomysskii prospects in the Gulf of Ob and estimate their influence on the ecological state of the basin of the Ob River and the Kara Sea on the whole.

Mesozooplankton biomass data from the Kapisigdlit an inner fjord branch of the Godthaabsfjord system, West Greenland, 2010

Sampling was conducted from March 24 to August 5 2010, in the fjord branch Kapisigdlit located in the inner part of the Godthåbsfjord system, West Greenland. The vessel "Lille Masik" was used during all cruises except on June 17-18 where sampling was done from RV Dana (National Institute for Aquatic Resources, Denmark). A total of 15 cruises (of 1-2 days duration) 7-10 days apart was carried out along a transect composed of 6 stations (St.), spanning the length of the 26 km long fjord branch. St. 1 was located at the mouth of the fjord branch and St. 6 was located at the end of the fjord branch, in the middle of a shallower inner creek . St. 1-4 was covering deeper parts of the fjord, and St. 5 was located on the slope leading up to the shallow inner creek. Mesozooplankton was sampled by vertical net tows using a Hydrobios Multinet (type Mini) equipped with a flow meter and 50 µm mesh nets or a WP-2 net 50 µm mesh size equipped with a non-filtering cod-end. Sampling was conducted at various times of day at the different stations. The nets were hauled with a speed of 0.2-0.3 m s**-1 from 100, 75 and 50 m depth to the surface at St. 2 + 4, 5 and 6, respectively. The content was immediately preserved in buffered formalin (4% final concentration). All samples were analyzed in the Plankton sorting and identification center in Szczecin (www.nmfri.gdynia.pl). Samples containing high numbers of zooplankton were split into subsamples. All copepods and other zooplankton were identified down to lowest possible taxonomic level (approx. 400 per sample), length measured and counted. Copepods were sorted into development stages (nauplii stage 1 - copepodite stage 6) using morphological features and sizes, and up to 10 individuals of each stage was length measured.